CHARACTERIZATION OF THE STRUCTURE OF THE L178H MUTANT OF APOLIPOPROTEIN A-I Megan Jessemyn Schifer Cochran B.A., Denison University, 2005 THESIS Submitted in partial satisfaction of the requirements for the degree of MASTER OF SCIENCE in CHEMISTRY (Biochemistry) at CALIFORNIA STATE UNIVERSITY, SACRAMENTO SUMMER 2012 © 2012 Megan Jessemyn Schifer Cochran ALL RIGHTS RESERVED ii CHARACTERIZATION OF THE STRUCTURE OF THE L178H MUTANT OF APOLIPOPROTEIN A-I A Thesis by Megan Jessemyn Schifer Cochran Approved by: __________________________________, Committee Chair Linda M. Roberts __________________________________, Second Reader Thomas J. Savage __________________________________, Third Reader Roy W. Dixon ____________________________ Date iii Student: Megan Jessemyn Schifer Cochran I certify that this student has met the requirements for format contained in the University format manual, and that this thesis is suitable for shelving in the Library and credit is to be awarded for the thesis. ___________________________, Graduate Coordinator Susan Crawford Department of Chemistry iv ___________________ Date Abstract of CHARACTERIZATION OF THE STRUCTURE OF THE L178H MUTANT OF APOLIPOPROTEIN A-I by Megan Jessemyn Schifer Cochran The top two leading causes of death worldwide in 2004 (stroke and ischemic heart attack) are both cardiovascular diseases (CVD). High-density lipoprotein and its major protein apolipoprotein A-I (apoA-I, 243 residues) are each inversely associated with risk of CVD and therefore believed to have cardio-protective properties. However, fibrillar apoA-I has been found in arterial plaque, indicating that fibrillogenesis of apoA-I may actually contribute to the development of CVD. Fourteen fibril forming variants of apoA-I are known whose mutations cluster in two regions of the protein's N-terminal helical domain, the "inside" cluster (residues 26107) and the "outside" cluster (residues 154-178). A mutant from the "inside" cluster, G26R, was previously characterized by members of our research group (Lagerstedt, J.O., Cavigiolio, G., Roberts, L.M., Hong, H.S., Jin, L.W., Fitzgerald, P.G., Oda, M.N., Voss, J.C. 2007. Biochemistry 46, 9693-9699). The G26R protein was found to exhibit an increase in beta-strand structure, N-terminal protease sensitivity and hydrophobic surface area compared to wild-type apoA-I. We proposed that the v alteration at position 26 disrupts the N-terminal helical bundle, destabilizing it and promoting the formation of misfolded fibrils. To determine whether mutations in the "outside" cluster produce similar effects on the protein, and therefore similar misfolded fibrils, the structure of the full-length L178H variant was studied using ANS binding, limited proteolysis, and intrinsic fluorescence quenching. In this thesis work, it is shown that the L178H mutation results in increased N-terminal protease sensitivity and hydrophobic surface area, but with no change in the nonpolar environment of the protein's tryptophan residues (Petrlova, J., Duong, T., Cochran, M., Axelsson, A., Morgelin, M., Roberts, L.M., Lagerstedt, J.O. 2012 J. Lipid Res. 53, 390-398). Thus, mutations from the two clusters result in similar structural effects on the protein. ANS binding was also used to analyze conformational changes over time in protein incubated at either 4oC or 37oC. L178H exhibited earlier and more substantial changes in hydrophobic surface area at both temperatures compared to wild-type apoA-I, which likely reflects the increased propensity of L178H to form fibrils. The effects of the L178H mutation are reviewed in the context of recent structural models of apoA-I. , Committee Chair Linda M. Roberts ______________________ Date vi ACKNOWLEDGMENTS First and foremost, I would like to thank my husband for his constant support and encouragement. Whenever I was discouraged, you were there to support me. Thank you for the many Saturday and Sunday lunches in the Union and the many weekend nights spent at home, so I could get up early the next morning. I know you had to sacrifice for me, and you never complained, instead always being my biggest supporter. Thank you. If not for Linda, I wouldn’t be here today. You let me work in your lab on the weekends to accommodate my schedule and put up with many calls and questions while at home with your family. You have been patient with my questions, flexible with my projects, and helpful with my thesis, especially from across the country. Thank you. I would like to thank my parents for not protesting when I wanted to move across the country for my Masters and my marriage. Both were worth it. Thank you. To Aunt Sara, for sparking my interest in science in the first place, thank you. Who knows what I would be today if not for you and your class? I’ll never forget that, “Chemistry is everywhere.” Thank you. To the other students in Dr. Roberts’ lab over the years, and there have been too many of you to name, thank you. For being there to talk, to vent, to discuss problems, to babysit me on the weekends, and to curse protein, it’s been quite an interesting seven years. Thank you. vii TABLE OF CONTENTS Page Acknowledgments ...................................................................................................... vii List of Tables ............................................................................................................... ix List of Figures............................................................................................................... x INTRODUCTION…. ................................................................................................... 1 MATERIALS AND METHODS ............................................................................... 38 RESULTS. .................................................................................................................. 46 DISCUSSION............................................................................................................. 65 CONCLUSION .......................................................................................................... 75 References ................................................................................................................. 77 viii vii i LIST OF TABLES Tables Page 1. Comparison of models of the lipid-free tertiary structure of apo A-I…..….....15 2. Summary of the apo A-I mutants found to date..………………………...…...34 3. Comparison of proteolytic cleavage fragment sequences ………………...….54 4. Comparison of estimated t ½ of ANS binding at various temperatures….........59 5. Comparison of Stern-Volmer constant, Ksv, of protein at various temperatures ……..……………………………………….………..................64 6. Comparison of relative fluorescence intensities of apo A-I proteins at 485 nm.……...…………………..…………………………………….............66 ix LIST OF FIGURES Figures Page 1. The arterial overview of the development of atherosclerosis ………………... 5 2. Mechanism of reverse cholesterol transport compared to forward transport…..8 3. The role of apo A-I in reverse cholesterol transport ……………………….….9 4. Structural organization of apolipoprotein A-I gene .…………………………11 5. Comparison of secondary structures of lipid-free apo A-I determined using HD-X and EPR…...............................…….………………………..…..14 6. Proposed model for lipid-free apo A-I ……….....……………………………17 7. Ribbon model of the first full length all-atom model of lipid-free apo A-I…..18 8. Comparison of apo A-I structure determined using EPR of the C-terminal domain and the full length structure…………………................………….....20 9. The crystal structure of 1-184 apo A-I ………...……………………………..21 10. Stereoviews of 1-43 apo A-I as a monomer, dimer, and tetramer……….….24 11. Models of lipid-bound apo A-I on discoidal HDL …………………………...26 12. The initial lipid-binding step of apo A-I mediated through helix 9 and 10 .... 27 13. Generic -sheet amyloid fibril structure, picture of actual -sheet amyloid fibrils, and a model of an-helical fibril supported by an electron micrograph .......................................................................................................30 14. Mechanisms of amyloid fibril formation……………………………………..31 15. Dimeric structure of apo A-I based on the apo A-I 1-43 structure……….... 35 16. L178H pilot expression ………………………………………………………47 17. Metal-ion affinity chromatography of L178H apo A-I ……………………....48 18. Analysis of proteolytic cleavage upon extended incubation…...……………. 50 19. Limited proteolysis of WT and L178H apo A-I ……...…………………...... 53 20. ANS binding to WT and L178H apo A-I...…………………………………..57 21. Percent loss of ANS binding at 485 nm in L178H and WT over 28 days…....58 22. Percent loss of intrinsic fluorescence of apo A-I proteins………...………….61 23. Stern-Volmer plot of intrinsic fluorescence quenching of L178H apo A-I at day 2 ………………………………………………………………………….63 x 24. Representation of chymotryptic hot spots in wild type, G26R, L178H apo A-I, and N-terminal deletion mutants………………………………….….......…...68 25. Position of amyloidogenic mutations in structural models of apo A-I……….70 26. Summary of important changes in WT and L178H apo A-I at 4ºC and 37ºC over 28 days because of helix ………………………………………………..74 xi 1 INTRODUCTION According to the World Health Organization (WHO), the top two leading causes of death worldwide in 2004 were two different cardiovascular diseases (CVD), ischemic heart disease and cerebrovascular disease, which together accounted for 19.9% of all deaths (145). That percentage is projected to increase to 26.3 % of all deaths worldwide from CVD in the year 2030. Another CVD, hypertensive heart disease, is projected to increase from 14th leading cause of death to the seventh. All CVDs together, including rheumatic heart disease, coronary artery disease (CAD), and others, caused 30.5 % of deaths worldwide in 2008 according to WHO and 34.2 % of deaths in the United States that same year (146). CVD is a leading target of medical and pharmaceutical intervention, and understanding the mechanisms and risk factors underlying CVD is critical in developing effective prevention and treatment. Lipoproteins have been used for decades to predict risk of CVD. Lipoproteins are molecules consisting of a layer of protein surrounding a lipid core that carry cholesterol and lipids to the liver and other body tissues. Lipoproteins vary by size, shape, protein content and density, and each is associated with CVD in a different way. The two most commonly known lipoproteins are high density lipoprotein (HDL, or "good" cholesterol) and low density lipoprotein (LDL, or "bad" cholesterol). The beneficial effects of HDL are well documented. For example, a 2-3% decreased risk of CAD is associated with an increased concentration of only 1 mg/dL blood plasma highdensity lipoprotein (HDL) (normal HDL blood level is > 40 mg/dL) (3). Conversely, 2 increased levels of low-density lipoprotein (LDL) are associated with an increased risk of CVD (3). An early survey known as the Framingham study showed that only a weak association exists for increased LDL levels and increased risk for CAD (122). Despite that, for over thirty years, levels of LDL and total cholesterol (TC) have been used to predict risk of CVD (135). In the Framinghan study, it was shown that low HDL levels are a major risk factor for each main manifestation of CAD, such as angina pectoris (chest pain), acute myocardial infarction (heart attack), and heart failure. Overall, HDL levels had a significant inverse correlation association with heart disease when other standard risk factors were taken into consideration (122). Roughly 70% of the protein in HDL is comprised by apolipoprotein A-I (apo AI), a flexible protein that is part of the apolipoprotein family, which includes many related proteins such as apo A, apo B, apo C, apo D, apo E, apo H, and apo M (1, 2, 84, 174). Within the past few years, it has been discovered that apolipoprotein A-I levels are as accurate as HDL levels when used for prediction of cardiac events (4, 5). It has been suggested that apo A-I levels are a better predictor of cardiovascular events than LDL levels (4). And more recently, the ratio of apo B/apo A-I was suggested to be a better indicator of the risk of CVD than total cholesterol/HDL which has been used by physicians for decades (189). Apo A-I's significance is reflected in the high prevalence of premature CAD exhibited in people with a mutation in the apo A-I gene that leads to severe HDL deficiency (79). 3 Apart from gender and age, the main predictor of CAD in a group of low-risk patients studied in 2001 was blood serum apo A-I level which is known to vary with age in females, but not males (91). In the INTERHEART study, it was determined that the strongest risk factors of heart attack were current smoking and apo B/apo A-I ratio and these results are the same for all ages, sexes, and regions of the world (93). To further underscore the importance of blood apo A-I levels, premature atherosclerosis, an inflammatory disease of the arteries, is found in the majority of patients with severe HDL and apo A-I deficiencies; it has been identified in people as young as 30 years old (3, 79) In fact, it may be initiated by low levels of apo A-I in otherwise low-risk patients (91). Atherosclerosis The main cause of CAD is atherosclerosis which is often referred to as hardening of the arteries. It is an inflammatory disease of the arteries which can lead to plaque formation (8, 104). Unstable plaques can cause narrowing of the arteries (stenosis), restriction in blood supply (ischemia), or a complete blockage (infarction) (81, 104). As atherosclerosis progresses, atherosclerotic lesions (atheroma) form. The initiation, propagation, and activation of these lesions in atherosclerosis occur largely because of immune mechanisms, in conjunction with metabolic risk factors (104). In fact, the process of atherosclerosis can be characterized by different types of arterial lesions that represent different stages in the inflammatory response. Such lesions may be present anytime in a person’s life with the earliest type of lesion, the purely 4 inflammatory fatty streak, sometimes occurring in infancy and childhood (81). These streaks can completely disappear, especially when occurring in childhood, or progress to form full-on atherosclerotic lesions (104). As with any other inflammatory response, an “injury” or other event must cause atherosclerosis. It is hypothesized that the “injury” for atherosclerosis could be elevated or modified LDL levels, free radicals caused by smoking, stress, or diabetes, genetic modifications, infectious microorganisms, or other unknown factors (81, 112, 123). Once the arterial injury occurs, the body tries to compensate to restore equilibrium in the arterial endothelium. These responses include increased endothelial permeability and adhesiveness, particularly to leukocytes and platelets, increased procoagulant properties in the endothelium, and formation of vasoactive molecules, inflammatory cytokines, and growth factors (81, 104). If this response is successful, the process can be reversed and the “injury” can disappear, as in the case of fatty streaks in children. However, if the response is unsuccessful, by not neutralizing or removing what caused the injury in the first place, the response itself can continue indefinitely (81). Continued inflammation then results in up-regulation of leukocyte and endothelial adhesion molecules, which can stimulate recruitment and adherence of monocytes and T cells at lesion-prone sites in the endothelium, such as the vascular side of the aortic valve, which is a critical part in the process of plaque formation (123). Then, as they are recruited, lipid-associated monocytes and macrophages (foam cells) together with T-lymphocytes form fatty streaks, the precursor to atherosclerotic lesions 5 (87, 57, 104). Next smooth muscle cells migrate and proliferate, then mixing with inflamed regions to form intermediate lesions. These lesions can then thicken the artery wall, for which it compensates by dilating the artery, called remodeling (177) (Figure 1). Figure 1. The arterial overview of the development of atherosclerosis (177). With cell accumulation and lesion progression, fibrous tissues tend to cover the lesion with a fibrous cap for protection of the lumen which may then form a necrotic core as a result of apoptosis and necrosis. The lesions continue to expand and eventually the artery cannot dilate anymore and the pathway ends up being blocked, altering the blood flow. At sites covering the lesion, the fibrous cap can rupture enabling the blood coming in contact with the plaque to coagulate. Platelets activated as a result of this 6 coagulation then lead to thrombus (blood clot) formation (81) (Figure 1). If the thrombus blocks a vessel, an acute myocardial infarction can occur, or alternately, the thrombus could also be reabsorbed. The healing response triggered by thrombosis can again stimulate smooth muscle migration, proliferation and extracellular matrix synthesis resulting in an even thicker fibrous cap (Figure 1). These thicker plaques may then be less susceptible to rupture and thrombosis, but instead can cause severe chest pain related to stenosis and ischemia. Apo A-I in Atherosclerosis Apo A-I influences development of atherosclerosis in many ways. First, it acts as an anti-inflammatory agent by preventing monocyte adherence to endothelial cells and their subsequent activation, which can prevent the entire process of atherosclerosis (10, 11). It can also stabilize antiatherogenic antioxidants like human paraoxonase/ arylesterase (9). By inhibiting procoagulant activity in erythrocytes, apo A-I protects against blood clots (12). Apo A-I can also bind and protect against lipopolysaccharides (LPS or endotoxin) by preventing an LPS-bound complex from binding a receptor on monocytes/ macrophages and thereby preventing initiation of the cytokine release found in atherosclerosis (52). This binding can also prevent the endotoxin-caused acceleration of atherosclerosis (179). Reverse Cholesterol Transport One of the most studied anti-atherogenic properties of apo A-I is reverse cholesterol transport (RCT) in which apo A-I promotes active transfer of excess 7 cholesterol from peripheral cells, particularly macrophage foam cells, to HDL and then to the liver for catabolism (13,14, 139) (Figure 2). Other than the obvious importance RCT plays in preventing atherosclerosis by cholesterol removal, it may also relate to inflammation. Recently, inflammation has been shown to retard and impair (sometimes by selective attenuation) RCT in vivo at multiple places along the pathway which has led to the possibility that impaired RCT may be a link between low grade inflammation and atherosclerosis (185). The reverse cholesterol transport pathway proceeds in four steps: cholesterol efflux from cells in peripheral tissues, lecithin: cholesterol acyl transferase (LCAT) -mediated esterification of HDL-associated cholesterol, receptormediated transfer of the ester to the liver, and clearance from the body in the liver for secretion in bile and feces (14, 185). 8 Figure 2. Mechanism of reverse cholesterol transport compared to forward transport. FC, free cholesterol; CE, cholesteryl ester; VLDL, very low density lipoprotein; HDL, high density lipoprotein. Arrows indicate the direction of net transport (14). Apo A-I plays a critical role in RCT (Figure 3). First, lipid-free apo A-I stimulates efflux of phospholipids (PL) and cholesterol in macrophages and other nonhepatic tissues by binding ATP binding cassette A1 (ABCA1, also called CERP) transport protein (67, 174). Apo A-I’s amphipathic α-helices bind to a hydrophobic site on ABCA1 thereby forming a time, temperature, and concentration dependent complex with it (98, 155). To form the complex, a helical segment of the protein inserts into an adjacent region of the plasma membrane bilayer, composed of free cholesterol (FC) and PL in a perturbed lipid domain created by ABCA1 activity (67, 98, 155). The initial 9 lipidation of apo-A-I, primarily by plasma membrane PL, leads to the formation of "lipid-poor” apo A-I (14, 97, 98, 155, 174). Figure 3. The role of apo A-I (labeled as A-I) in reverse cholesterol transport (165). CE, cholesteryl ester. FC, free cholesterol. Continued acquisition of lipid converts “lipid poor” apo A-I to discoidal (also called nascent) HDL. While the structure of synthetic discoidal HDL has been thoroughly investigated (described below), the structure of lipid-poor apo A-I is not completely understood. FC in discoidal HDL is converted to cholesteryl esters (CE) and lyso-phosophatidylcholines by LCAT on the surface of HDL by a transesterification reaction involving an Asp-His-Ser catalytic triad in the active site of LCAT (14, 65). The esterification results in migration of CEs into the particle interior which leads to formation of large spherical HDL particles. Spherical HDL then transports the cholesterol to the liver (13-15) where apo A-I (as part of HDL) interacts with scavengerreceptor BI (SR-BI), causing the liver to uptake the cholesterol into cells (16, 110). Much of the cholesterol is then converted to bile acid and excreted. 10 HDL and apo A-I are prime candidates for further study to be used in CVD prevention and treatment. Because apo-A-I comprises most of the protein in HDL, elucidating its structure is essential in determining HDL function. However, progress toward understanding HDL and its function in preventing CVD has been slow due to a lack of high resolution structural and structure-related functional information for both HDL and its major protein, apo A-I. Apolipoprotein A-I Structure Exon Structure The apo A-I gene resides on the long arm of chromosome 11 in a multi-gene cluster of about 22 kilobases with apo C-III, apo A-IV, and apo A-V (30, 31, 50). The gene structure of apo A-I is composed of four exons separated by three introns, removed during splicing, similar to most other human apolipoproteins (Figure 4A). The first exon is untranslated. The latter three-fourths of Exon 2 codes for signal peptides. Exon 3 codes for signal peptides, the prosegment which is cleaved before becoming mature apo A-I, and amino acids 1-43 in the mature apo A-I. Exon 4 codes for amino acids 44-243 in apo A-I (1). 11 Figure 4. A. Structural organization of apolipoprotein A-I gene. Boxes represent exons. Open bars represent untranslated regions. Shaded bars represent the signal sequence region. Checked region represents the prosegment. Solid bars represent regions coding mature protein. Horizontal lines represent introns. The numbers below represent the sizes of introns or exons in number of nucleotides. Drawing is not to scale (50, 61). B. Placement of amphipathic helices of apo A-I according to analysis by SAD. Exon 3 (1-43) is labeled G* and Exon 4 helices (44-243) are labeled H1 through H10 (212). Lipid-free protein structure Primary and secondary structure Although lipid-free apo A-I constitutes less than 10% of plasma apo A-I, it has received intense study because of its importance in reverse cholesterol transport. It is important to understand the lipid-free structure because this form initiates lipidbinding, may interact with cellular receptors such as ABCAI, and is likely to be the form from which amyloid structure develops. Apo A-I is synthesized in the liver and small intestine in the form of preproapo A-I which has 267 residues (50, 119, 120, 125). The first eighteen amino acids form a hydrophobic signal sequence peptide which undergoes co-translational cleavage by signal peptidase of the endoplasmic reticulum to yield the 249-residue proapo A-I (119, 125). The remaining six amino acids are cleaved extra-cellularly by a protease to generate mature plasma apo A-I 12 (119, 120, 125). The amino acid sequence of mature human apo A-I contains only 18 of the 20 different amino acids (127), lacking Ile and Cys. Apo A-I (as well as many other apolipoproteins) contains a series of highly homologous repeating amphipathic helices; an amphipathic helix contains polar and nonpolar faces oriented opposite each other along the helix’s long axis (151). The apo A-I sequence was analyzed using computer programs developed to identify different classes of amphipathic helices by the Segrest group (73). These studies identified within residues 44-243 ten contiguous 11- and 22- residue amphipathic helices (which are often numbered 1-10), punctuated by proline residues similar to other apolipoproteins (Figure 4B) (1, 147, 152). Helices 3 and 9 are comprised of 11 residues while the other eight are 22-residues long (147). While sequence analysis has been helpful in understanding the role of the amphipathic helix in apo A-I structure and function, the protein’s dynamic nature has made it difficult to obtain definitive structural information for the lipid-free protein. This is evidenced by the conflicting structural details emerging from a variety of methods. The structure of lipid-free apo A-I has been studied by limited proteolysis, circular dichroism (CD), X-ray crystallography, electron paramagnetic resonance spectroscopy (EPR), Forster resonance energy transfer (FRET) studies, hydrogen deuterium exchange (HD-X), nuclear magnetic resonance (NMR), chemical cross linking, and mass spectrometry (2, 33, 36, 39, 70, 149, 193, 212, 224). In spite of intensive study using these methods, it has been difficult to firmly establish either the 13 secondary or the tertiary structure of the lipid-free protein. For example, while CD measurements consistently indicate lipid-free apo A-I has a helical content of 50-60 %, the placement of helices has not been conclusively established (33, 41, 70). Furthermore, the existence of a small but significant amount of beta structure has been proposed by some (36, 39, 224) but disputed by others (193). By CD, about 8% of the protein has beta structure which could be important in the development of amyloid structure in some variants and under some conditions (33, 36, 39, 40, 41, 70, 224). One of the more recent and promising techniques for measuring the secondary structure of lipid-free apo A-I is EPR spectroscopy using spin-labeled protein. The motion of the label at different positions along the sequence can reveal, through its periodicity, the secondary structure of a segment of protein. This information, coupled with the solvent accessibility of the spin label to different quenchers, reveals whether a sequence of residues is helical, beta structure, or neither. EPR analysis of apo A-I revealed the positions of α helices (residues 6-34 and 50-98), β strands (residues 4049), or areas without either (residues 35-39), which are assumed to be random coils (36, 39, 224). In addition to secondary structure, tertiary structure can be determined when EPR is used with dipolar coupling, discussed below (36). HD-X coupled with mass spectrometry was also used in secondary structure analysis. In this technique, hydrogen is replaced by deuterium in the amides of the protein backbone. After this exchange is carried out, the protein is cleaved at various sites resulting in overlapping peptide fragments which are then separated by HPLC. 14 The amount of exchange that occurred can then be determined by analysis of the peptide fragment using mass spectrometry because the deuterium is heavier than the hydrogen. That information can be used to determine solvent accessibility and therefore protein secondary structure. In this analysis, 48 ± 3% of lipid-free apo A-I was determined to have helical structure, which is in the range reported using CD studies (193). The location of helix according to HD-X and EPR can be compared (Figure 5). The helical content of the EPR and HD-X structures is similar, but the locations are quite different. The helical placements differ the most in the C-terminus. In addition the EPR study places β-strands in both termini whereas the HD-X study has none. This is a particularly important controversy to resolve because the EPR authors implicate βstrand structure in critical apo A-I functions, including lipid-binding. In addition, establishing β-strand structure, if any, is critical for understanding amyloid formation. This diagram shows the H-DX determined helical regions appear to be the most in line with those predicted by Rogers, et. al in 1998 using limited proteolysis (70, 224) Figure 5. Comparison of secondary structures of lipid-free apo A-I determined using HD-X and EPR. Light gray boxes with border represent α helices, dark grey boxes represent β strands, and light grey borderless boxes represent random coil/ β turn. Arrows represent sites displaying intermolecular interaction (224). 15 Tertiary structure A number of models of lipid-free apo A-I tertiary structure have been proposed using data from a variety of techniques, including limited proteolysis (69, 70), FRET (149), X-ray crystallography (212), chemical cross-linking coupled with mass spectrometry (40), and EPR (36, 39, 224). Collectively, these studies suggest the lipidfree protein has two domains, an amino-terminal helix bundle from residues 1-190 and a more loosely folded and less structured, smaller carboxy-terminal domain with some helix and/or beta-strand structure. The various models that have been proposed are summarized in table 1. Table 1. Comparison of models of the lipid-free tertiary structure of apo A-I Model Main Features Techniques Borhani, et al Proposed to represent lipidCrystallization bound conformation (44-243) Rogers, et al Model proposes helix bundle Denaturation, analytical from 1-190, unstructured ultra centrifugation, carboxy- terminal domain limited proteolysis Brouillette, et Supports globular helixSpin labeling, FRET al bundle and not helical hairpin Davidson, et First all- atom model, Cross linking chemistry, al depicting a helical bundle high resolution mass structure with termini close spectrometry, molecular to each other modeling Voss, et al Dynamic solution structure, Spin label EPR contains beta-sheet spectroscopy Mei, et al Half-circle two-domain Crystallization dimer structure of helical repeats, truncated protein (1-184). References 7 (1997) 70 (1998) 149 (2005) 40 (2005) 36, 39, 224 (2007, 2012) 212 (2011) 16 Limited proteolysis involves the addition of a protease to the protein and analysis of the cleavage pattern that occurs. It is assumed the protein is loosely folded and/or has a flexible structure at the cleavage positions (206). Several limited proteolysis experiments have been performed on apo A-I using different proteases. Together, these studies reveal a major cleavage point around residue 190 (Y192 when chymotrypsin is used, E191 when S. aureus V8 is used) which is the junction between proposed amino and carboxy-terminal domains (69). A series of studies on full-length and terminal deletion mutants employed limited proteolysis along with stability analysis and analytical centrifugation to develop one of the first models of lipid-free apo A-I (Figure 6). While proposed more than a decade ago, many aspects of this model are upheld by recent research using a variety of techniques. In it, an equilibrium exists between a bundle of six helices and a helical hairpin with five helices. In both models, the carboxy terminus is unstructured. 17 Figure 6. Proposed model for lipid-free apo A-I. This is presented in equilibrium between helical bundle (A) and helical hairpin (B) (70). Cylinders represent helices. The carboxy and amino termini are identified as C and N respectively. Solid circles represent the locations of methionine residues. Open arrow heads represent the positions of cleavage in methionine-reduced apo A-I. Dashed lines represent probable secondary structure which may or may not be helices (70). FRET was used to distinguish between the helical bundle and the helical hairpin conformations (149). FRET measures the energy transfer between two fluorophores, in this case tryptophan and the external fluorescent probe (AEDANS, aminoethylaminonaphthalene-1-sulfonic acid). The energy transfer is distance dependent. The distance between the tryptophan and the probe would be quite different in a helical bundle conformation than in a helical hairpin. The FRET data supports a discrete helical bundle conformation for lipid-free apo A-I comprising residues 1-186 18 as part of a single folded domain. The FRET data does not support the existence of a helical hairpin in solution. In 2005, the first all-atom full-length apo A-I structure was proposed based on cross-linking of lysine residues combined with high resolution mass spectrometry (Figure 7) (40). After chemical cross-linking, the sample was digested with trypsin, the digests were separated by liquid chromatography, and their masses were determined by mass spectrometry. These masses were used to identify the various cross-links that were then put into a molecular homology model that was built using proteins with > 30% sequence homology. The resulting model is a helical bundle structure with both termini close to each other as was also proposed in the model based on FRET analysis (Figure 7). Figure 7. Ribbon model of the first full length all-atom model of lipid-free apo A-I (40). Spirals represent helices, lines represent coils. 19 This model does uphold previously proposed structures with its helical bundle and its unstructured carboxy-terminus. However, the authors of this study point out the limitations of this technique due to the flexible nature of apo A-I in solution. Because of the dynamic nature of apo A-I, this model may represent one of several solution structures. Despite this limitation, the technique does identify residues close enough to each other for a long enough time period to cross link and these points of contact should be considered in developing models of the protein. EPR spectroscopy has been used to develop a tertiary structure model of lipidfree apo A-I based on side-chain mobility, quencher/ solvent accessibility, and dipole coupling of spin-labeled apo A-I (39, 224) (Figure 8). β–strands are located from residues 20-25, 120-129, 150-158, and 214-220 and helices are located in residues 1419, 26-51, 55-85, 92-98, 102-115, 130-148, 159-188, 200-205, and 224-231. The β– strands are found anti-parallel to each other and could possible exist in one β–sheet while the helices are found in an amphipathic helical bundle. 20 Figure 8. Comparison of apo A-I structure determined using EPR of the Cterminal domain (panel A) and the full length structure (39, 224). A, The model contains the tertiary structure of the C-terminal domain according to EPR. B, the “betaclasp” model of lipid-free apo A-I consisting of four anti-parallel beta-strands represented by arrows (maybe in a single sheet) surrounded by an amphipathic helical bundle with each helix represented by a spiral. The model is on the left and the schematic design is on the right (224). A crystal structure of residues 1-184 has been recently published (Figure 9) (212). The structure is composed of a half-circle dimer with a backbone of two elongated antiparallel proline-kinked monomers composed of two helices each. The Nterminal domain of one molecule forms a four-helix bundle with the carboxy-terminal region of the other molecule. The center of the molecule is flexible and the bundles may unfold to bind lipid. This crystal structure is a good resolution, at 2.2 angstroms, but it has a helical content of approximately 80%, much higher than has been predicted using various other methods. This may be due to the nature of the crystallization 21 process or to the fact that the protein’s unstructured region (by solution methods) is deleted (residues 185-243). Figure 9. The crystal structure of 1-184 apo A-I. A, The overall structure of the monomer of 1-184 apo A-I. B, The model shows the dimerization of the two monomers (212). Though much progress has been made in determining the structure of apo A-I in recent years, an all-atom model that agrees with all experimental data is still lacking. It has not been possible to firmly establish even the secondary structure of the protein. In particular, the various methods employed vary greatly in their proposed secondary structure for the carboxy-terminus. The high helix content indicated by crystallization is probably not reflective of the solution structure. However, the two solution methods (EPR and HD-X) show little agreement for the secondary structure in the carboxyterminus. The various techniques reach closer agreement for the amino-terminal domain where extensive helix is postulated, particularly in the first 100 residues. However, the exact position of helical segments remains in dispute. As mentioned 22 before, it is difficult to identify an exact structure due to the dynamic nature of the protein. In summary, while the structure of apo A-I has yet to be definitively determined, some aspects are consistently revealed by a variety of methods. The secondary structure of lipid-free of apo A-I in solution has been found by multiple methods to be approximately 50-55% α helical (33, 70, 36, 193). The protein has two domains, with a helix bundle and possibly a small segment of beta strand located in the N-terminal domain while the C-terminus is either a combination of helix, sheet, and random coil or simply random coil, and is less structured than the N-terminus. In most models, the N- and C- termini are close to each other. This may be important in the self-association properties apo A-I possesses and could be a factor in amyloid formation, as discussed further below. Lipid Bound Structure Nascent HDL When lipid free apo A-I has lipid added to it, it forms a lipid-protein complex with a discoidal shape and is referred to as nascent discoidal HDL (or preβ-2 HDL) which is a critical intermediate in the formation of spherical HDL (131, 225). Discoidal HDL is typically 10 nm (or 100 Å) in diameter but its size can vary depending upon its protein content and lipid composition (96, 174, 225). Most HDL particles consist of either two or four molecules of apo A-I. The size and shape of the 23 apo A-I monomers make it suitable for wrapping around the particle perimeter (discoidal or spherical), which can have diameters between 80 and 120 Å (190, 212). A low-resolution crystal structure of ∆1-43 discoidal apo A-I was determined in 1997 which led to a model of lipid-bound protein (7). The structure contains four apo A-I molecules arranged in an anti-parallel fashion. The monomer structure within the tetramer contains a long alpha helix punctuated by kinks caused by proline residues, leading to a sharply curved horseshoe, or an almost closed circle (Figure 10 A). The monomer’s dimensions are 125 x 80 x 40 Å, but the curve of the ∆1-43 molecule is such that the N- and the C- termini are only 23 Å apart (7). 24 Figure 10. Stereoviews of Δ1-43 apo A-I as a monomer in A, a dimer in B, and a tetramer in C. The N- and C- termini are labeled (7). The monomer then self-associates to form a tightly associated antiparallel dimer (Figure 10 B) overlapping enough to form a closed elliptical ring instead of an open horseshoe. Because of its antiparallel association and the length of the helices, 25 helix 1 (using the helix numbering applied by the Segrest group, referring to the 10 amphipathic α -helices identified by their computer programs, figure 4B) on one monomer overlaps with helix 9 on the second monomer. The two helix 5’s from each monomer overlap. Two apo A-I dimers associate to form a tetramer ring that is 135 x 90 Å on the outside with an inner diameter of 95 x 50 Å (Figure 10 C). The formation of tetramers is probably due to the absence of lipid in the structure because the dimers orient to sequester their hydrophobic faces from exposure to water (7). This appears to be a good model of lipid-bound protein for several reasons. First, ∆1-43 discoidal apo A-I exhibits similar lipid-binding properties to those of native apo A-I as determined by measuring surface activity, lipid association of 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles, and lipid association with plasma lipoproteins (68). Second, the dimensions of the dimer are close to those that would be needed to wrap around a lipid disc of approximately 100 nm. Based on the ∆1-43 structure, the Segrest group proposed a computational model that retained some features of the crystal structure but added modifications to improve the wrapping of the protein around a lipid-disc (90). The Segrest model, also referred to as the double belt model, consists of two antiparallel apo A-I molecules that extend with the long axes of the helices parallel to the plane of the lipid bilayer (187, 190). Experimental data has since been found to support this model including cross-linking studies coupled with mass spectrometry, IR experiments, EPR, and FRET studies (2, 131, 160). These experiments led to models 26 which are essentially minor variations of the double belt model, except for the proposal of a hairpin structure where two monomers fold on themselves and are then wrapped around the disc. This would again give an antiparallel association of amphipathic helices (Figure 11). The spherical model of HDL is less well-developed than discoidal HDL, but presumably contains overlapping antiparallel helices (7). Figure 11. Models of lipid-bound apo A-I on discoidal HDL including (A) the double belt and (B) the hairpin (32). Stability and Lipid Binding Conformational Stability The N-terminus is required to maintain the lipid-free structure because the helix-bundle structure is lost when residues 1-43 are removed, presumably due to disruption of the N-terminal helix bundle. This deletion may also cause loss of a stabilizing interaction between N- and C- termini (76, 175). Experimental evidence including CD, chemical denaturation, and fluorescence spectroscopy of mutants shows that the C- and N-terminal regions are in close proximity, supporting an interaction between the C-terminus and the N-terminal helix bundle (76, 175). It has been suggested that disrupting the interaction between the termini is important for lipidbinding (36, 39, 70, 175). Because the C-terminus is relatively flexible and has a high 27 affinity for lipid, this interaction may be relatively easy to disrupt when lipid is present (36, 39, 76, 175). Figure 12. The initial lipid-binding step of apo A-I mediated through helix 9 and 10. The biding of helices 9 and 10 is triggered by presence of lipid. This releases helix 1 for binding, following by the remaining helices (33, 187). Lipid Binding The C-terminal domain has been found to be critical in driving apo A-I to bind lipid with high affinity (80, 147, 154). The helices of highest lipid affinity, 9 and 10, bind lipid first. The helices with the next highest affinity, 1 and 2, bind next, followed by the binding of the rest of the apo A-I helices to the lipid (Figure 12) (80, 147). Not only does the C-terminus serve as a site of initial lipid contact, but it also is a source of energy to drive the process of lipid association (39). It is proposed that both intermolecular as well as intramolecular interhelical interactions play a part in this binding (147, 154). While helices 9, 10, 1, and 2 have the highest lipid affinity, they 28 aren’t the only helices with any lipid affinity. Even when the extreme N- and C-termini are cleaved, the remaining residues (44-186) can still bind lipid (132). However, in normal apo A-I, it is likely that the presence of lipid triggers disruption of the N-C interaction. It is believed that, in addition to an intramolecular interaction, intermolecular interaction is prevalent and leads to aggregated lipid-free protein. The physiological relevance of this, if any, is only now being investigated. It is suggested apo A-I selfassociation is important for key steps in reverse cholesterol transport such as lipid binding (226). However, intermolecular interaction presents an opportunity for undesired aggregation and the possible development of amyloid protein. Amyloid Apo A-I Some proteins aggregate in an organized or semi-organized fashion to produce an amyloid conformation. Amyloid was originally defined based on the ability of protein aggregates to bind to dyes applied to histological specimens. Now, it usually refers to aggregated protein that has a cross-beta structure (discussed below). However, even this application of the term is becoming outdated as polymorphisms are being discovered in amyloid protein associated with different conditions including Alzheimer’s, Huntington’s, Parkinson’s, and cataracts. It is becoming increasingly clear that most proteins can form amyloid or some kind of fibril structure under the right conditions (95). In recent years, a number of amyloidogenic variants of apo A-I 29 have been identified (141). These proteins form amyloid deposits in various tissues throughout the body including nerves, skin, heart, liver, kidney, testicles and larynx. All amyloid fibrils have a long rigid structure in common, while other aspects of amyloid fibrils such as their secondary structure and length can differ greatly depending on the protein from which they’re formed (Figure 13). Initially it was believed that fibrils could only be formed from β –sheets, but it has more recently been determined α-helical fibrils exist as well. Many protofilaments that form fibrils have a cross-β structure, meaning the β-sheets in the protofilament, and therefore the fibril, run perpendicular to the fibrillar axis (49, 88) (Figure 13 B). 30 Figure 13. Generic β-sheet amyloid fibril structure, picture of actual β-sheet amyloid fibrils, and a model of an α-helical fibril supported by an electron micrograph. A, β-sheets make up the four (shown here) protofilaments. The β-sheets run perpendicular to the fibrillar axis (49). B, an atomic force microscope image of bundle of fibrils of the immunoglobulin light chain variable domain (47). C, a model of an α-helical fibril in which a bar represents a helix. The helix is then flipped vertically and horizontally, polymerized, and twisted to form α-helical protofilaments where the helical axis is actual perpendicular to the fibrillar axis (195). The proposed structure is supported by an electron micrograph of the protofilament. The formation process of this long rigid amyloid structure has been investigated (Figure 14). One possible explanation for the process of amyloid formation is that after proteins are synthesized on the ribosome, they then begin to 31 form some type of secondary structure, whether random coil, α-helix, or β-sheets, and are referred to as partially folded (47). These partially folded molecules can then aggregate under certain conditions and have either monomers or protein aggregates (also called protofibrils) added to them to form protofilaments which are units, 2-5 nm in diameter (47, 88). Typically, mature amyloid fibrils consist of 2-6 protofilaments associated together, sometimes twisted, to form fibrils about 4-13 nm in diameter (47). Figure 14. Mechanisms of amyloid fibril formation (88). An additional step not shown in the figure occurs between the β-structured aggregates and the amyloid fibrils called the protofilament. 32 The initial instigator of amyloid formation is unknown but may involve a pH change, a temperature change, or other environmental factors. A very delicate balance exists in the protein’s environment to form the correct conformation as opposed to a fibrillogenic, sometimes fatal, one. Once formed, one fibril’s existence can greatly increase amyloid formation because it is accelerated when seeded (88). In other words, the first amyloid fibril takes the most time to form and each subsequent fibril forms more quickly, similar to the growing of a crystal (95). Once a fibril is formed, it is generally linked to disease. Yet, it is debated as to how amyloids actually cause disease. One debate is whether the fibrillar aggregates or their precursors, oligomers and protofibrils, cause disease (95). One study supporting the protofibrils hypothesis states that because no correlation exists between number of amyloid fibrils present at autopsy and severity of Alzheimer’s or Parkinson’s disease, perhaps protofibrils cause disease by their toxic, pore-like morphology (44). Studies have been performed to test this hypothesis where amyloid fibrils were injected in the brains of mice. Some of those mice then began to exhibit amyloid accumulation and symptoms similar to Alzheimer’s Disease, insinuating amyloid fibrils, not their precursors, are actually the cause of the disease (158). Apo A-I’s fibrils are often associated with disease. Apo A-I fibrils are caused by different mutations but they can also be formed by WT apo A-I under the right conditions (141, 227). 33 Apo A-I Amyloidogenic Mutants Four different apolipoproteins have been found to be amyloidogenic in vivo (aposerum AA, A-I, A-II, and A-IV), a fifth is amyloidogenic in vitro (C-II), and a sixth exists in amyloid deposits as a minor component (E) (141). Mutations in apo A-I have been discovered for more than 30 years (Table 2). According to a 2008 paper, over 70 mutations have been found so far (129). Some of these mutants cause amyloidosis and some have an effect on HDL and apo A-I levels, but these are not mutually exclusive. Many mutants reported have caused amyloidosis with, or without, CVD (Table 2). Because of the role of apo A-I in HDL production, mutations in the apo A-I gene often disrupt both apo A-I and HDL production (133, 129). The first four amyloidogenic apo A-I mutations discovered involved an addition of a positive charge (113) (Table 2). At one point, this charge change was thought to be a reason for the amyloid formation. Eventually, additional mutations were found with similar introduction of positive charges without forming amyloids and amyloidogenic mutations were discovered without a difference in charge (53). One possible explanation for why some mutations cause amyloid formation in apo A-I and others don’t is that amyloidogenic mutations alter the interaction between the C- and N- terminal domains. This disruption leaves an unstable partially folded N-terminal helix bundle, which appears to be important in fibril formation (175). 34 Table 2. Summary of the apo A-I mutants found to date (37, 55). Mutation Fibril Location Arg173Cys (Milano) None Other Notes Hypertriglyceridemia and decreased HDL without atherosclerosis Gly26Arg Nerves, Kidney, 1st report of apo A-I (Iowa) Liver amyloidosis; low HDL and apo A-I, neuropathy, +1 charge difference between amino acids Leu60Arg Kidney Non-neuropathy amyloidosis Del.60-71; Liver Non-neuropathy amyloidosis Ins Val/Thr and low HDL and apo A-I levels Del. 70-72 Kidney Extensive deposits in liver and spleen but no adverse clinical effects Leu90Pro Skin Cardiomyopathy; 1st neutralneutral substitution Leu174Ser Heart, Joints, 1st time fibril fragment doesn’t Nerves include mutation Arg173Pro Skin, Heart, Larynx Amyloidosis, decreased LCAT activation Leu178His Heart, Larynx, Skin 1st time fibrils have full length apo A-I and TTR fragments Ala175Pro Visceral, Larynx Infertility Leu178Pro Leu75Pro Larynx, Heart, Abdominal Fat Testicles, Liver Premature CAD and increased risk of CAD complications, Infertility, enlarged testicles, hypo-gonadism, macroorchidism Year Reference Discovered 1980 133, 83, 130 1988 126, 128, 113, 143 1992 1996 153 114 1998 72 1999 53, 71 1999 54, 181 1999 15, 92 2000 43 2002 82 2004 134, 181 2008 62 Amyloidogenic apo A-I mutations tend to be located in two clusters within the protein sequence, both involving the amino terminal helix bundle domain (46). The first cluster spans residues 26-107 (Table 2) and contains “inside” mutations because they occur mainly within the fragments of protein (1-90) found in deposits in vivo 35 (229). These mutations tend to lead to formation of apo A-I amyloid in the liver and kidneys. Of these mutations, only the G26R mutation is associated with decrease in HDL. Mutations are also found in a small cluster from residues 154-178 (the “outside” mutations) and tend to lead to amyloid formation in the larynx, heart, and skin. Although the two clusters are separated in the sequence, they are in pretty close proximity in lipid-bound apo A-I (Figure 15). Figure 15. Dimeric structure of apo A-I based on the apo A-I Δ1-43 structure. In dark grey are side chains of residues mutated in amyloidosis when the mutations are outside V93. The light grey dots are mutated residues found within the peptide 1-93. The bright white dot represents Val 93, which is a common site of cleavage in fragments (141). One amyloidogenic mutant associated with cardiac and larynx amyloidosis and, less frequently, with skin lesions is L178H in which the leucine residue at position 178 has been mutated to a histidine residue, replacing a bulky nonpolar residue with a polar and weakly basic one (43). Interestingly, another mutation occurs at the same position but involves proline instead of histidine and has a completely different clinical 36 manifestation (134). This mutation still leads to laryngeal amyloidosis like L178H, but also has been associated with amyloids in the heart (181). L178P was assumed to be associated with systemic amyloids when they were found even in abdominal fat (181). The L178P mutation has been known to cause endothelial dysfunction, changes in arterial wall thickness, premature CAD and increases risk of CAD complications, which is very different from L178H (134). The L178H mutant is unique in that, when it was discovered in 2000, it was the first amyloid apo A-I mutant whose fibrils contained full-length protein (43). Other mutants found prior to this produced amyloid deposits containing only amino-terminal fragments from residue 1 to about 90, commonly 1-93. It was thought at one time that mutations had to occur within the amino-terminal 90 residues to produce an amyloid conformation, but this was shown to be false in 1999 when L174S was found to produce the 1-93 fragment (54, 113). It is now clear that mutations in both of the clusters can lead to amyloid containing the ~ 90 residue N- terminal fragment as the fragment has been found in the amyloid stemming from multiple apo A-I mutations (92, 53, 126, 124, 43). Our group, in collaboration with researchers at UC Davis, Children’s Hospital, Oakland Research Institute, and Lund University in Sweden, published the first structural analysis of an amyloid variant from the 1-90 cluster, G26R. In it, the G26R mutation was found to have decreased lipid-binding ability when compared to wild 37 type apo A-I, amino-terminal protease sensitivity, and increased β strand secondary structure. FOCUS OF THIS RESEARCH A number of amyloidogenic mutations in apo A-I have been identified which cluster in two regions of the protein. Furthermore, in addition to amyloid produced by mutation, WT apo A-I protein has been found in some amyloid plaques. Because plaques are an early step in many CVD, it is important to elucidate the mechanism of amyloid formation to try to understand and prevent plaque formation and therefore CVD. One full-length apo A-I amyloidogenic mutant has already been studied, G26R, and the sum of its structural differences. This mutation is inside the 1-100 cluster. In this work, apo A-I with a mutation in the 173-178 region, L178H is characterized using ANS binding, limited proteolysis, and intrinsic fluorescence studies. In this work, it is shown that the L178H mutations, like G26R, lead to increased amino-terminal protease susceptibility and increased hydrophobic surface area. This suggests the two proteins have a similar tertiary structure that may promote amyloid fibril formation. 38 MATERIALS AND METHODS Recombinant Protein Production Transformation Dr. John Voss (University of California, Davis) generously provided both WT and L178H apo A-I as 6X-His TAG L178H cDNA fusions in the pNFXex plasmid (109). The plasmid was transformed into One Shot® Top10F’ (for long-term plasmid storage) and BL21(DE3)pLysS (for expression) E. coli chemically competent cells (both Invitrogen). Cells and DNA were thawed on ice and aliquotted into pre-cooled sterile centrifuge tubes with 1 µL DNA (or water for blank) and 20 µL desired cells. Mixtures were stirred gently with pipet tip and incubated on ice for five minutes. Mixtures were then heated at 42° C for 45 seconds and put back on ice for 2 minutes. 180 µL of sterile room temperature SOC media (a nutrient rich media used for protein culture) was added and samples were put in a shaking 37° C incubator at 250 rpm for 45 minutes. Fifty µL of the transformation mixtures were plated on a LB plate with 50µL of 50 mg/mL ampicillin (AMP) to select for transformants bearing the ampR gene. The plates with BL21(DE3)pLysS E. coli competent cells also had 34 µL of 34 mg/mL chloramphenicol (CAM) to select for retention of the pLysS plasmid. The plates were incubated at 37º overnight. Individual colonies were then chosen for expression. 39 Inoculation and Expression Four 125 mL Erlenmeyer flasks containing 50 mL of sterile LB media, pH 7.2, 35 µg/mL CAM, and 50µg/mL AMP were inoculated with 1 colony from an agar plate or from a frozen glycerol stock made of 8% sterile glycerol stored at -80º C. The inoculated flask was then incubated at 37º C overnight for 12-16 hours while shaking at 250 rpm. Four one L flasks with 500 mL of sterile LB media, 35 µg/mL CAM, and 50µg/mL AMP were then inoculated with the entire contents of the overnight culture and grown at 37ºC while shaking at 250 rpm until the OD600nm was greater than 0.6, usually 4-6 hours. Then, 500 µL of 0.5 M Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce the expression of apo A-I. After the addition of IPTG, flasks were incubated again at 37ºC shaking at 250 rpm for three hours. The cells were then pelleted by centrifugation in an SLA-1500 rotor at 8000 RPM for 15 minutes at 4º C. Cell pellets were frozen in an acetone/dry ice bath and stored at -20º C until ready for purification. Protein Purification L178H cell pellets were thawed in an ice bath. Then 500 µL protease inhibitor cocktail p2714 (Sigma), 10 mL phosphate buffered saline (PBS, 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.2), and one mL 1% triton-x-100 were added to each pellet before shaking over ice for 20 minutes. Pellets were then lysed using sonication at 60% maximum in six separate 30-seconds bursts with 60 second breaks on ice in 40 between bursts. The resulting solution was centrifuged at 27,000 * g for 20 minutes at 4º C. The pellets were discarded and the resulting supernatant was filtered through a 0.45 µm syringe filter. The total volume of clarified cell lysate was determined and guanidine and imidazole were added to make the final solution 3M guanidine and 40 mM imidazole. A 10 cm x 1 cm chromatography column was loaded with 10 mL His·Bind® resin (Novagen) and allowed to settle to 5 mL final bed volume. The column was then rinsed with 20 mL deionized water, charged with 30 mL charge buffer (400 mM NiSO4) and washed with 30 mL binding buffer (1X PBS, 3 M guanidine, 40 mM imidazole). The crude cell extract was then loaded onto the column and the flow through was collected in bulk except for the last 1.5 mL which was collected separately. The column was then washed with 70 mL binding buffer with the first sixteen 1.5 mL fractions collected separately and the rest of the buffer collected in bulk. The protein was then eluted from the column with 35 mL elution buffer (1X PBS, 500 mM imidazole) supplemented with 100 µL protease inhibitor cocktail p8849 (Sigma) and the first 13 1.5 mL fractions were collected for analysis with the rest of the sample collected in bulk. The column was then stripped using 36 mL strip buffer (0.5M NaCl, 100 mM ethylenediaminetetraacetic acid (EDTA), 20 mM Tris-HCl, pH 8.0) with the first 1.5 mL collected separately and the rest collected in bulk. To determine protein concentration, protein was usually diluted 1:1 or 1:10 in 6 M guanidine and an absorbance spectrum was collected. The absorbance at 280 nm 41 was taken in triplicate samples and averaged and then inserted into the Beer-Lambert Law (A=εbc) to determine protein concentration using 1.13 cm2/mg as the apo A-I extinction coefficient and 1 cm as the path length (33). The purity of the protein was determined by SDS-PAGE on 0.75 mm 15% Tris-Glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) gels (Jule Inc.) or 8-16% Precise (Pierce) gels. Low weight molecular weight markers previously mixed with 5x sample buffer (Bio Rad) were used as standard. Samples were prepared by combining 10 µL sample of a fraction with 10 µL 5x sample buffer. Gels were run at 120 V at 4° C until the leading edge was right above the bottom of the gel rig, approximately 90 minutes. Gels were then stained in coomassie blue for at least 30 minutes followed by destain overnight. Structural Analysis Limited Proteolysis Limited proteolysis experiments were performed on both WT and L178H apo A-I. Before proteolysis, protease inhibitor was dialyzed out of the sample into PBS for 3 hours. Then between 5 and 40 µg of protein were digested at 37ºC with a 1:2000 (wt:wt) ratio of chymotrypsin to apo A-I protein for times ranging from 1 minute to 4 hours. Reactions were quenched with 5µL protease inhibitor cocktail cat. # p8849 (Sigma). Five to 15 µL 5X sample buffer were then added to digests before freezing at -20° C. Resulting digests were later analyzed by SDS-PAGE. 42 To obtain sufficient proteolyzed fragments for sequencing, selected limited proteolysis digests were separated on SDS-PAGE gels along with pre-stained standards, then transferred via electroblotting to a PVDF membrane. The blotting apparatus was layered top to bottom as follows: cathode, two pieces Beckman blotter paper soaked in Towbin solution (0.025M tris, 0.192M glycine, 20% Methanol, pH 8.0), gel briefly soaked in Towbin, 0.45 µm pore size Millipore polyvinylidene fluoride (PVDF) membrane soaked in methanol and then Towbin, two more Towbinsoaked blotting papers, anode. The blotter paper and PVDF membrane were cut to the exact size of the gel before soaking in appropriate solutions. Electroblotting was performed using a TE70 blotting apparatus (Hoefer Scientific) at 30 V at 4° C for 90 minutes. Transferred proteins were visualized by staining the PVDF membrane with in Coomassie blue for 2 minutes followed by destaining in methanol/acetic acid for 10 minutes. Membrane was then air dried before bands of interest were excised and sent for sequencing. Protein fragment size was determined by measuring migration distances of proteolytic fragments on three similar limited proteolysis gels from three different batches of protein (i.e. protein produced in three different expressions). The retention factor (Rf) was determined by dividing the migration distance by the length of the gel, measured from bottom of well to dye front. A standard curve was created by plotting the Rf vs. log of the molecular weights of the known standards. The molecular weights of the fragments were then predicted from their Rf using the regression line equation. 43 The Rf and the molecular weights of the uncleaved protein was similarly determined. The calculated molecular weight of the fragment was subtracted from the calculated molecular weight of the full-length protein to determine the size with the small fragment removed by proteolysis. Because the intact protein’s calculated molecular weight was not exactly the known molecular weight of 28.1 kDa but 30 kDa instead, the size of the larger proteolysis fragment was established by subtracting the smaller fragment from 28 kDa. For example, if a fragment’s calculated molecular weight was 24.3 kDa, the 24.3 was subtracted from 30 kDa. The difference of 5.7 was then subtracted from 28 so the molecular weight of this fragment would be reported as 22.3 kDa. This calculated molecular weight was then divided by 115 (the average molecular weight of an amino acid) to calculate the number of residues in each fragment . Since the N-terminal sequence of the fragment was known from Edman sequencing, the residue at the cleavage site and its place in the protein (residue number) was known as well. The number of residues in the fragment were then added to the residue number of the N-terminal cleavage site and the C-terminus was determined for all three gels. Since proteases cleave at specific residues and chymotrypsin cleaves on the carboxyl side of tyrosine, tryptophan, and phenylalanine, the sites of cleavage with residue specificity closest to the c-terminus fragments were assumed to be the correct positions. 44 ANS Binding ANS (1-anilinonaphthalene-8-sulfonate) (Sigma-Aldrich) binding experiments were performed on solutions containing 50 µg/mL protein and 250 µM ANS (>100 molar excess) in a 2 mL final volume (68). Bovine serum album (BSA) was used as a positive control (50 µg/mL BSA and 250 µM ANS with a 2 mL final volume). Data were collected on a Shimadzu RF- 5301 PC Spectrofluorophotometer. ANS fluorescence was obtained as an emission spectrum from 410-560 nm with 395 nm excitation wavelength using excitation and emission slit widths of 5 nm and 3 nm, respectively. All samples were run in triplicate on 10 protein preps, though only 8 were used due to undesired protein cleavage. Intrinsic Fluorescence The intrinsic fluorescence of the protein was monitored over time. Data were collected on the Shimadzu RF- 5301 PC Spectrofluorophotometer. Fluorescence was obtained as an emission spectrum from 310-400 nm with a 295 nm excitation wavelength and slit widths of 5 nm (excitation) and 3 nm (emission). Intrinsic fluorescence quenching experiments were carried out on protein at 0.02 mg/mL concentration. KI was added from 2M stock (with 1mM Na2S2O3 to prevent oxidation of I-) to give final concentrations varying from 0.01M to 0.5M. KCl was added to keep the total ionic strength 0.5M. The Stern Volmer constant, Ksv, was determined using the equation Fo/F = 1 + Ksv (Q) where Fo and F are the fluorescence intensity at 331 nm 45 in the absence and presence of quencher, respectively, and Q is the concentration of I-. All samples were run in triplicate on separate protein preps. 46 RESULTS L178H Expression and Purification To conduct a structural analysis of L178H apo A-I, it was first necessary to obtain the pure protein in high yield. The L178H cDNA in a pNFXex plasmid (109) was obtained from the laboratory of Dr. John Voss at UC Davis. It was transformed into One Shot® Top10F’ cells because of their excellent transformation efficiency and for production of DNA for sequence analysis. For protein expression, the plasmid was transformed into BL21(DE3)pLysS cells which have an IPTG-inducible gene for T7 RNA polymerase that can also lower the expression of background genes while not interfering with the level of expression of the target IPTG-inducible gene. To determine the optimal expression conditions, a pilot expression of L178H was performed in which the temperature of incubation and the length of time after induction with IPTG was varied. Cell lysates were prepared by sonication of cell pellets resuspended with Triton x100 and guanidine. Protein expression was evaluated by analyzing aliquots of cell lysate by SDS-PAGE. Incubating the cells at 37° C for three hours after IPTG induction produced the most protein (Figure 16). 47 Figure 16. L178H pilot expression. Arrow indicates migration position of apo A-I (29kDa). Lanes are: 1, molecular weight markers; 2-5, incubation at 25ºC for 0, 1, 2, and 3 hours after IPTG induction; 6-9, incubation at 37º C for 0, 1, 2, and 3 hours after IPTG induction. Large scale expressions using approximately 2 L of media were then carried out to obtain sufficient protein for structural analysis. Cell lysates from these experiments were loaded onto a nickel ion affinity column and eluted by increasing imidazole concentration from 40 mM to 500 mM. Aliquots of column fractions were subjected to SDS-PAGE to detect the elution of L178H protein, which was usually most abundant in fractions 2-6 (Figure 17A). 48 Figure 17. Metal-ion affinity chromatography of L178H apo A-I. A, Example of protein not cleaved at time of purification. Lanes as follows: 1, molecular weight marker; 2, flow through; 3-5, binding fractions 4, 7, and 10; 6-10, elution fractions 2, 4, 6, 8, and 10. Solid arrow designates L178H apo A-I. B, Example of protein cleaved at time of purification. Lanes as follows: 1, molecular weight marker; 2, flow through; 3-5, binding fractions 2-4; 6, elution fraction 1. Hollow arrow designates cleaved apo A-I. In some purifications, the protein was cleaved (Figure 17 B). This is a known problem due to the high protease susceptibility of apo A-I (38,69). To minimize nonspecific proteolysis, protease inhibitor several times more concentrated than recommended was used throughout the purification process. In some cases, guanidine was added to the cell lysate to both prevent protein aggregation and to help denature residual proteases. However, addition of guanidine did not significantly reduce nonspecific proteolysis (data not shown). To minimize proteolytic degradation over time, the protein was lyophilized and stored at -80º C following purification. However, the resolubilized protein frequently exhibited extensive proteolysis and was highly insoluble. Therefore, lyophilization was not used in subsequent preparations. 49 The proteolysis problem was exacerbated by long incubations carried out to examine conformational changes over time (up to one month). Protein that was intact at time of purification was sometimes found to be cleaved days or weeks later. To establish the quality of the protein for structural analysis, aliquots were taken periodically throughout a typical 28-day incubation period and analyzed by SDSPAGE to determine whether the protein was still intact at the end of the study (Figure 18). Sometimes protein remained intact through 28 days and sometimes it didn’t; the basis of this inconsistency is unclear. The degree of degradation was also determined, not surprisingly, to be temperature dependent. In one experiment, the integrity of the protein was monitored over 28 days at three different temperatures. The protein remained intact at 4° C but was degraded almost completely by day four when incubated at 25 and 37° C. All the experiments reported in this thesis are from protein that was demonstrated to be fully intact by SDS-PAGE analysis. 50 Figure 18. Analysis of proteolytic cleavage upon extended incubation. Lanes are as follows: 1, molecular weight markers; 2 – 4, protein incubated for 4 days after purification and incubated at 4º C, 25º C, and 37º C, respectively; 5-7, day 7 after purification incubated at 4º C, 25º C, and 37º C, respectively. Structural Analysis of L178H Apo A-I The structure of lipid-free apo A-I has been extensively studied, both as plasma apo A-I isolated from HDL as well as WT apo A-I produced in various prokaryotic and eukaryotic expression systems. Due to the dynamic nature of this protein, a complete understanding of its structure remains elusive. However, some consistently observed features include about 55% -helix content, organization of residues 1-190 into an Nterminal helix bundle with a more loosely structured C-terminal domain (residues 191243), and a small, but significant amount of -strand structure within the N- and Ctermini. 51 There are two clusters of amyloid mutations in apo A-I, both within the Nterminal helix bundle formed from residues 1-190. One cluster occurs within residues 26-107 ("inside" mutations) and the other in residues 154-178 ("outside" mutations). In conjunction with John Voss’ group at UC Davis and Michael Oda at Children’s Hospital Oakland Research Institute, we characterized a mutant in the first cluster, G26R, and found it causes some of the helices in the N-terminus to convert to random coil and beta-strand structures, leading to the formation of fibrils (38). In addition, the G26R protein has a much higher sensitivity to protease digestion in the amino terminus, presumably due to increased β-strand structure beyond residue 26. Here, we analyzed the effect of a mutation in the second cluster, L178H, on apo A-I structure using limited proteolysis, ANS binding, and intrinsic fluorescence. Limited Proteolysis Limited proteolysis involves the addition of a small amount of protease to a protein for a short time. Initial cleavage sites indicate positions in the protein that have increased flexibility (206) or loosely folded structure, and thus more accessibility to the protease. Despite lipid-free apo A-I’s susceptibility to cleavage compared to typical globular proteins, limited proteolysis can still be used to examine the protein’s structure because the protease is added in very small amounts; for apolipoproteins, a ratio of about 1:2000 (w/w) is used compared to 1:50 or 1:20 (depending upon the protease used) for lipid-bound apo A-I (69). Previous studies have employed limited proteolysis to explore the structure of wild type lipid-free apo A-I (38, 69). Two 52 proteases, chymotrypsin and Staphylococcus aureus V8 protease, were used to show that major cleavage occurs near residue 190 with additional minor cleavage occurring in the middle of the protein and the extreme carboxy-terminus. The same pattern of cleavage was observed by others using chymotrypsin and elastase (211). We also employed limited proteolysis to detect structural changes resulting from the G26R mutation. In contrast to wild type, digestion with either Staphylococcus aureus V8 protease or chymotrypsin produced cleavage primarily in the N-terminus of the protein (Y18, E 34, and F57). Here, limited proteolysis was performed to examine the effect of the L178H mutation on the structure of apo A-I. WT and L178H were each treated with a 2000:1 (w/w) protein to chymotrypsin ratio. Samples were incubated with enzyme for periods ranging from one minute to four hours and the resulting digests were analyzed by SDSPAGE (Figure 19, Table 3). The cleavage pattern of wild type protein appeared very similar to that reported in previous studies (38, 69). Based on N-terminal sequencing and size analysis of the proteolytic fragment, the major cleavage product corresponds to the fragment D1- Y192 (WTb) and the most prominent minor product corresponds to the fragment D1-F229 (WTa) (38). 53 Figure 19. Limited proteolysis of A. WT and B. L178H apo A-I. Both A and B, lanes as follows: 1, molecular weight markers; 2-8, 0, 1, 5, 30, 60, 120, and 240 minutes of exposure to chymotrypsin. Bands that were sequenced are indicated by arrows in B. The fragments indicated by the arrows are (top to bottom), LHa, LHb, LHc. The cleavage pattern of L178H is superficially similar to that exhibited by WT with bands close in size to intact protein constituting the major fragments. Both 54 L178H and WT apo A-I exhibit cleavage patterns which show cleavage occurring at or before one hour and very little full length protein remaining after 4 hours. However, several differences are evident: there are more bands close in size to intact protein with the one closest in size (LHa) appearing slightly sooner in L178H relative to WT, and a greater abundance of smaller fragments in L178H. Sequence analysis reveals that the three bands LHa, b, and c are all produced by cleavage in the N-terminus (Table 3). Table 3. Comparison of proteolytic cleavage fragment sequences. Band Name Sequence1 Fragment ID2 Number of Residues MW Estimation in kDa LH a VDVLK V19-D243 224 24.9 LH b DRVKD D9-Y192 184 21.8 LH c SKLRE S58-E235 177 20.4 WT a MHHHH M1-F2253 224 NR4 WT b MHHHH M1-Y1923 191 NR4 Iowa a VDVLK V19-D2433 224 NR4 Iowa b SKLRE S58-D2433 185 NR4 1, Edman analysis of Western Blot fragment. 2, Determined from Nterminal sequence and molecular weight estimation. 3, reference 38. 4 Not Reported. Two bands, here numbered one and two, were excised from a PVDF blot of a gel containing protein digested for 30 minutes. Edman sequencing revealed two distinct sequences within the first band. The first sequence, LHa, was VDVLK 55 indicating cleavage at Y-18. The second sequence, LHb, was DRVKD, indicating cleavage at W-8. Two sequences were also found in the second band. One was the LHb sequence found in the first band. The second sequence found in band two, LHc, was SKLRE which indicates cleavage at F-57. Overall, the cleavage pattern of L178H is much more similar to that of G26R than to wild-type apo A-I, with major cleavage occurring in the N-terminus of the protein. Amyloid structure in proteins develops over time. To determine timedependent changes in structure, limited proteolysis was conducted on samples incubated for different periods of time. A monomeric concentration of protein was used to prevent dimers of L178H from forming. Limited proteolysis was performed on both WT and L178H protein stored at 4º C, 25º C and 37º C for one month. Samples incubated at 25º C and 37º C degraded and could not be evaluated. For both WT and L178H protein stored at 4º C, the proteolysis pattern at day 28 appeared the same as day 0 indicating little or no change in protease susceptibility at this temperature. For L178H protein stored at 25º C and 37º C, the proteolysis pattern at day 2 appears the same as day 0. However, by day 7, protein had degraded which prevented analysis of structures brought on by the mutation at these incubation temperatures. ANS Binding The hydrophobic binding of ANS has been used to evaluate changes in protein tertiary structure (200, 201); ANS exhibits increased fluorescence intensity and a blue shift in the emission wavelength from approximately 500 nm to between 460 and 470 56 nm when introduced into a nonpolar environment (176). ANS binds to proteins by both its nonpolar anilinonaphthalene and negatively charged sulfonate groups. Despite the ability to bind via its negative charge, it is generally understood that ANS is a probe for hydrophobic environments (176, 200). It has frequently been used to probe the molten globule state of proteins because ANS binds to the molten globule state but not to fully folded or to unfolded proteins (176). As applied to apolipoproteins, ANS binding has been used to report tertiary structure changes or changes in lipid-binding sites (68, 175, 202). Since neither the complete tertiary structure nor specific lipidbinding sites in apo A-I are fully known, ANS data is mostly qualitative in nature and is used to follow changes in structure. To analyze the effect of the L178H mutation on the hydrophobic surface exposure of apo A-I, the ANS binding of L178H and WT apo A-I were compared. At day 0, the intensity of the fluorescence of ANS bound to L178H apo A-I is more than three times higher than ANS bound to the same concentration of wild type apo A-I (Figure 20). The maximum emission wavelength of ANS bound to WT is 484 nm compared to 473 nm for L178H, a blue shift of 11 nm from WT (Figure 20). Therefore, L178H apo A-I has a greater exposure of hydrophobic surface than wild type apo A-I. A similar increase in ANS binding was previously observed for the G26R protein (38). 57 Figure 20. ANS binding to WT and L178H apo A-I. The standard deviation of both WT (triangles) and L178H (squares)at 480 nm was ±8 arbitrary fluorescence units. The ANS binding collected over a period of time was compared relative to the day 0 value. ANS binding was used to assess amyloid formation over time because as amyloids are formed, hydrophobic surface exposure may be expected to change. The fluorescence intensity of ANS binding of protein incubated at 4º C and 37º C from day 0 to day 28 was determined. The undesired proteolysis previously described leading to degradation of protein was again observed in this experiment. Protein stored at 4º C typically stayed intact over the entire 28-day incubation period while protein incubated at 37º C over 28 days degraded in some experiments, but not others. Only experiments involving intact protein as demonstrated by SDS-PAGE analysis are shown here 58 except for experiments repeated at 37ºC where fluorescence results are similar to those of experiments where SDS-PAGE showed no degradation. A B Figure 21. Percent loss of ANS binding at 485 nm in L178H and WT over 28 days. Data was collected from multiple L178H protein samples. A. 4ºC and B. 37ºC. A. ◆ LH sample 5; ■ LH sample 6; X LH sample 16; ▲ LH sample 18; * WT protein. B. ■ LH sample 14; ▲ LH sample 16; ◆ LH sample 18; *WT protein. 59 At 4º C, ANS binding of wild type protein remained constant over the 28-day period of incubation (Figure 21). In contrast, L178H protein exhibited a large decrease in ANS binding ability between days 14 and 21, with an approximate t1/2 of 18 days. This suggests that a structural change, possibly formation of either pre-fibrillar oligomers or fibrils, was exhibited by L178H that limited the exposure of the ANS binding site around day 18 (Table 4). In contrast to incubation at 4º C, ANS binding by WT protein decreased at 37ºC. The decrease was largest between days 2 and 6 with an estimated t1/2 of 4 days, reaching a maximum decrease of 50% that persisted for an additional two weeks. L178H protein incubated at 37º C had its largest decrease in ANS binding ability a few days later, approximately double that of WT protein (Table 4). Four curves representing four different protein purifications are shown in Figure 21B. The curves have different shapes, which precludes accurate curve-fitting. However, visual examination of the two curves with the longest incubation times (LH sample 14 and 18) yield an estimated t1/2 of 10 days. In these same samples, the final ANS fluorescence intensity was higher for WT than for L178H indicating a greater loss of hydrophobic surface area in L178H. Table 4. Comparison of estimated t1/2 of ANS binding at various temperatures. Protein 4º C 37º C L178H 18 days 10 days WT No change 4 days 60 These results indicate that WT and L178H apo A-I incubated at 37º C exhibit loss of ANS binding, and that this occurs at a more rapid rate at 37º C for L178H than at 4ºC. Although this data does not report on fibril formation, it does show that structural changes occur in either protein incubated at 37º C, leading to reduced exposure of hydrophobic surfaces over time. Quenching of Intrinsic Fluorescence Apo A-I contains tryptophans at positions 8, 50, 72, and 100. Since all the tryptophans are within the first 100 amino acids of the protein, intrinsic fluorescence reports on the structure of the N-terminal helix bundle domain, (residues 1-190). To analyze structural changes in the two proteins over time, the intrinsic tryptophan fluorescence of L178H was measured at 4º C and 37º C and of WT at 4º C over a 2-4 week period (Figure 22). The maximum fluorescence intensity at 331 nm was plotted versus time of incubation. 61 Figure 22. Percent loss of intrinsic fluorescence of apo A-I proteins. ◆ (diamond) LH sample 19 at 4º C and ▲ LH sample 19 at 37º C. ■ LH sample 20 at 4ºC and X LH sample 20 at 37º C. *WT sample 3 at 4º C. All data reported here had SDS-PAGE gel evidence to prove the protein was intact at time of data collection. The intrinsic fluorescence of both the L178H and WT proteins is unchanged when incubated at 4º C over a 28-day period (Figure 22). However, incubation at 37º C led to a 60% decrease in intrinsic fluorescence of L178H between days 2 and 7 and an 80% decrease by day 14. It appears to be mostly quenched by day 7 with little additional change at day 14. These data indicate the L178H protein undergoes structural changes between days 2 and 7 that result in quenching of tryptophan fluorescence. 62 To further investigate the tryptophan environment of the two proteins, iodide, a tryptophan fluorescence quencher, was used. It was added in various concentrations to the incubated protein and the resulting fluorescence was measured. A Stern-Volmer plot was made in which the initial fluorescence divided by the quenched fluorescence is plotted versus the concentration of the quencher. The slope is the Stern-Volmer constant, KSV. A straight line is expected if the protein is homogenously emitting and only has one fluorophore (Figure 23). Fluorescence of most proteins is heterogeneous because of their multiple tryptophan residues. Since multiple tryptophan molecules usually exist in different environments, it is expected that quenching tryptophan in apo A-I would not necessarily give a straight trendline in Stern-Volmer plots. 63 Figure 23. Stern-Volmer plot of intrinsic fluorescence quenching of L178H apo AI at day 2. L178H was studied at 4º C ◆ and 37º C ■ and WT was at day 0 ▲ at 4º C. The KSV of WT at 4º C and L178H at 37º C are both 1.5, whereas the KSV of L178H at 4º C is 1.7, the same as a double truncation apo A-I mutant containing residues 44-186 (Table 5). The calculated KSV of WT in this experiment is the same as that reported elsewhere (132). For the WT protein, the trendline is the least linear with an R2 value of 0.88 whereas the R2 values of the trendlines of the L178H protein incubated at 4º C at 37º C are 0.97 and 0.99. 64 Table 5. Comparison of Stern-Volmer constant, Ksv, of protein at various temperatures. Protein Ksv R2 L178H, 4º C 1.7 0.97 L178H, 37º C 1.5 0.99 WT 1.5 0.88 WT1 1.5 Apo A-I (44-186) 1 1.7 Human Plasma2 1.4 1, reference 132. 2, Roberts, L.M. unpublished observations. 65 DISCUSSION Amyloidogenic mutations in human apo A-I cluster in two regions within the amino-terminal helix bundle from residues 26-107 (termed "inside" mutations) and 154-178 (termed "outside" mutations). The effects of a mutation from the 1-90 cluster, G26R, were investigated in a collaboration between the laboratory of John Voss at UC Davis and our group (38). This mutation leads to increased susceptibility to proteolysis in the N-terminus, increased hydrophobic surface area, and increased strand content in the N-terminus. The goal of this research was to determine, through analysis of L178H apo A-I, if mutations from the two clusters lead to similar structural effects. The overall conclusion from this research is that L178H mutation produces structural changes similar to those observed for G26R. This suggests there may be a common underlying mechanism for initiating fibril formation among mutations from the two clusters. Part I Structural Effects ANS binding is substantially increased in L178H compared to WT as evidenced by a 3-fold increase in the intensity and a 4 nm blue shift (figure 20) indicating more hydrophobic surfaces for binding to ANS in L178H than in WT. G26R exhibited a similar, if somewhat lower, increase in ANS binding (Table 6). Thus, both the G26R and L178H mutations lead to an increase in accessible hydrophobic surface area. 66 Table 6. Comparison of relative fluorescence intensities of apo A-I proteins at 485 nm. WT G26R L178H 1.0 1.91 3.4 1, Hilt, S. and Roberts, L.M. unpublished observations. Studies on apo A-I with single-site mutations engineered into the protein for structural analysis show that mutations in the N-terminal helix bundle domain (1-190) generally lead to an increase in ANS binding whereas those that occur in the unstructured C-terminal domain lead to a decrease in ANS binding. The ANS fluorescence in the presence of N- terminal single site mutants of apo A-I averaged about 1.5 times that of WT apo A-I with an average of a 4 nm blue shift in wavelength of maximum fluorescence (103). In contrast, the maximum fluorescence intensities of ANS bound to C-terminal single site mutants were about 0.5 that of WT showing that those mutations decreased the exposed hydrophobic surfaces of the molecule. Combining an N-terminal mutation at Y18P with one of more C-terminal mutations increased the intensities back to 1.5 that of WT (180). ANS binding was also studied in truncation mutants. When the entire Cterminus (187-243) was removed from apo A-I, the intensity of the mutants was approximately half that of WT apo A-I (33, 70). Combining a similar carboxy-terminal truncation with a Y18P mutation increased the ANS fluorescence intensity to 1.5 that of WT (180). 67 Given these results, it is likely that both G26R and L178H mutations alter interactions within the N-terminal helix bundle that lead to increased hydrophobic surface exposure and therefore, increased accessibility to ANS. Increased hydrophobic surface exposure may be a requirement to initiate fibril formation (234). Disruption of the helix bundle by both mutations is further supported by the limited proteolysis results. Both G26R and L178H led to an increase in N-terminal protease susceptibility, with major cleavage occurring at Y18 and F57 for both proteins. This is in contrast to WT protein, where the major cleavage occurs between the two domains (at Y192 when chymotrypsin is used) (Figure 24). An additional cleavage at W8 was detected in L178H, which may indicate more accessibility in L178H vs G26R. However, an exhaustive analysis of minor and major cleavage products of both proteins using LC-MS would be needed to determine if this is a significant difference. G26R and L178H are both point mutations that result in N-terminal protease sensitivity. N-terminal truncations, which would similarly disrupt the helix bundle, also exhibit N-terminal protease susceptibility. Two truncations have been studied by our group, Δ1-23 (184) and Δ1-43, whose crystal structure was used to develop models of lipid bound protein (7). All four of these mutations, G26R, L178H, Δ1-23, and Δ143, have a cleavage site at F-57 showing that perturbations to the helical domain all result in similar protease susceptibility. 68 Cleavage also occurred in the C-terminal domain of L178H as evidenced by fragments containing the intact N-terminal sequence. Based on molecular mass, the cleavage positions are most likely Y192 and F229, but this would need to be confirmed by mass spectrometry. Thus, the C-terminal domain retains a protease susceptibility in L178H and G26R similar to that of WT (38) and the main effect of these mutations is within the N-terminal helix bundle domain. Figure 24. Representation of chymotryptic hot spots in wild type, G26R, L178H apo A-I, and N-terminal deletion mutants. (38, 69, 184) Arrows signify cleavage sites determined by sequence analysis and estimated mass of proteolysis fragments. 69 While it is clear from both ANS binding and limited proteolysis data that L178H disrupts the helix bundle domain, intrinsic fluorescence experiments show the tryptophan environment remains relatively nonpolar. L178H compared to WT protein had a similar wavelength of maximum intensity and exhibited only a very slightly increased accessibility to quenching by iodide (Table 5). Since the quenching of peptide fluorescence by iodide can yield Ksv values of 20 M-1 or more and range from about 2 to about 9 M-1 for proteins in the presence of urea, an increase from 1.5 to 1.7 is essentially insignificant and the tryptophans of L178H remain in a non-polar environment, despite alterations to the helix bundle (230, 231). Structural models can be used to examine the locations of these two mutations and how, despite their separation into two clusters, they might produce similar effects. The different models of lipid-free apo A-I were described in the introduction. Here, we refer to the two most recent models, the EPR model of full-length apo A-I published by Lagerstedt and co-workers (224) and the crystal structure of 187-243 apo A-I published by Mei and Atkinson (229). Each model has some drawbacks. The EPR model does not agree with all of the experimental data for apo A-I, particularly in the placement of beta-strands, while the crystal structure is from a C-terminal truncation mutant. However, both models are consistent with some features of apo A-I in solution determined by a variety of methods by different groups of researchers, as discussed in the Introduction, and can be used as a reference point for interpreting structural changes brought about by mutation. In the crystal structure of the dimeric C- 70 terminal truncated mutant (Figure 25), residues 26 and 178 are very near each other spatially. Figure 25. Position of amyloidogenic mutations in structural models of apo A-I. A and B, Crystal structure of 185-243 (229). The amyloidogenic mutation sites and neighboring residues are shown as stick figures. C, EPR model of full-length apo A-I. L178H is represented as a sphere and G26R by a light grey arrow (228). The dimeric structure serves as a model for the monomeric structure if it is assumed the monomer folds back on itself to produce the same inter-helical interactions that are in the dimer (229). According to the Atkinson group, the bundle-opening effect produced by G26R is caused by the loss of a flexible glycine in the structure and by replacement of an attractive interaction with a positively charged residue causing a repulsive interaction. The Atkinson group postulates that L178H destabilizes the bundle by adding a polar residue in the middle of the structure therefore disrupting the packing at the bottom of the bundle. Based on the position of amyloidogenic mutations within the crystal structure, these researchers further hypothesize that the general effect of amyloidogenic mutations is to disrupt the salt-bridge interactions in 71 the helical bundle increasing the solvent exposure which renders the protein more susceptible to protease cleavage. Experiments performed in this thesis support this theory as both L178H and G26R increased protease accessibility within the N-terminal helix bundle. The EPR model proposed by Lagerstedt et al, developed from the full-length protein in solution, resembles the crystal structure of 185-243 apo A-I in that it contains a helix bundle and extensive helix-helix interactions involving numerous salt bridges. However, its topology is quite different and it contains about 18% beta-strand structure. The authors of this model do not address the relationship of their model to the two clusters of amyloidogenic mutations. However, we note here that this model also places G26 and L178 in close proximity (Figure 25C). Therefore, the structural similarity of G26R and L178H are consistent with a close spatial location of these residues in two disparate models of apo A-I. In conclusion, the thesis research shows that the L178H mutation, like the G26R mutation, produces a disruption to the N-terminal helix bundle. Such disruption, particularly when it involves increased hydrophobic surface exposure, is likely to lead to increased aggregation, and from there, to the development of fibrillar, and possibly amyloid, structure. Part II. Development of Aggregated Structure The changes occurring in WT and L178H apo A-I over time presented in this thesis and elsewhere by our group (228) are summarized in Figure 26. ANS binding 72 of both WT and L178H decreased upon incubation at 37oC with the t1/2 of WT occurring earlier (4 days vs 10 days) but to a lesser extent than L178H. ANS binding also decreased in L178H but not WT when incubated at 4oC. In general, a decrease in ANS binding suggests loss of hydrophobic binding sites, as might be brought about by a conformational change. Because ANS binds both pre-fibrillar and fibrillar states of proteins (200, 233), it is not possible to use this method alone to distinguish between monomeric, pre-fibrillar, and fibrillar states. However, this data can be compared to circular dichroism data obtained by our research group in collaboration with the Lagerstedt group (228). Circular dichroism data show that L178H incubated at 37o C acquires helical, rather than beta-strand, structure over time, with a sigmoidal function that has a t1/2 of about 12 days. The transitional region in this curve may represent prefibrillar species that aggregate into helical fibrils by about the third week of incubation at 37o C; samples incubated for 1 month showed clear evidence of fibril formation where WT did not (228). The change in ANS binding for L178H incubated at 37o C shows a similar t1/2 of about 10 days (Figure 21B). WT also exhibited a sigmoidal loss in ANS binding when incubated at 37o C. This occurred with a shorter t1/2 of 4 days, and a smaller decrease in intensity than in L178H. No change occurred in the secondary structure of WT over a 28 day period (228). WT apo A-I has been shown to form aggregated fibrils in vivo and after oxidation (186) in vitro. Researchers investigating the aggregation of WT apo A-I before and after oxidation reported no aggregation occurring for un-oxidized apo A-I 73 in a variety of solutions conditions based on Thioflavin T binding (ThT binds to crossbeta structure), light scattering, and electron microscopy. However, samples were incubated only up to 96 hours for ThT binding and researchers did not report whether data from light scattering and electron microscopy experiments were collected past day 0. Our results indicate that while ANS changes in WT are smaller than those in L178H, their occurrence suggests structural changes that may lead to aggregation, and further investigation into this behavior is needed. Neither WT nor L178H incubated at 4o C exhibited change in intrinsic fluorescence and WT fluorescence did not change at 37o C (Figure 22). However, L178H exhibited a large decrease in fluorescence within the first week of incubation at 37o C. There aren't enough data points to fit these curves but given the trends, the t1/2 must occur by day 5. The magnitude of the intensity decrease suggests the fluorescence change is not due to solvent exposure as unfolded L178H retains much of its fluorescence (235). Instead, the fluorescence is specifically quenched by interaction with a quencher being brought into close proximity. At least eight different amino acid side chains (237) and even the peptide bond (236) are capable of quenching tryptophan fluorescence. Therefore, identification of the quencher source requires more defined structural information. 74 Figure 26. Summary of important changes in WT and L178H apo A-I at 4º C and 37º C over 28 days because of helix (228). 75 CONCLUSION The structure of the L178H mutant of apo A-I was studied over a 28-day period at both 4º C and 37º C by using ANS binding, intrinsic fluorescence, and limited proteolysis experiments. Through those experiments, the L178H protein was observed to undergo structural changes within the first two weeks of incubation at 37o C that may be due to formation of pre-fibrillar oligomers and/or fibrils. These may result from the increase in exposed hydrophobic surface area evidenced by increased ANS binding in L178H compared to WT at day 0-2. An increase in hydrophobic surface area is often associated with the beginning of fibril formation (47, 88, 234). Changes in both ANS binding and intrinsic fluorescence were correlated with increased -helix and the formation of helical fibrils. In addition, we have shown that L178H was found to have different N-terminal protease susceptibility when compared to WT, but similar to the amyloidogenic apo A-I mutant, G26R. This may be due to the nearness of these residues to each other in the protein, as illustrated by two recent structural models of apo A-I derived from different experimental methods. In collaboration with John Voss and Jens Lagerstedt, our group discovered L178H forms helical fibrils (228) while G26R forms beta-sheet fibrils (38). The structural similarity of the proteins presented in this thesis does not provide an easy explanation for this difference. Mutations from either cluster may disrupt the helix bundle in a similar fashion, creating a looser, more exposed structure which can then aggregate. Cluster position may dictate which fibril type then forms. 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