Exam in protein chemistry NKED15/TFKE46 2013-03-15 NOTE question 4 is worth 23 points!! 1 a) Keratin is based on a seven residue or heptad repeat. Describe the properties of this sequence that favour coiled-coil structures. (2p) b) Several connective tissue diseases have been identified as arising from mutations in genes encoding collagen chains. Most common are single base mutations that result in the substitution of glycine by a different residue. Describe the molecular basis of these diseases using your knowledge of structure and stabilizing interactions in collagen. (3p) c) Membrane proteins are often divided in two groups depending on their position in the membrane. What are the names for these two groups? (2p) d) Only a few percent of all protein structure coordinates deposited in the Protein Data Bank comes from membrane proteins. What is the reason for the low percentage of determined structures of membrane proteins? (2p) d) Hydropathy plots of two different membrane proteins of similar size were performed (Figure 1). Which hydropathy profile belongs to which protein? Give an explanation to the different hydropathy profiles. (4p) 1 A B C D Figure 1A-D Three-dimensional structure of two membrane proteins and it corresponding hydropathy plot. 2. a) Enzyme catalysis is often very efficient which can be exemplified by ester hydrolysis. The enzyme catalyzed reaction had a kcat = 1.1 x 103 s-1. The corresponding uncatalyzed reaction had a rate constant of 1.2 x 10-3 M-1s-1. Calculate the catalytic effect and give comments to the result. (3p) b) Draw an energy profile for an enzyme catalyzed reaction and illustrate what difference in energy levels that can be calculated from kcat/Km. (3p) c) For an enzyme it was suspected that an active-site Tyr was forming a critical H-bond with substrate in the transition state of the enzymatic reaction. By site-directed mutagenesis this Tyr was replaced by a Phe and then kcat/Km-values were measured for the wild type and mutant. The values turned out to be 88 M-1s-1 for the wild type and 0.1 M-1s-1 for the mutant. Do these data give support to the assumed H-bond stabilization of the transition state? Support your conclusions with relevant calculations. (kobs = kBT/h exp(-ΔG#/RT); where kobs is a rate constant, R=1.99 cal/moldegree, h=1.58x1034 cals, kB=3.3x10-24cal/degree, T=298 K). (4p) 2 3 a) Discuss why proteins are only marginally thermodynamically stable (5 – 15 kcal/mol). Moreover, how comes that the contribution to the protein stability from a H-bond is only approx. 1 kcal/mol, when the corresponding bond between two water molecules is 5 kcal/mol? (2p) b) How can a chaperone like GroEL prevent a folding molecule from aggregation? In what state is the folding protein most prone to aggregation and why is that? (2p) c)The folding mechanism of the SH3 domain has been studied by φ-value analysis. Describe how ΔΔG# can be determined for the transistion state between I and F (TS(IF) in Figure 2).(NOTE: Figure 2 (without figure text) is also available in colour in Appendix 1) (2p) 3 Figure 2. Changes in ΔG used to calculate the φ-value, along with changes in free energy upon point mutation, ΔΔG, along the folding pathway.Values of G are referenced with respect to the unfolded state U that is arbitrarily assigned a value of 0. TS(UI) and TS(IF) denote the rate-limiting transition states between states U,I and I,F respectively. Inset for the A39V/T47S/N53P/V55L mutant shows the pair of DG profiles ('pseudo-wt' and mutant) from which ΔΔG values are obtained, with the free energies of U states both assigned arbitrarily to 0. I is a folding intermediate and the top figure shows just in a general way how the comparisons are made for a general state denoted A. The mutations A39V/N53P/V55L do not affect the protein at all compared to the wildtype and the protein variant with these mutations are regarded as a pseudowild-type form. Thus, the mutations to consider (shown in bold face) here are from top to botton in the figures above: T47S, R40T, L3A, E5V and F20L. 4 d) What can be concluded from the φ-values about the formation of the β-turn between βstrands 3 and 4 (as probed by positions 40 and 47 (Figure 3)) as well as the folding of β-strand 1 (as probed by positions 3 and 5 (Figure 3))? (2p) e) From NMR-data there is evidence that the intermediate I has a non-native hydrophobic core. What does the F20L mutant tell us regarding that in this study? (2p) Figure 3. Schematic representation of the secondary structure of a homology model of theA39V/N53P/V55L Gallus gallus Fyn SH3 domain (the 'pseudo-wild-type' in this study) in the native state F, featuring the characteristic SH3 domain β sandwich fold formed by the terminal (strands β1, β5) and the approximately orthogonal central β-sheets (strands β2, β3, β4), along with α 310-helical turn. Residues mutated for φ-value analysis are shown in ball-and-stick representation. 5 4 ) Methyltransferases is a large protein family with different functions in the cell, e.g detoxification of substances. A protein structure of one of the member of this family is illustrated below showing the three-dimensional structure, sequence and topology diagram (Figure 4 A, B, and C) A B 6 C Figure 4 A) Three-dimensional structure of a methyltransferase, B) topology diagram of the same methyltransferase and C) secondary structure and amino acid of the same methyltransferase. a) The above figure (Figure 4A) shows the structure of a well-known protein motif. What is the protein motif called? (2p) b) To function this protein bind a cofactor called S-adenosylmethionine illustrated below (figure 5). Suggest a binding site for the cofactor using the topology diagram illustrated above (figure 4B) (2p) Figure 5 Structure of S-adenosylmethionine c) Two emission spectra of the methyltransferase using an excitation wavelength of 295 nm under native conditions (0 M GuHCl) and denatured condition (6M GuHCl) were performed (figure 6). What conclusion can be drawn regarding the positions of the Trp 7 in the methyltransferase? Trp fluorescence is often used to monitor stability of a protein. Why is this method not suitable in this case? (4p) Fluorescence intensity A.U 14000 12000 10000 8000 6000 4000 2000 0 320 340 360 380 400 Wavelength (nm) Figure 6. Emission spectra of methyltransferase under native ( filled circle) and denatured (open cicle) conditions. d) Instead of using Trp fluorescence, an alternative method was used. The extrinsic fluorescent probe, ANS (figure 7) was added to the samples and the thermal stability was monitored (Table 1). Calculate the stability of the protein and interpret the data in terms of structural changes. (3p) Figure 7 Extrinsic fluorescent probe, Anilino-naphtalene sulphonate, ANS 8 Table 1 Experimental data monitoring the fluorescence intensity at 475 nm at various temperatures Temperature 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 Fluorescence Intensity at 475 nm 27560 27620 26350 26690 26030 26140 26280 25700 24690 23820 22740 22080 20240 19070 16430 13470 9440 7570 4740 3490 3520 3430 3170 2180 2410 1870 1190 1070 500 140 190 180 e) To calculate the binding of the cofactor to the protein a ligand binding assay was performed using equilibrium dialysis. Describe the principle of two other methods you can use to measure ligand binding. 9 (4p) f) To analyse the binding of a ligand to the protein an equilibrium dialysis experiment was performed. The protein concentration was kept constant at 4 mg/ml and the molecular weight of the protein was 40000 Da. The experimental data for the ligand binding assay is illustrated below (Table 2). Calculate the dissociation constant and number of binding sites. Give an explanation of what type of binding it is. (4p) Table 2 Experimental data from equilibrium dialysis Total ligand concentration (mM) 0.010 0.020 0.050 0.075 0.100 0.150 0.200 0.400 0.700 1.000 Bound ligand concentration (mM) 0.005 0.009 0.021 0.030 0.039 0.047 0.058 0.067 0.075 0.099 g) The mutation Y240C interacts with a residue in the vicinity and cause stabilization of the protein. Give a reasonable cause for this stabilization and identify the interacting residue. (4) 10