BioChem Ch 7
Structure-Function Relationships in Proteins

Diseases can be caused by changes in protein structure that affect protein’s ability to bind other molecules and carry out its function; can be caused by conformational changes in proteins that affect solubility and degradability


In amyloidosis (AL), Ig chains form insoluble protein aggregate (amyloid) in organs and tissues
Alzheimer disease and familial amyloid polyneuropathy – neurodegenerative diseases characterized by deposition of amyloid

Prion diseases result from misfolding and aggregation of normal cellular protein

Sickle cell anemia – mutation in Hgb, principally affects quaternary structure of Hgb and its solubility, not its ability to bind O
2

Sequence of amino acids and primary structure determines way protein folds into 3D structure (native conformation); once folded, 3D structure of protein forms binding sites for other molecules, thereby dictating function of protein in body o Protein must fold in such a way that it is flexible, stable, able to function in correct site in cell, and

 capable of being degraded by cellular enzymes
Rigidity of peptide backbone determines types of secondary structure that can occur
In globular proteins such as myoglobin, tertiary structure generally forms densely packed hydrophobic core with polar amino acid side chains on outside

Quaternary structure – 2 or more subunits, each composed of polypeptide chain

Tertiary structure of globular protein can be made up of structural domains (regions of structure that fold independently); multiple domains can be linked together to form functional protein o Within domain, combo of secondary structural elements forms fold, such as nucleotide-binding fold or actin fold (folds defined by similarity in several different proteins)

Assembly of globular polypeptide subunits into multisubunit complex (quaternary structure) can provide opportunity for cooperative binding to ligands, form binding sites for complex molecules (antigen binding to Ig), and increase stability of protein

Polypeptide chains of fibrous proteins such as collagen aligned along axis, have repeating elements, and are extensively linked to each other through hydrogen and covalent bonds

Proteins form binding sites for specific molecules (ligands) or for another protein; affinity of binding site for ligand characterized quantitatively by association or affinity constant (K a
) or its dissociation constant (K d
, such that K d
= 1/K a
)

Primary structure of protein dictates way it folds into tertiary structure, which is stable conformation that is identical to shape of other molecules of same protein (native conformation)

Chaperonins act as templates to overcome kinetic and thermodynamic barrier to reaching stable conformation

Prion proteins cause neurodegenerative diseases by acting as template for misfolding

Simplest test for detecting protein in urine is use of specific reagent test strips coated with tetrabromophenol blue, buffered at pH 3.0; with protein, indicator dye is no longer as responsive to changes in pH unlike in absence of protein; yellow color indicates that protein not detectable; as protein levels increase, yellow color changes, going to green, then bluish green; useful qualitative test but to be more quantitative, different dyes
(which react specifically with particular amino acid side chains) used o Different tests required to detect presence of specific protein in urine or sera
Descriptions of Protein Structure

Proteins generally grouped into major structural classifications: globular proteins, fibrous proteins, and transmembrane proteins o Globular proteins – usually soluble in aqueous medium and resemble irregular balls o Fibrous proteins – geometrically linear, arranged around single axis, and have repeating unit structure o Transmembrane proteins – consist of proteins that have one or more regions aligned to cross lipid membrane o DNA-binding proteins (member of globular protein family) sometimes classified separately

Forces involved in protein folding into final conformation primarily noncovalent interactions; include ionic interactions, hydrophobic effect, hydrogen bonding, and van der Waals interactions

Requirements of Three-Dimensional Structure

Protein must create binding site specific for just one molecule or group of molecules with similar structural properties; specific binding sites of protein usually define biologic functions

3D structure must exhibit degrees of flexibility and rigidity required to carry out specific function; some rigidity essential for creation of binding sites and stable structure, but appropriate degree of flexibility and mobility in structure enables protein to fold as it is synthesized and to adapt as it binds other proteins and small molecules

Conformation must be stable, with little tendency to undergo refolding into form that can’t fulfill its function or that precipitates in cell

Must have structure that can be degraded when damaged or no longer needed in cell

Almost every region of primary structure participates in fulfilling one or more of above requirements through chemical properties of peptide bonds and individual amino acid side chains
Three-Dimensional Structure of Peptide Backbone

Amino acids joined sequentially by peptide bonds between carboxyl group of one amino acid and amide gropu of next amino acid in sequence – usually assumes trans configuration in which successive α-carbons and their R groups located on opposite sides of peptide bond

Polypeptide backbone can bend in very restricted way; peptide bond itself is hybrid of 2 resonance structures, one of which has double-bond character, so carboxyl and amide groups that form bond must remain planar; as consequence, peptide backbone consists of sequence of rigid planes formed by peptide groups

Rotation within certain allowed angles (torsion angels) can occur around bond between α-carbon and α-amino group and around bond between α-carbon and carbonyl group; rotation subject to steric constraints that maximized distance between atoms in different amino acid side chains and prohibit torsion angles that place side-chain atoms too close to each other
Secondary Structure

Secondary structures – regions within polypeptide chains forming recurring, localized structures

Both α-helix and β-sheet contain repeating elements formed by hydrogen bonding between atoms of peptide bonds

Other regions form irregular, nonrepetitive secondary structures such as loops and coils

α-helix common secondary structural element of globular proteins, membrane-spanning domains, and DNAbinding proteins; has rigid stable conformation that maximizes hydrogen bonding while maintaining allowed rotation angles of polypeptide backbone o Peptide backbone formed by hydrogen bonds between each carbonyl oxygen atom and amide hydrogen of amino acid residue located 4 residues farther down the chain ; each peptide bond connected by H bonds to peptide bond 4 AA residues ahead of it and 4 AA residues behind it o Core of helix tightly packed, thereby maximizing association energies between atoms o Trans side chains of AAs project backward and outward from helix, thereby avoiding steric hindrance with polypeptide backbone and with each other o Proline, because its N is part of its cyclic structure, cannot form appropriate bond angles to fit into αhelix, so proline is the helix breaker

β-sheets – H bonding usually occurs between regions of separate neighboring polypeptide strands aligned parallel to each other; carbonyl oxygen of one peptide bond is H-bonded to N of peptide bond on adjacent strand; optimal H bonding occurs when sheet is bent (pleated) o β-sheet described as parallel if polypeptide strands run in same direction (as defined by amino and carboxy terminals), and antiparallel if they run in opposite directions o Antiparallel strands often same polypeptide chain folded back on itself, with simple hairpin turns or long runs of polypeptide chain connecting strands o Amino acid side chains alternate between extending above or below plane of sheet o Parallel sheets tend to have hydrophobic residues on both sides of sheets; antiparallel sheets usually have hydrophobic side and hydrophilic side o Frequently, sheets twist in one direction o In antiparallel sheet, atoms involved in H bonding are directly opposite each other o In parallel sheet, atoms involved in H bonding slightly skewed from one another, such that one amino acid is H-bonded to 2 others in opposite strand


Bends, loops, and turns are irregular secondary structures that do not have repeating element of H bond formation; characterized by abrupt change of direction and often found on protein surface o β-turns – short regions that usually involve 4 successive AA residues; often connect strands of antiparallel β-sheets o Surface of large globular proteins usually has at least one omega loop (structure with a neck like Ω)

LDH typical of globular proteins, which average 31% α-helical structure and 28% β-sheet structure o Helices of globular domains have average span of 12 residues, corresponding to 304 helical turns
(though many are longer) o Sheets are average of 6 residues long and 6 wide (2-15 strands); generally twist to the right rather than life flat o Most globular domains contain motifs (relatively small arrangements of secondary structure recognized in many different proteins (for example, certain β-strands connected with α-helices to form βαβαβ structural motif) o Remaining polypeptide segments connecting helices and sheets have coil or loop conformation; some of these have names, and some appear disordered or irregular; nonregular regions (coils) are stabilized through specific H bonds dictated by primary sequence of protein and do not vary from one molecule of protein to another of same protein o Irregular coils, loops, and other segments usually more flexible than relatively rigid helices and sheets; often form hinge regions that allow segments of polypeptide chain to move as compound binds or to
 move as protein folds around another molecule
Amyloid fibril structure composed of repeated β-sheets aligned orthogonally (perpendicular) to axis of fiber o In amyloid diseases, amyloid derived from different protein that has changed its conformation to that of amyloid repeated β-sheet structure o Once amyloid deposition begins, it proceeds rapidly as if fibril itself were promoting formation and deposition of more fibrils (seeding) o Different clinical presentations of each disease result from differences in function of native protein and site of amyloid deposition
Tertiary Structure


Creates specific and flexible binding sites for ligands

Tertiary structure of protein is folding pattern of secondary structural elements in to 3D conformation
Maintains residues on surface appropriate for protein’s cellular location, polar residues for cytosolic proteins, and hydrophobic residues for transmembrane proteins

3D structure of protein flexible and dynamic, with rapidly fluctuating movement in exact position of AA side chains and domains; fluctuations wiggle without unfolding o Allow ions and water to diffuse through structure and provide alternative conformations for ligand binding

Forces that maintain tertiary structure are H bonds, ionic bonds, van der Waal’s interactions, hydrophobic effect, and disulfide bond formation

Tertiary structure of large complex protein often described in terms of physically independent regions
(structural domains); each domain formed from continuous sequence of AAs in polypeptide chain that are folded into 3D structure independently of rest of protein, and domains are connected through simpler structure such as loop o Structural features of each domain can be discussed independently of another domain in same protein, and structural features in one domain may not match that of other domains in same protein

Folds – relatively large patterns of 3D structure that have been recognized in many proteins, including proteins from different branches of phylogenetic tree o Characteristic activity associated with each fold, such as ATP binding and hydrolysis (actin fold) or NAD + binding (nucleotide-binding fold)

Actin fold – ATP bound in middle of cleft of actin fold by AA residues contributed by domains on both sides; ATP binding promotes conformational change that closes cleft; once bound ATP cleaved to ADP and phosphate o Actin fold found in actin (polymerizes to form cytoskeleton), heat shock protein 70 (hsp70 – uses ATP energy in changing conformation of other proteins), and hexokinase (catalyzes phosphorylation of glucose – these three look nothing like each other, but actin folds are almost superimposable

 Amount of sequence identity they have in common is consistent with membership in same fold family and establishes that they are homologs of same ancestral protein
 In all these proteins, ATP binding results in large conformational changes that contribute to
 function of protein
Nucleotide-binding fold – formed by one domain; fold is binding site for NAD + or, in other proteins, molecules with generally similar structure (e.g., riboflavin); many proteins that bind NAD+ or NADP+ contain very different fold from separate fold family o These 2 different NAD + -binding folds arise from different ancestral lines and have different structures, but have similar properties and function (believed to be product of convergent evolution)

Most globular proteins soluble in cell; in general, core of globular domain has high content of AAs with nonpolar side chains out of contact with aqueous medium (hydrophobic effect) o Hydrophobic core densely packed to maximize attractive van der Waals forces, which exert themselves over short distances o Charged polar AA side chains generally located on surface of protein, where they form ion pairs (salt bridges, ionic interactions) or are in contact with aqueous solvent; charged side chains often bind inorganic ions to decrease repulsion between like charges
 When charged amino acids located on interior, they are generally involved in forming specific binding sites
 Polar uncharged AA side chains usually found in surface of protein, but may occur in interior, Hbonded to other side chains
 Cystine disulfide bonds (bond formed between 2 cysteine sulfhydryl groups) sometimes involved in formation of tertiary structure

Transmembrane proteins, such as β
2
-adrenergic receptor, contain membrane-spanning domains and intracellular and extracellular domains on either side of membrane o Many ion channel proteins, transport proteins, neurotransmitter receptors, and hormone receptors contain similar membrane-spanning segments that are α-helices with hydrophobic residues exposed to lipid bilayer – connected by loops containing hydrophilic AA side chains that extend into aqueous medium on both sides of membrane o In β
2
-adrenergic receptor, helices clump together so extracellular loops form surface that acts as binding site for hormone adrenaline – binding site sometimes called functional domain even though it is not formed from continuous segment of polypeptide chain
 Once adrenaline binds to receptor, conformational change in arrangement of rigid helical structures transmitted to intracellular domains that form binding site for heterotrimeric G protein (GTP-binding protein); thus receptors require both rigidity and flexibility to transmit signals across PM o Transmembrane proteins usually have several post-translational modifications that provide additional chemical groups to fulfill requirements of 3D structure
 Amino terminus of β
2
-adrenergic receptor (residues 1-34) extends out of membrane and has branched high-mannose oligosaccharides linked through N-glycosidic bonds to amide of asparagines; part of receptor anchored in lipid PM by palmitoyl group that forms thioester with
–SH residue of cysteine; -COOH terminus, which extends into cytoplasm, has several serine and threonine phosphorylation sites that regulate receptor activity
Quaternary Structure

Association of individual polypeptide chain subunits in geometrically and stoichiometrically specific manner o Oligomers – proteins that have a few subunits (unspecified) combined to make one protein

Subunits of particular protein always combine in same number and in same way because binding between subunits dictated by tertiary structure, which is dictated by primary structure, which is determined by genetic code

Protomer – unit structure within protein composed of multiple, nonidentical subunits (Hgb is tetramer of α
2
β
2
, but one α-β pair can be called a protomer)

Oligomer – multisubunit protein composed of identical subunits (i.e., F-actin)

Multimer – generic term for complex with many subunits of more than one type


Insulin composed of 2 nonidentical polypeptide chains attached to each other through disulfide bonds between chains; subunits of globular proteins generally not held together by disulfide bonds, but regions of same chain may be connected by disulfide bonds that form as chain folds o Insulin fits generalization because it is synthesized as single polypeptide chain, which forms disulfide bonds; subsequently, proteolytic enzyme in secretory vesicles clips polypeptide chain into 2 nonidentical subunits o Generally each subunit of most protomers and oligomers synthesized as separate polypeptide chain o In fibrous proteins, which have regular sometimes repeating sequence of AAs, interchain and intrachian covalent binding serves different functions
 In collagen, extensive interchain binding provides great tensile strength

Contact regions between subunits of globular proteins resemble interior of single subunit protein: contain closely packed nonpolar side chains, H bonds involving polypeptide backbones and their side chains, and occasional ionic bonds or salt bridges

Subunits of globular proteins rarely held together by interchain disulfide bonds and never by other covalent bonds; fibrous and other structural proteins may be extensively linked to other proteins through covalent bonds

Assembly into multisubunit structure increases stability of protein; increase in size increase number of possible interactions between AA residues, and therefore makes it more difficult for protein to unfold and refold; as a result, many soluble proteins composed of 2-4 identical or nearly identical subunits

Multisubunit structure enables protein to exhibit cooperation between subunits in binding ligands or to form binding sites with high affinity for large molecules; different subunits can have different activities and cooperate in common function
Quantitation of Ligand Binding

Association constant (K a
) is equilibrium constant for binding reaction of ligand (L) and protein (P)
[LP]/[L][P] = K a
= 1/K d o The tighter the binding of ligand to protein, the higher the K a
and lower the K d o K a
useful for comparing proteins produced by different alleles or describing affinity of receptor for different drugs
Structure-Function Relationships in Myoglobin and Hemoglobin

Myoglobin and hemoglobin are 2 oxygen-binding proteins with very similar primary structures o Myoglobin is globular protein composed of single polypeptide chain that has one O
2
-binding site o Hemoglobin is tetramer composed of 2 different types of subunits; each subunit has strong sequence homology to myoglobin and contains O
2
-binding site

Tetrameric structure of Hgb facilitates saturation with O
2
in lungs and release of O
2
as it travels through capillary beds

When amount of O
2
bound to myoglobin or Hgb plotted against P
O2
, hyperbolic curve obtained for myoglobin but sigmoidal for Hgb (myoglobin is much more to the left than Hgb)

Myoglobin present in heart and skeletal muscle; can bind O
2
released by Hgb, which it stores to meet demands of contraction; as O
2
used in muscle cell for generation of ATP during contraction, it is released from myoglobin and picked up by cytochrome oxidase (heme-containing enzyme in electron-transport chains that has even
 higher affinity for O
2
than myoglobin)
Myoglobin readily released from skeletal muscle or cardiac tissue when cell damaged; large injuries to skeletal muscle that result from physical crushing or lack of ATP production result in cellular swelling and release of myoglobin and other proteins into blood o Myoglobin passes into urine (myoglobinuria) and turns urine red because heme (which is red) remains covalently attached to protein o During AMI, myoglobin is one of first proteins released into blood from damaged cardiac tissue; however amount released not high enough to cause myoglobinuria o Because myoglobin not present in skeletal muscle and heart as tissue-specific isozymes, and amount released from heart much smaller than amount that can be released from large skeletal muscle injury, myoglobin measurements not specific for MI o Because of lack of specificity in myoglobin measurements, cardiac markers of choice for detection of MIs are heart isozyme of troponins (I and/or T), and CK


Tertiary structure of myoglobin consists of 8 α-helices connected by short coils (globin fold); unusual for a globular protein because it has no β-sheets; helices create hydrophobic O
2
-binding pocket, containing tightly bound heme with Fe 2+ in its center o Negatively charged propionate groups on porphyrin ring interact with arginine and histidine side chains from Hgb, and hydrophobic methyl and vinyl groups that extend out from porphyrin ring interact with hydrophobic AA side chains from Hgb, positioning heme group within protein o About 16 different interactions between myoglobin AAs and different groups in porphyrin ring o In binding pocket of myoglobin, O
2
binds directly to Fe 2+ on one side of planar porphyrin ring; Fe 2+ able to chelate 6 different ligands (4 of ligand positions in plane and taken by central N’s in planar porphyrin ring and 2 perpendicular to this plane: one taken by N on a histidine (proximal histidine) which extends down from myoglobin helix and other taken by O
2
, CO, or remains empty)
 Proximal histidine of myoglobin and Hgb sterically repelled by heme porphyrin ring, so when histidine binds Fe 2+
 When O
2
in middle of ring, it pulls Fe 2+ above plane of ring
binds on other side of ring, it pulls Fe 2+ back into plane of ring – pull of O
2
moves proximal histidine toward porphyrin ring, which moves helix containing proximal histidine, causing conformational change that has no effect on function of myoglobin o Movement of one helix of Hgb leads to movement of other helices in that subunit, including one in corner of subunit that is in contact with different subunit through ionic interactions (salt bridges); loss of salt bridges induces conformational changes in all other subunits, and all 4 subunits may change in concerted manner from original conformation to new conformation

Sickle cell anemia – caused by wrong quaternary structure; vaso-occlusive crises caused by polymerization of
HbS molecules into long fibers that distort shape of RBCs into sickle cells o Substitution of hydrophobic valine for glutamate in β
2
chain creates hydrophobi knog on surface of deoxygenated HbS that fits into hydrophobic binding pocket on β
1
subunit of a different Hgb molecule o Third HbS molecule, which binds to first and second molecules through aligned polar interactions, binds a fourth HbS through its valine knob, and polymerization continues until long fibers formed o Polymerization of Hgb molecules highly depends on concentration of HbS and is promoted by conformation of deoxygenated molecules o At 100% O
2
saturation, even high concentrations of HbS will not polymerize o RBC spends longest amount of time at lower O
2
concentrations of venous capillary bed, where polymerization most likely initiated

Cooperativity of O
2
binding in Hgb comes from conformational changes in tertiary structure that take place when O
2
binds; changes from T (tense) state with low affinity for O
2
to R (relaxed) state with high affinity for O
2 o Breaking salt bridges in contacts between subunits is energy-requiring process and, consequently, binding rate for first O
2
is very low o When next O
2
binds, many Hgb molecules containing one O
2
will already have all 4 subunits in R state, so rate of binding much higher o With 2 O
2
molecules bound, even higher percentage of Hgb will have all 4 subunits in R state (positive cooperativity – responsible for sigmoidal O
2
saturation curve)
Structure-Function Relationships in Immunoglobulins

Immunoglobulins – all have similar structure: 2 identical small polypeptide chains (light chains) and 2 identical large polypeptide chains (heavy chains); chains joined to each other by disulfide bonds

IgG (γ-globulins) – most abundant Ig in human blood; have attached oligosaccharides that participate in targeting protein for clearance from blood o Both light and heavy chains consist of domains (immunoglobulin fold) which is collapsed number of βsheets (β-barrel)

Both light and heavy chains contain variable regions and constant regions; variable regions of L and H chains interact to produce single antigen-binding site at each branch of Y-shaped molecule

Each population (clone) of B cells produces antibody with different AA composition in variable region that is complementary to structure of antigen that elicits that response

K d
of antibodies for their specific antigens is extremely small, so antigen binds very tightly with almost no tendency to dissociate and can be removed from circulation as antigen-antibody complex ingested by macrophages


Constant domains that form Fc part of antibody important for binding antigen-antibody complex to phagocytic cells for clearance and other aspects of immune response

Can prove that Igs produced by single clone by doing electrophoresis and looking for spike of lots of Igs in same area (plasma cell dyscrasia)

In amyloidosis/AL, amyloid formed from degradation of products of λ or κ-light chains that deposit most frequently in ECM of kidney and heart, but also may deposit in tongue o In other types of amyloidosis, amyloid arises from other proteins and deposits in characteristic organ
 Amyloid associated with chronic inflammatory conditions, such as rheumatoid arthritis, derived from serum protein (serum amyloid A) produced by liver in response to inflammation; deposits most frequently in kidney o Treatment is only partially successful – most efficacious approach involves stem cell transplantation in concert with immunosuppression; if patient not candidate for transplantation, then drugs such as melphalan (antineoplastic agent) and dexamethasone (steroid) can be used o Renal and cardiac transplantation have been performed with some success

Antibodies useful for radioimmunoassay (radioactive-tagged antibodies bind to whatever you want, then separate that from solution and measure bound radioactivity); useful for measuring small amounts of hormones present in blood for diagnosis of endocrine diseases
Protein Folding

Sequence of AA side chains (determined by primary structure) dictates fold pattern of 3D structure and assembly of subunits into quaternary structure

Under certain conditions, denatured proteins can refold into their native conformation, regaining original function

Proteins can be denatured with organic molecules such as urea that disrupt H-bonding patterns (both of protein and water) and convert protein to soluble random coil; many simple single-subunit proteins such as ribonuclease denatured this way will fold spontaneously back to native conformation if brought back to physiologic conditions o Even complex multisubunit proteins containing bound cofactors can sometimes renature spontaneously under right conditions o Very little difference seen in energy state of native state and several other stable conformations that protein might assume o If misfolded proteins do not precipitate into aggregates, they can be degraded in cell by proteolytic reactions or even refolded o Not all proteins fold into native conformation on their own; as protein starts folding, it passes through many high-energy conformations that slow process (kinetic barriers) that can be overcome by heatshock proteins, which use energy provided by ATP hydrolysis to assist in folding process
 Heat-shock proteins have different families with different activities
 Hsp70 proteins bind to nascent polypeptide chains as their synthesis is being completed to keep uncompleted chains from folding prematurely; also unfold proteins before insertion through membrane of mitochondria and other organelles
 Hsp60 family called chaperonins; unfolded protein fits into barrel cavity that excludes water and serves as template for folding process; hydrolysis of several ATP molecules used to overcome energy barriers to reaching native conformation o Cis-trans isomerase and protein disulfide isomerase participate in folding
 Cis-trans isomerase converts trans peptide bond preceding proline into cis conformation, well suited for making hairpin turns
 Disulfide isomerase breaks and reforms disulfide bonds between –SH groups of 2 cysteine residues in transient structures formed during folding process; after protein folded, cysteine-SH groups in close contact in tertiary structure can react to form final disulfide bonds

Primary structure of mature insulin different from that of its precursor (preproinsulin) o Proinsulin forms 3D structure, then C-peptide removed from protein by proteolytic cleavage, thereby altering primary structure of protein; change in primary structure does not allow denatured mature insulin to refold into an active conformation


Amino acids on proteins undergo wide range of chemical modifications not catalyzed by enzymes, such as nonenzymatic glycosylation or oxidation o Lead to loss of function and denaturation of protein, sometimes to form that cannot be degraded in cell o Nonenzymatic glycosylation – glucose present in blood, interstitial fluid, or intracellular fluid, binds to exposed amino group on protein; 2-step process forms irreversibly glycosylated protein
 Proteins that turn over very slowly in body, such as collagen or Hgb, exist with significant fraction present in glycosylated form
 Because reaction is nonenzymatic, rate proportionate to concentration of glucose present, and indivduals with hyperglycemia have much higher levels of glycosylated proteins o Collagen and other glycosylated proteins in tissues further modified by nonenzymatic oxidation and form additional cross-links; net result is formation of large protein aggregates (advanced glycosylation end products (AGEs); AGEs accumulate with age, even in individuals with normal blood glucose levels

Rate of irreversible nonenzymatic glycosylation of Hgb and other proteins directly proportional to glucose concentration to which they are exposed over last 4 months; danger of sustained hyperglycemia is, over time, many proteins become glycosylated and subsequently oxidized, affecting solubility and ability to function o Glycosylation of collagen in heart results in cardiomyopathy in patients with chronic uncontrolled DM o Glycosylation of Hgb has little effect on its function

Proteins can be denatured by changes (pH, temp, solvent) that disrupt ionic, H, and hydrophobic bonds o At low pH, ionic bonds and H bonds formed by carboxylate groups disrupted, and at alkaline pH, H and ionic bonds formed by basic AAs would be disrupted o Proteins denatured by gastric juice of stomach (pH 1-2); pH cannot break peptide bonds, disruption of native conformation makes protein better substrate for digestive enzymes o Temperature increases vibrational and rotational energies in bonds, affecting energy balance that goes into making stable 3D conformation (i.e., albumin under heat converts to denatured white precipitate instead of translucent native state) o Protein precipitates can sometimes be dissolved by amphipathic agents (urea, guanidine hydrochloride, or sodium dodecylsulfate (SDS) that form extensive H bonds and hydrophobic interactions with protein o Hydrophobic molecules can denature proteins by disturbing hydrophobic interactions in protein; longchain fatty acids can inhibit many enzyme-catalyzed reactions by binding nonspecifically to hydrophobic pockets in proteins and disrupting hydrophobic interactions (long-chain fatty acids and other highly hydrophobic molecules have their own binding proteins in cell)

Prion proteins cause neurodegenerative disease by acting as template to misfold other cellular prion proteins into form that cannot be degraded o Prion = proteinaceous infectious agent o Prion diseases can be acquired through infection (mad cow disease) or from sporadic or inherited mutations (Creutzfeldt-Jakob disease (CJD)) o Mad cow disease in UK (new-variant CJD); growth-hormone inoculations in U.S. and France (iatrogenic or doctor-induced CJD); ritualistic cannibalism in Fore tribespeople (Kuru) – all rare but have publicity o Prion protein normally found in brain and is encoded by gene normally component of human genome; disease-causing form of prion protein has same AA composition but is folded into different conformation that aggregates into multimeric protein complexes resistant to proteolytic degradation o Normal conformation of prion protein is PrP c and disease-causing form is PrP Sc (sc for prion disease scrapies in sheep) – although PrP Sc and PrP c have same AA composition, PrP Sc conformer substantially enriched in β-sheet structure compared with normal PrP c conformer, which has little or no β-sheet structure and about 40% α-helix
 Both conformations have similar energy levels
 Spontaneous refolding of PrP proteins into PrP Sc conformation prevented by large activation energy barrier that makes conversion extremely slow (3000-4000 years), so very few (if any)
PrP Sc normally formed in lifetime
 Infectious disease occurs with ingestion of PrP Sc dimers in which prion protein already folded into high β-structure; PrP Sc proteins act as template to lower activation energy barrier for conformational change, causing native proteins to refold into PrP Sc conformation much more

rapidly (like chaperonins); refolding initiates cascade as each new PrP Sc formed acts as template for refolding other molecules
 As number of PrP Sc molecules increase in cell, they aggregate into multimeric assembly resistant to proteolytic digestion; once aggregate begins to form, concentration of free PrP Sc decreases, shifting equilibrium between PrP and PrP Sc to produce more PrP Sc , which leads to further aggregate formation and shifting equilibrium further in that direction o Prion diseases categorized as transmissible spongiform encephalopathies (neurodegenerative diseases characterized by spongiform degeneration and astrocytic gliosis in CNS); frequently protein aggregates and amyloid plaques seen o Familial prion diseases caused by point mutations in gene encoding Pr protein; diseases’ names related to different mutations and clinical syndrome o Familial Creutzfeldt-Jakob disease (fCJD) arises form inherited mutation and has autosomal dominant pedigree; typicall presents in 4 th decade of life; mutation lowers energy required for protein to fold into
PrP Sc conformation, thereby conversion occurs more readily
 Lowering activation energy for refolding by mutation decreases time of spontaneous generation to 30-40 year prodromal (early symptom indicating disease) period o Sporadic CJD may arise from somatic cell mutation or rare spontaneous refolding that initiates cascade of refolding into PrP Sc conformation – this accounts for 85% of all cases of CJD)
Basics of Hemoglobin Cooperativity

All subunits of Hgb contain 8 helices (labeled A through H), with A representing helix at amino-terminal end

Deoxygenated form of Hgb stabilized by o Asp 94 of F helix of β-chain, through carboxylate side chain, forms salt bridge (ionic interaction) with charged imidazole group of His 146, which is carboxy-terminal AA in β-chain o Carbonyl carbon of Val 98 forms H bond with tyrosine hydroxyl group at position 145 (next-to-last AA in
β-chain); this positions helices F and H in close proximity o Free carboxylate group of His 146 (of β-chain) forms salt bridge with ε-amino group of side chain of Lys
40 of corresponding α-chain; salt bridge allows communication between these 2 subunits and places βchain H helix close to α-subunit

In β-chain, proximal His 92 (8 th AA of F helix or F8) forms coordinate covalent bond with iron in heme, and in so doing, pulls iron slightly out of plane of heme ring (all occurring during deoxygenated state)

O
2
binds to iron in bent conformation, triggering movement of iron into plane of heme ring o Because iron also covalently linked to histidine F8, entire F helix and FG corner also move o Asp 94 in F helix – because it is moved (because of movement of F helix), it can no longer form salt bridge with imidazole of His 146, which weakens interactions between F and H helices o Valine 98 in FG corner of β-subunit, so as that moves, H bond formed between Val 98 and Tyr 145 (at end of H helix) also broken o Caused in part by loss of interactions between F and H helices, H helix moves, and in so doing, breaks ionic interaction between His 146 and Lys 40 of α-subunit; this leads to rotation of one αβ-dimer relative to other αβ-dimer, and will allow O
2
to bind more readily to other subunits

Disruption of bonds (sequence described above) has to occur for O
2
to bind, which is why it takes high concentration of O
2
to get first O
2
bound to Hgb o At low O
2
levels, O
2
can dissociate from iron, which allows T form to re-form and allows salt bridges and
H bonds, which stabilize deoxygenated form, to reform o If O
2
concentration high such that iron continuously occupied with O
2
, oxygenation of Hgb will occur better as events of oxygenation more likely to occur