Proteins As food source of amino acids source of calories (energy) determine physical properties An Introduction to Protein tructure In the body structural contractile/motion enzymes enzyme inhibitors 2 transport/binding lipid trafficking receptors transporters antibodies electron carriers hormones (some) tarting at The Beginning G.M. mith, F 0 order amino acid composition Primary sequence econdary repeating structural patterns defined by ϕ, ψ and ω angles Motifs common combinations of secondary structure Tertiary overall fold Domains portions of proteins that fold independently Quaternary subunit structure ther nonprotein components - 3 + " ' Glycine G 7.2% occurrence 3 Alanine A 7.2% occurrence 3 3
3 3 2 2 erine 6.8% ysteine 1.9% 3 3 istidine 2.3% Asparagine 4.3% 3 3 2 2 2 3 Threonine T 5.9% 2 2 3 Methionine M 2.2% (usually considered non-polar) Glutamine Q 4.3% 3 2 2 2 Aspartate D 5.3% 3 2 Lysine K 5.9% 3 2 2 2 2 3 Glutamate E 6.3% 3 2 2 Arginine 5.1% 3 2 2 2 2 2 Valine V 6.6% 3 Phenylalanine F 3.9% 3 2 Leucine L 9.1% 3 3 3 3 2 Tyrosine Y 3.2% 3 2 Isoleucine 5.3% I 3 3 3 2 3 Tryptophan W 1.4% 3 2
2 2 2 Proline P 5.2% 2 residue pka (K/D) residue pka (K/D) Ile I ---- 4.5 Trp W ---- -0.9 Val V ---- 4.2 Tyr Y 10.07-1.3 Leu L ---- 3.8 Pro P ---- -1.6 Phe F ---- 2.8 is 6.0-3.2 ys 8.33 2.5 Asn ---- -3.5 Met M ---- 1.9* Gln Q ---- -3.5 Ala A ---- 1.8 Asp D 3.86-3.5 Gly G ---- -0.4 Glu E 4.25-3.5 Thr T ---- -0.7 Lys K 10.53-3.9 er ---- -0.8 Arg 12.48-4.5-2 -terminus Lysine - -terminus Aspartate Glutamate - 2 Asparagine Glutamine - erine Threonine - ysteine (Methionine) -guanidino Arginine -imidazole istidine -φ Phenylalanine Tyrosine -indole Tryptophan -aliphatic Ala, Val, Leu, Ile - 2 -terminus Lysine Ionization (gain of a proton, - 3+ ) Good nucleophile (if deprotonated,- 2 ) Acylation Alkylation (substitution rx) ondensation with carbonyl groups Michael additions trong -bond acceptor (if deprotonated) or donor Metal coordination (if deprotonated) - -terminus Aspartate Glutamate Ionization (loss of a proton) Weak nucleophile an be attacked by nucleophile best if activated trong -bond acceptor at either or donor (if protonated) Metal coordination - 2 Asparagine Glutamine either acidic nor basic an be attacked by nucleophile hydrolysis nitrogen exchange is moderately nucleophilic glycosylation trong -bond donor or acceptor = acceptor 2 = donor or weak acceptor Metal coordination (rare?)
- erine Threonine (Tyrosine) Good nucleophile Acylation Alkylation (substitution rx) ondensation with carbonyl groups trong -bond donor or acceptor Metal coordination Elimination (not Tyr) - ysteine Excellent nucleophile, especially if deprotonated Acylation Alkylation (substitution rx) Michael additions xidizable Disulfide formation igher oxidation states Ionization (loss of a proton) trong -bond donor (if protonated) or acceptor Metal coordination, tight binding to soft metals Elimination -- Methionine xidation Weak -bond acceptor Metal coordination (cytochromes c) Elimination Very weak nucleophile Most adducts do not lead to products -guanidino Arginine Positively charged to very high p Moderate nucleophile Acylation Alkylation (substitution rx) ondensation with carbonyl groups trong -bond donor -imidazole istidine Good nucleophile if deprotonated Acylation Alkylation (substitution rx) Ionization (gain of a proton) trong -bond acceptor (if deprotonated) or donor Metal coordination -φ Phenylalanine Tyrosine Electrophilic substitution e.g., nitration oxygenation/oxidation iodination
-indole Tryptophan -aliphatic Ala, Val, Leu, Ile Electrophilic substitution e.g., nitration oxygenation Iodination xidation Good -bond donor But indolic proton is not acidic Free radical reactions? Backbone All free amino acids can be decarboxylated to form the corresponding amine e.g., tryptamine, histamine, tyramine 3 + - r deaminated to form an -ketoacid or -hydroxyacid (rare) e.g., -ketoglutarate, oxaloacetate, pyruvate Generally, by specific enzymes involved in the synthesis or degradation of the amino acid. " ' Deacylation, acylation of -Terminus Proteolysis Methylation Phosphorylation idechain modification for crosslinking onversion of sidechains to prosthetic groups Attachment of prosthetic groups Attachment of lipids Glycosylation Deformylation of formyl methionyl proteins Acetylation of cytoplasmic proteins of eukayrotes (60-90%)! -Acetyltransferases + 3 2 3 oa Generally: Gly, Ala, er, Thr, or + Met-Asp, Met-Glu, or Met-Asn oa 2 deformylase (e.g., Methionyl aminopeptidase) Myristoylation leavage of signal peptides and additional proteolytic processing + - 2 + ( oa! -MyristoylProtein 3 2 ) 11 2 + Myristoyl oa oa
arboxyl Modifications Amidation, especially peptide hormones Usually removal of an -terminal Gly 2 + 2 Glycolic acid Membrane anchors Protein 2 2 P 6Man!1 2Man!1 - Gal!1 Gal!1 2Man!1 4Glc 2!1 Gal!1 Gal!1 2 P = FA - 1 P 3 -linked glycophosphoinositol FA 2 2 = P 3 exoses Galactose Mannose Glucose Deoxyhexoses L-Fucose exosamines -Acetylglucosamine -Acetylgalactosamine ialic Acid Acylneuraminic Acid Pentoses Xylose L-Arabinose 2 3 -Acetylneuraminic acid (AA) 3 Lactate 3 2 pyruvate 2 3 2 -Acetylmuramic Acid 2 AA 3 Thr 133 Gal 2 2 AA 3 3 Gal Ac 3 2 2 2 3 GlcAc-"1-Asn 2 2 2 Gal-"1-yl 2 2 2 2 3 GlcAc-!1-er 2 Xyl-"1-er Lysyl xidase 2 2 2 2 2 Lysyl residue 2 2 2 2 2 2 Allysyl residue 2 2 2 2 2 4 + + 2 reduction + 2 2 2 2 2 Lysyl residue 2 2 2 Allysyl residue Lysinonorleucine crosslink 2 2 2 2 2 2 2 2
Aldol condensation between two allysines 2 2 2 + 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ' ' 2 2 2 3 Allysine + Lysine Desmosine Disulfide Bonds in Lysozyme ys- 2 - + - 2 -ys ys- 2 --- 2 -ys + 2 + + 2e - (don t forget these) peptide peptide 2 Topaquinone Tryptophylquinone Fe: lone irons; 2-iron, 4-iron clusters; hemes FeMo clusters Zn: zinc enzymes; zinc fingers a: parvalbumin and the EF hand Mg: chlorophyll u, i, (from amine oxidases)
Iron lusters Ferredoxins, IPIP s and aconitase 3 2 ys 2 Fe Fe 2 ys ys 2 2 ys Fe Fe ys 2 2 ys 3 3 ys 2 Fe Tetrahedral Zn 2+ Fe 2 ys ys Zn 2 Many enxymes (alcohol dehydrogenase shown) ys is All cysteines + acid-labile sulfur ys, carboxylates, at least one is* 2 2 2 2 Fe 3 2 Unfolded proteins bind water build viscosity little else Folded proteins have surfaces, crevices binding sites Folding information is in the sequence Potential pitfalls misfolding aggregation helper systems exist The rdered Lattice of Ice -bonding in liquid water is much less ordered exokinase V = -µ 1 µ 2 (2cos Θ 1 cos Θ 2 - sin Θ 1 sin Θ 2 ) / r 3 V = µ 1 µ 2 / r 3 V = -µ 1 µ 2 / r 3 V = -2µ 1 µ 2 / r 3
ydrogen Bonds between water and other donors and acceptors Type of bond typical distance, r ydroxyl-hydroxyl 2.8Å ydroxyl-carbonyl 2.8 Amide-carbonyl 2.9 Amide-hydroxyl 3.0 Amide-imidazole 3.1 r ' r bond angle, ~155-160 not critical r = 2Å+0.15Å peptide bond ~120 kcal/mole disulfide bond ~80 electrostatic interaction 10-20 -bonds 1-5 van der Waals attraction 3-5 other dipole attraction weaker. hothia Water can -bond extensively without regard to orientation. There is always another water molecule to -bond to. Enthalpy is low and entropy is high. At an interface, water orients itself to maximize -bonding. Entropy is lower. The solute should present the smallest surface possible.
Alcohol Dehydrogenase with bound water in a crystal If hydrophobic effect drives proteins to fold and the structure of water mediates the hydrophobic effectwhat happens if the structure of water is altered? The ofmeister series: competing theories: haotropes and lyotropes Arakawa and Timasheff: preferential exclusion/hydration Denaturants Anions F - P 3-3 2-4 3 - l - Br - I - - ations ( 3 ) 4 + ( 3 ) 2 + 2 + 4 K + a + s + Li + Mg 2+ a 2+ Ba 2+ Lyotropic haotropic structure formers structure breakers Arakawa & Timashef seem to be winning the argument mpnds/ions that are preferentially excluded from the hydration sphere of proteins are stabilizers Why? (T. ecord, Protein ci. 10, 2485-2497) Urea is preferentially partitioned into the first hydration sphere Unfolded proteins have more surface area larger hydration sphere (associate with backbone (~13% of backbone is on the surface). o high [urea] favors more hydration sphere. Gu l behaves similarly, but also interacts with charged sidechains Glycine betaine and glycerol partion out of the hydration sphere The phenomenon of salting out has another component: the Activity of water as a solvent may be changed because water becomes occupied in solvation of other solutes. There is not enough free water to solvate proteins. (What would you use as a salting out agent, a chaotrope or a lyotrope?)
auses proteins to fold, hydrophobic inside, hydrophobic outside. trengthens alt Bridges (formation of an ionic bond between charged side-chain groups (e.g., Glu and Lys). an be disrupted by high ionic strength of the medium. May not be too important on the surface of a protein but can be very important if they occur inside the protein. -bonds contribute to the stability significantly, but after water is excluded from the core. ow, how do they fold? They fold into three-dimensional structures. To fold, the linear chain must bend. " ' Where and how can it bend? What are the constraints?.. exokinase onstraints: What conformations are possible? otations about the peptide bond Amide bond = ω Amide esonance ω is locked in at 0 or 180
ovalent bond lengths & energies - 1.54Å 83. kcal - 1.47 (1.32) 99-1.43 86 = 1.22 179-1.09 104-1.00 93 An Amide Bond -bond acceptor -bond donor onformation - minimalists view ingle bond rotations - dihedral angles α α φ α ψ φ α ψ ω
!! "! " # is Trans low cis- trans- isomerization! # a. b. c. More $ ovalent (single bond) van der Waals 0.77Å -3 2.0 Å 0.66 1.40 0.70 1.5 1.04 1.85 <.28 (2) 1.2 n-1 - β β - n+1
amachandran Diagram Experimental amachandran Diagram - Glycine 180 Allowed (degrees) 0 Partially Allowed Allowed -180-180 0 (degrees) 180 Experimental amachandran Diagram - o Gly or Pro Experimental amachandran Diagram - Pro α - elix a 3.613 helix Proline is a helix-breaker but participates in formation of other motifs 3.6 residues per turn 5.4 Å per turn ("pitch") 1.5Å per residue ("rise"), 18 residues = 27Å In helices In turns
Periodicity Along the elix A elical Wheel 360 per turn 3.6 residues/turn 100 /residue hiffer & Edmundson ydrophobic Moment µ = {[Σ n n sin(δn)] 2 + [Σ n n cos(δn)] 2 } 1/2 Eisenberg β - heet The β - heet also has Polarity ote The beta strand is an element of secondary structure. The beta sheet is actually tertiary structure, since it involves folding together of distant parts of the protein. heet onsecutive residues, alternate sides 0 1 elix 3.6 residues per turn, or 100 /residue o, every 3rd, 4th or 7th residue is on the same side GTEKMAELIAKGIIEG 0 1 2 3 4 5 6 7 8 11 14 15 2 3 4 7 11 15 14
ommon β-bulge Beta Turns Usually in Antiparallel sheet auses a bend Alters the polarity of the surface Features: 4-residue turns = i often -bonded to i+3 i+3 i i i+1 i+2 i+1 i+3 i+2 Features: 3-residue turns = i often -bonded to i+2 Gamma Turns i+2 i i+1 Dihedral Angles in Turns β φ i + 1 i + 2 ψ φ ψ I -60-30 -90 0 I 60 30 90 0 II -60 120 80 0 II 60-120 -80 0 III -60-30 -60-30 III 60 30 60 30 IVa(cis) -60 120-90 0 IVb(cis) -120 120-60 0 γ φ ψ turn 70 to 85-60 to -70 Inverse -70 to -85 60 to 70 econdary tructure and amachandran's Plot ψ (degrees) 180 0-180 -180 Extended hain β trand in parallel sheet Polyproline elix ollagen elix β trand in antiparallel sheet Type II turn 3 10 elix α elix 0 φ (degrees) α elix (left) Type II turn 180 Prediction of econdary tructure The hou-fasman ules - looked at 29 proteins. They were 38% α-helix, 20% β-sheet, 32% β-turn Determined frequency of occurrence; how often is amino acid X in a helix (fα), β-strand (fβ), a turn (f t )? Define Pα = fα/.38, Pβ = fβ/.2, Pt = f t /.32 The potential of each amino acid to from helix, etc. 4 helix-formers out of 6 => α-helix 3 β-formers out of 5 => β-strand ome of each? ompare aveg Pα to aveg Pβ?
Pα Glu 1.51 Met 1.45 Ala 1.42 Leu 1.21 Lys 1.16 Phe 1.13 Gln 1.11 Trp 1.08 Ile 1.08 Val 1.06 Asp 1.01 is 1.00 Arg 0.98 Thr 0.83 er 0.77 ys 0.70 Tyr 0.69 Asn 0.67 Pro 0.57 Gly 0.57 hou-fasman Parameters Pβ Val 1.70 Ile 1.60 Tyr 1.47 Phe 1.38 Trp 1.37 Leu 1.30 ys 1.19 Thr 1.19 Gln 1.10 Met 1.05 Arg 0.93 Asn 0.89 is 0.87 Ala 0.83 er 0.75 Gly 0.75 Lys 0.74 Pro 0.55 Asp 0.54 Glu 0.37 Pt Asn 1.56 Gly 1.56 Pro 1.52 Asp 1.46 er 1.43 ys 1.19 Tyr 1.14 Lys 1.01 Gln 0.98 Thr 0.96 Trp 0.96 Arg 0.95 is 0.95 Glu 0.74 Ala 0.66 Met 0.60 Phe 0.60 Leu 0.59 Val 0.50 Ile 0.47 hou and Fasman had the right idea, but... The energy of the fold may contain contributions from more global interactions Use the global fold, not local propensities as your guide Example: PED 1) Find proteins with substantial sequence homology to yours 2) Make allowances for insertions/deletions 3) heck for catastrophic substitutions 4) Give the protein the same secondary structure as the family 5) ope you re right depends on how well relatives of your protein are represented in the database Backbone never forms knots idechains pack together o spaces, no holes (helices aren t hollow) ometimes waters, ions Partial specific volumes similar (.720 v.760 cc/g) rosslinks () Posttranslational modifications Glycosylation Acylation Prosthetic Groups ther crosslinks