Amino Acids, Peptides, Protein Structure

Amino Acids, Peptides, Protein Structure
Andy Howard Biochemistry Lectures, Spring January 2019

Let’s study amino acids, peptides, & proteins!
We’ll talk a bit more about amino acids themselves, and oligomers and polymers of amino acids We’ll discuss structure Then we’ll talk about methods of dealing with proteins 01/24/2019 Amino acids, Peptides, Proteins

Topics for today Tertiary & Quaternary Structure The 20 amino acids
Main-chain chemistry Side-chain chemistry Peptide bonds Disulfides Primary & Secondary Structures Helices and Sheets Protein structure Structural proteins Globular proteins Tertiary & Quaternary Structure 01/24/2019 Amino acids, Peptides, Proteins

Properties of the amino acids
We presented the structures of the 20 ribosomally encoded amino acids on Tuesday. Today we’ll explore their properties, starting with their acid-base properties and moving on to other characteristics. 01/24/2019 Amino acids, Peptides, Proteins

Chirality Remember: any carbon with four non-identical substituents will be chiral Every amino acid except glycine is chiral at its alpha carbon Two amino acids (ile and thr) have a second chiral carbon: C 01/24/2019 Amino acids, Peptides, Proteins

Ribosomally encoded amino acids are L-amino acids
All have the same handedness at the alpha carbon The opposite handedness at the alpha carbon gives you a D-amino acid Inverting the handedness at the beta carbon in ile and thr without also inverting the handedness at the alpha carbon gives something non-biological 01/24/2019 Amino acids, Peptides, Proteins

We do care about D-amino acids
There are D-amino acids in many organisms Bacteria incorporate them into structures of their cell walls Makes those structures resistant to standard proteolytic enzymes, which only attack amino acids with L specificity 01/24/2019 Amino acids, Peptides, Proteins

The CORN mnemonic for L-amino acids
Imagine looking from the alpha hydrogen to the alpha carbon The substituents are, clockwise: C=O, R, N: 01/24/2019 Amino acids, Peptides, Proteins

Abbreviations for amino acids
3-letter and one-letter codes exist All the 3-letter codes are logical Most of the 1-letter codes are too 6 unused letters, obviously U used for selenocysteine O used for pyrrolysine B,J,Z are used for ambiguous cases: B is asp/asn, J is ile/leu, Z is glu/gln X for “totally unknown” 01/24/2019 Amino acids, Peptides, Proteins

Nomenclature http://www.chem.qmul.ac.uk/ iupac/AminoAcid/A2021.html
Wikipedia article is pretty complete too 01/24/2019 Amino acids, Peptides, Proteins

Acid-base properties -amino acids take part in a variety of chemical reactivities, but the one we’ll start with is acid-base reactivity The main-chain carboxylate and amine groups can undergo changes in protonation Some side chains can be protonated or deprotonated as well 01/24/2019 Amino acids, Peptides, Proteins

Letters A-F: acid-base properties
Amino Acid Side-chain 3-lett abbr. 1-let pKa, COO- pKa, NH3+ alanine CH3 ala A 2.4 9.9 * asx B cysteine CH2SH cys C 1.9 10.7 aspartate CH2COO- asp D 2.0 glutamate (CH2)2COO- glu E 2.1 9.5 phenylalanine CH2-phe phe F 2.2 9.3 01/24/2019 Amino acids, Peptides, Proteins

Letters G-L Amino Acid Side-chain 3-lett abbr. 1-let pKa, COO-
pKa, NH3+ glycine H gly G 2.4 9.8 histidine -CH2-imidazole his 1.8 9.3 isoleucine CH(Me)Et ile I 2.3 Ile/leu * lex? J lysine (CH2)4NH3+ lys K 2.2 9.1 leucine CH2CHMe2 leu L 9.7 01/24/2019 Amino acids, Peptides, Proteins

Letters M-S met M 2.1 9.3 asn N 8.7 pyl O 2.2 9.1 pro P 2.0 10.6 gln Q
methionine (CH2)2-S-Me met M 2.1 9.3 asparagine CH2-CONH2 asn N 8.7 pyrrolysine see above pyl O 2.2 9.1 proline (CH2)3CH (cyc) pro P 2.0 10.6 glutamine (CH2)2CONH2 gln Q arginine (CH2)3-guanidinium arg R 1.8 9.0 serine CH2OH ser S 9.2 01/24/2019 Amino acids, Peptides, Proteins

Letters T-Z threonine thr T 2.1 9.1 Sec U 1.9 10.7 val V 2.3 9.7 trp W
CH(Me)OH thr T 2.1 9.1 selenocysteine CH2SeH Sec U 1.9 10.7 valine CH(Me)2 val V 2.3 9.7 tryptophan CH2-indole trp W 2.5 9.4 unknown Xaa X tyrosine CH2-Phe-OH tyr Y 2.2 9.2 glu/gln (CH2)2-COX glx Z 01/24/2019 Amino acids, Peptides, Proteins

Remembering the abbreviations
A, C, G, H, I, L, M, P, S, T, V easy F: phenylalanine sounds like an F R: talk like a pirate D,E similar and they’re adjacent 01/24/2019 Amino acids, Peptides, Proteins

One-letter abbreviations, concluded
N: contains a nitrogen W: say tryptophan with a lisp Y: second letter is a Y Q: almost follows N, and gln is like asn You’re on your own for K,O,J,B,Z,U,X 01/24/2019 Amino acids, Peptides, Proteins

Do you need to memorize these structures?
Yes, for the 20 major ones (not B, J, O, U, X, Z) The only other complex structures I’ll ask you to memorize are: DNA, RNA bases Ribose, glucose, deoxyribose, glyceraldehyde Cholesterol, stearate, palmitate, oleate A few others I won’t enumerate right now. 01/24/2019 Amino acids, Peptides, Proteins

How hard is it to memorize the structures?
Very easy: G, A, S, C, V Relatively easy: F, Y, D, E, N, Q Harder: I, K, L, M, P, T Hardest: H, R, W Again, I’m not asking you to memorize the one-letter codes, but they do make life a lot easier. 01/24/2019 Amino acids, Peptides, Proteins

Main-chain acid-base chemistry
Deprotonating the amine group: H3N+-CHR-COO- + OH-  H2N-CHR-COO- + H2O Protonating the carboxylate: H3N+-CHR-COO- + H+  H3N+-CHR-COOH Equilibrium far to the left at neutral pH First equation has Ka=1 around pH 9 Second equation has Ka=1 around pH 2 01/24/2019 Amino acids, Peptides, Proteins

Why does a main-chain pKa depend on the side chain?
Opportunities for hydrogen bonding or other ionic interactions stabilize some charges more than others More variability in the amino terminus, i.e. the pKa of the carboxylate group doesn’t depend as much on R as the pKa of the amine group 01/24/2019 Amino acids, Peptides, Proteins

When do these pKa values apply?
The values given in the table are for the free amino acids The main-chain pKa values aren’t relevant for internal amino acids in proteins The side-chain pKa values vary a lot depending on molecular environment: a 9.4 here doesn’t mean a 9.4 in a protein! 01/24/2019 Amino acids, Peptides, Proteins

Relating pKa to percentage ionization
Derivable from Henderson-Hasselbalch equation If pH = pKa, half-ionized: [HA] = [A-] One unit below: 91% at more positive charge state, 9% at less + charge state One unit above: 9% / 91% 01/24/2019 Amino acids, Peptides, Proteins

Ionization of Leucine pH 1.3 2.3 3.3 8.7 9.7 10.7 %+ve 91 50 9
% zwitterion %-ve Main species NH3+–CHR–COOH NH3+–CHR–COO- NH3+– CHR– COO- NH2– CHR– COO- 01/24/2019 Amino acids, Peptides, Proteins

Side-chain reactivity
Not all the chemical reactivity of amino acids involves the main-chain amino and carboxyl groups Side chains can participate in reactions: Acid-base reactions Other reactions In proteins and peptides, side-chain reactivity is more important because main chain is locked up! 01/24/2019 Amino acids, Peptides, Proteins

Acid-base reactivity on side chains
Asp, glu: side-chain COO-: Asp sidechain pKa = 3.9 Glu sidechain pKa = 4.1 That means that at pH = 5.1, glutamate will be ~90.9% charged Lys, arg: side-chain nitrogen: Lys sidechain –NH3+ pKa = 10.5 Arg sidechain =NH2+ pKa = 12.5 01/24/2019 Amino acids, Peptides, Proteins

Acid-base reactivity in histidine
It’s easy to protonate and deprotonate the imidazole group Remember: Since pKa ~ 6, it’s mostly in the neutral form at pH=7 01/24/2019 Amino acids, Peptides, Proteins

Cysteine: a special case
The sulfur is surprisingly ionizable Within proteins it often remains unionized even at higher pH Side-chain pKa ~ 8.3 01/24/2019 Amino acids, Peptides, Proteins

Why isn’t that a big deal?
It turns out many cysteines are involved in disulfide bonds (-S—S-), so the sulfhydryls aren’t available for ionization This is particularly true under oxidizing conditions 01/24/2019 Amino acids, Peptides, Proteins

Ionizing hydroxyls X–O–H  X–O- + H+
Tyrosine is easy, ser and thr hard: Tyr pKa = 10.5 Ser, Thr pKa = ~13 Difference due to resonance stabilization of phenolate ion: 01/24/2019 Amino acids, Peptides, Proteins

Resonance-stabilized ion derived from tyrosine
01/24/2019 Amino acids, Peptides, Proteins

Other side-chain reactions
Little activity in hydrophobic amino acids other than van der Waals Sulfurs (especially in cysteines) can be oxidized to sulfates, sulfites, … Nitrogens in his can covalently bond to various ligands Hydroxyls can form ethers, esters Salt bridges (e.g. lys - asp) 01/24/2019 Amino acids, Peptides, Proteins

Phosphorylation ATP donates terminal phosphate to side-chain hydroxyl of ser, thr, tyr Some phosphorylation of H, D, E too ATP + Ser-OH  ADP + Ser-O-(P) Often involved in activating or inactivating enzymes Under careful control of enzymes called kinases (add (P)) and phosphatases (remove (P)) 01/24/2019 Amino acids, Peptides, Proteins

Angles, angles, angles… By now you’re accustomed to thinking about bond angles, which involve three atoms that are covalently attached We also need to deal with torsion angles, which involve 4 covalently-attached atoms 01/24/2019 Amino acids, Peptides, Proteins

Torsion angles Involve four consecutive points
1 1 Torsion angles 3 2→3 2 4 4 Involve four consecutive points Imagine standing such that you’re looking down the line segment connecting the second and third of those four points Then the torsion angle is the angle between the (1→2) segment and the (3 → 4) segment 01/24/2019 Amino acids, Peptides, Proteins

Peptides & proteins Peptides are oligomers of amino acids
Proteins are polymers Dividing line is a little vague: ~ aa. All are created, both formally and in practice, by stepwise polymerization Water eliminated at each step 01/24/2019 Amino acids, Peptides, Proteins

Growth of oligo- or polypeptide

The peptide bond The amide bond between two successive amino acids is known as a peptide bond The C-N bond between the first amino acid’s carbonyl carbon and the second amino acid’s amine nitrogen has some double bond character 01/24/2019 Amino acids, Peptides, Proteins

Double-bond character of peptide

The result: planarity! This partial double bond character means the nitrogen is sp2 hybridized Six atoms must lie in a single plane. 01/24/2019 Amino acids, Peptides, Proteins

Which six atoms are constrained?
First amino acid’s alpha carbon Carbonyl carbon Carbonyl oxygen Second amino acid’s amide nitrogen Amide hydrogen Second amino acid’s alpha carbon 01/24/2019 Amino acids, Peptides, Proteins

Rotations and flexibility
Planarity implies  = 180º, where  is the torsion angle through the N-C bond Free rotations are possible about N-C and C-C bonds Define  = torsion angle through N-C Define  = torsion angle through C-C We can characterize main-chain conformations according to ,  01/24/2019 Amino acids, Peptides, Proteins

Cis- vs. trans- peptides
Determined by positions of successive alpha carbons If 2 successive alpha carbons are on opposite sides of the peptide bond, it’s trans; if they’re on the same side, it’s cis Fig. courtesy of Voet & Voet, Biochemistry 01/24/2019 Amino acids, Peptides, Proteins

Why trans peptides are more common
Trans is much more common because the side chains are less likely to overlap Exception: amide nitrogen of proline—cis is only a little more interfering than trans Figure courtesy of Wikimedia 01/24/2019 Amino acids, Peptides, Proteins

Ramachandran angles G.N. Ramachandran 01/24/2019
Amino acids, Peptides, Proteins

Preferred Values of  and 
Steric hindrance makes some values unlikely Specific values are characteristic of particular types of secondary structure Most structures with forbidden values of  and  turn out to be errors 01/24/2019 Amino acids, Peptides, Proteins

How far from 180º can w vary? Remember partial double bond character of the C-N main-chain bond That imposes planarity In practice it rarely varies by more than a few degrees from 180º. Aromatic amino acids are the most likely to have non-planar peptides 01/24/2019 Amino acids, Peptides, Proteins

Ramachandran plot Cf. figures in text
If you submit a structure to the PDB with Ramachandran angles far from the yellow regions, be prepared to justify them! 01/24/2019 Amino acids, Peptides, Proteins

How are oligo- and polypeptides synthesized?
Formation of the peptide linkages occurs in ribosome under careful enzymatic control Polymerization is endergonic and requires energy in the form of GTP (like ATP, only with guanosine): GTP + n-length-peptide + amino acid  GDP + Pi + (n+1)-length peptide 01/24/2019 Amino acids, Peptides, Proteins

What happens at the ends?
Usually there’s a free amino end and a free carboxyl end: H3N+–CHR–CO–(peptide)n–NH–CHR–COO- Cyclic peptides do occur Cyclization doesn’t happen at the ribosome: it involves a separate, enzymatic step. 01/24/2019 Amino acids, Peptides, Proteins

Reactivity in peptides & proteins
Main-chain acid-base reactivity unavailable except on the ends Side-chain reactivity available but with slightly modified pKas. Terminal main-chain pKavalues modified too Environment of protein side chain is often hydrophobic, unlike free amino acid side chain 01/24/2019 Amino acids, Peptides, Proteins

iClicker question 1 1. What’s the net charge on ELVIS at pH 7? (a) 0
(b) +1 (c) -1 (d) +2 (e) -2 01/24/2019 Amino acids, Peptides, Proteins

iClicker question 2 2. Leucine is one of the more common amino acids in proteins. In a typical protein, I would expect the leucine content to be about: (a) 53%; (b) 7%; (c) 5%; (d) 3% (e) We do not have enough information to know. 01/24/2019 Amino acids, Peptides, Proteins

Disulfides In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains 01/24/2019 Amino acids, Peptides, Proteins

What could this do? Can bring portions of a protein that are distant in amino acid sequence into close proximity with one another This can influence protein stability 01/24/2019 Amino acids, Peptides, Proteins

Proteins have definable structures!
This isn’t intuitively obvious Many biomolecules are much more flexible in terms of the number of conformations they actually take on in the real world Why are protein structures definable? They’re big enough to have an interior Hydrophobic in, hydrophilic out imposes order 01/24/2019 Amino acids, Peptides, Proteins

Levels of Protein Structure (CF&M §4.1-4.5)
We conventionally describe proteins at four levels of structure, from most local to most global— primary, secondary, tertiary, and quaternary 01/24/2019 Amino acids, Peptides, Proteins

The four levels of structure
Primary: linear sequence of peptide units Secondary: main-chain H-bonds that define short-range order in structure Tertiary: three-dimensional fold of a single polypeptide Quaternary: Folds of multiple polypeptide chains to form a complete oligomeric unit 01/24/2019 Amino acids, Peptides, Proteins

Not all proteins have all four levels of structure
Monomeric proteins don’t have quaternary structure Tertiary structure: subsumed into 2ndry structure for many structural proteins (keratin, silk fibroin, …) Some proteins (usually small ones) have no definite secondary or tertiary structure; they flop around! 01/24/2019 Amino acids, Peptides, Proteins

What does the primary structure look like?
-ala-glu-val-thr-asp-pro-gly- … Can be determined by amino acid sequencing of the protein Can also be determined by sequencing the gene and then using the codon information to define the protein sequence Amino acid analysis means percentages; that’s less informative than the sequence 01/24/2019 Amino acids, Peptides, Proteins

Components of secondary structure
, 310,  helices pleated sheets & strands that comprise them Beta turns More specialized structures like collagen helices 01/24/2019 Amino acids, Peptides, Proteins

An accounting for secondary structure: phospholipase A2

What does this PLA2 look like?
Predominantly helical, with one pair of beta strands, some ”coil” Active site near pair of helices in middle Agkistrodon PLA2 14kDa monomer, EC PDB 1MC2, 0.85Å 01/24/2019 Amino acids, Peptides, Proteins

Alpha helix (CF&M §4.3) 01/24/2019 Amino acids, Peptides, Proteins

Characteristics of  helices
Hydrogen bonding from amino nitrogen to carbonyl oxygen in the residue 4 earlier in the chain 3.6 residues per turn Amino acid side chains face outward ~ 10 residues long in globular proteins 01/24/2019 Amino acids, Peptides, Proteins

What would disrupt this?
Not much: the side chains don’t bump into one another Proline residue will disrupt it: Main-chain N can’t H-bond The ring forces a kink Glycines sometimes disrupt because they tend to be flexible 01/24/2019 Amino acids, Peptides, Proteins

Other helices NH to C=O four residues earlier is not the only pattern found in proteins Other helical structures differ in that they have connections between amine N and C=O that is 3 or 5 residues earlier 01/24/2019 Amino acids, Peptides, Proteins

Amino Acids, Peptides, Protein Structure

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