Secondary Structure of Proteins

4th lecture

Cheese crystals

in parmesan; tyrosine

Monosodium glutamate (MSG)

umami taste from specific amino acid receptors; originally isolated from dashi; literally no medical risk

N-terminus to C-terminus

standard for writing primary sequences (don’t be a Knarf)

Levels of protein structure (4)

primary, secondary, tertiary, quaternary

Levinthal's paradox: Do proteins fold by sampling all possible conformations?

protein folding landscape has stable conformations that aren’t its native; lots and lots of testing has to be done then, but proteins fold spontaneously and fast!

Anfinsen’s Experiment: RNase A Folding

1972 Nobel Prize in Chemistry

postulated that the native structure of a protein is the thermodynamically stable structure; it depends only on the amino acid sequence and on the conditions of solution, and not on the kinetic folding route

hydrogen bonding disrupted with urea and disulfide bridges disrupted by beta mercaptoethanol → refolds with urea removed (and trace mercaptoethanol added - undos wrong disulfide bonds)

disulfide exchange

exchange salt-bridge for two thiols

Urea hydrogen bond disruption

disrupts the hydrophobic effect, preventing water from properly interacting with the protein during folding

protein folding

1. alpha helices form

2. hydrophobic collapse

3. beta sheets

4. plus or minus a few kinetic traps

   1. boom native state

Bond rotations in peptide backbone

specify protein secondary structures

amide bond

always draw trans (more stable by ~ 8 kJ/mol) UNLESS proven to be cis (Proline residues have ~10% cis conformation)

Φ bond rotations

n to alpha carbon; four atoms to track

ψ bond rotation

alpha carbon to carbonyl carbon; four atoms to track

Ramachandaran Plot

shows accessible (low energy) ψ and Φ conformations; Dark areas are “allowed”, white areas denote high energy conformations; Calculate using PDB structures and a calculator (code) or from one of several programs (e.g., pymol, VMD)

Ramachandran plot and secondary structure

match with the dihedrals of specific secondary structures

Average dihedral angles for secondary structures

>30% residues have alpha helical values of Φ & ψ; then beta strands; unfolded proteins have lots of PII conformation (collagen)

Types of secondary structure

1. helix

2. sheets

3. loops/random coils

right-handed (alpha) helix

All N-H and C=O pairs make H-bonds except the first 3 N-H (at N-terminus) and last three C=O (at C-terminus); Residue "i" C=O makes H-bond with residue "i+4" N-H; common; i, i+3 and i+4 residues reside on the same face of the alpha-helix so they can interact with side chains at i+3/i+4 positions using salt-bridges, hydrogen bonding, pi-stacking or van der Waals interactions

Secondary structure

regular local folds of the backbone; motifs

guidelines for generating secondary structures in proteins

1. Avoid steric clashes

2. Maximise the number of hydrogen bonds

3₁₀ helix

10 atom H-bonds, from i to i+3

α helix

13 atoms H-bonds; i to i+4; most stable and abundant helix in proteins

π helix

16 atoms H-bonds; i to i+5

helical wheel

represents the alpha helix; Heptad Repeat: left-handed twist makes 3.5 residues/turn

helix preferences

1. Helix propensity is favored by Ala, destabilized by Gly or Pro.

2. Leu stabilizes helix more than I or V. Crowding Cβ is less favorable.

3. Side chains interact at spacing of i,i+3 and i,i+4: acid base bridges (E,D to K,R) stabilize helix, hydrophobic groups too.

4. P is a tertiary amine - cannot do hydrogen bonding - breaks helices

Ozempic (semaglutide)

Modified peptide analog of Glucagon-like peptide-1 (GLP-1) that has stabilised helices (basically is!); Lowers blood glucose; Significant weight loss; development inspired by Gila monsters

β-Strand

often twisted; Distance between adjacent amino acids ~ 3.4 Å

strand/sheet preferences

Enriched in Val, Ile, Thr (branched amino acids); Tyr, Trp, Phe (aromatic); Cys - provides space

Ramachandran plot

Consecutive dihedral angles of (Φ, ψ) of -120° and 120°

parallel β-Sheet

hydrogen bonds; pleated so atoms can connect

anti-parallel β-Sheet

hydrogen bonds; pleated so atoms can connect; connected by short turns or loops

Silk

fibrous protein consisting of three subunits, one of which consists almost entirely of β-sheets, which collectively give silk its strength; cocoon of the Japanese silkworm

β-Sheet

Connecting β−strands through loops and turns into a channel

Gly and Pro are common in turns. Why?

totally change connectivity and stereochemistry

Random loops/coils Ramachandran plot

various phi/psi angles

Polyproline Helices

PPII from repeating proline trans amide bonds - 3 residues per turn

PPI from repeating proline cis amide bonds - rare, generally observed only in organic solvents.

ω deihedral angle

amide bond; can be 0 or 180

“Random” Coil

No well-defined secondary or tertiary structure; Conformations of polypeptide rapidly interconvert between multiple different states; small energy differences among backbone conformations compared with kT (thermal energy)

Coiled-Coils

Helix-helix contacts that wrap around each other in a left-handed spiral

Parallel Coiled-Coil

Heptad Repeat: left-handed twist makes 3.5 residues/turn; a/d always in contact, likely hydrophobic; g/e additional interactions, likely salt-bridge

Anti-Parallel Coiled-Coil

a/d’ and d’/a; g/g’, e/e’

Helix Bundle

hydrophobic core

Collagen

most abundant protein in mammals (25 to 35%); main component of connective tissue; triple helix; repeat of Pro-Hyp-Gly

Super-secondary Structures

2+ secondary structures combined

• Helix-turn-helix

• Helix-loop-helix

• β-α-β

• Greek key

  • Beta Barrel