3D Structures of Proteins

Structure Hierarchy

primary: chemical structure

secondary: local structural elements

tertiary: long-range interactions overall fold single polypeptide chain

quaternary: interactions between polypeptide chains

Backbone Configuration

peptide bond can be cis or trans with cis being the most common (xxx-Pro)

the plane of each peptide bond shares one alpha carbon with the next peptide bond plane

rotation of the psi and phi angles changes the relative position of the amide planes → some configurations are NOT possible

• this causes steric hindrance (BAD)

• peptide bonds to gly or pro residues are sometimes found in unallowed regions due to the small size of gly side chain and ring structure of pro

Structure Hierarchy

primary: chemical structure

secondary: local structural elements

tertiary: long-range interactions overall fold single polypeptide chain

quaternary: interactions between polypeptide chains

Backbone Configuration

peptide bond can be cis or trans with cis being the most common (xxx-Pro)

the plane of each peptide bond shares one alpha carbon with the next peptide bond plane

rotation of the psi and phi angles changes the relative position of the amide planes → some configurations are NOT possible

• this causes steric hindrance (BAD)

• peptide bonds to gly or pro residues are sometimes found in unallowed regions due to the small size of gly side chain and ring structure of pro

Secondary Structure Elements

alpha helis

beta sheet

beta turn

loops

alpha-Helix Structure by Linus Pauling

right handed helix with 3.6 residues per turn

pitch of 5.4 angstroms per turn

held together by H bonds between C=0 residue n to NH of residue n+4

helix propensity is the tendency of certain amino acids to occur more frequently in alpha-helices

amino acid side chains point outward from the axis

proline, lots of charged residues, and lots of bulky residues are bad

Beta Sheets

two or more separate strands of polypeptide H bonded together through peptide bond groups

could be parallel (not straight H bonds) or antiparallel (straight H bonds) orientations, could be mixed

strands are not flat but puckered and slightly twisted to the right as a beta pleated sheet

beta bulges happen when residues don’t line up

turns can occur between strands

strands in a beta sheet are connected by tight turns (beta-turns) or large loops

• beta turns always have pro at position 2 and connect anti-parallel beta strands

• type 2 beta turns always have gly at position 3

Sequence and Secondary Structure

pro rarely occurs in secondary structure

different residues have different helical propensities

side chain interactions can disrupt or distort secondary structure

Sequence Affects Helix Stability

not all polypeptide sequences adopt alpha-helical structures

small hydrophobic residues like Ala and Leu are strong helix formers

pro is a helix breaker because can’t rotate around N-Ca bond

gly is a helix breaker because the small R-group supports other conformations (too much free rotation)

Circular Dichroism Analysis

measure molar absorption difference of left and right circularly polarized light

peptide bond in each secondary structure has characteristic CD spectrum

chromophore in chiral environment make characteristic signals

spectrum can be deconvoluted to give a measure of %helix, % sheet, and % coil

Protein Tertiary Structure

overall spatial arrangement of atoms in a polypeptide chain or protein

two major classes:

• fibrous proteins: typically insoluble, made from a single secondary structure long extended structures, many polypeptides, often structural

• globular proteins: water-soluble, lipid-soluble membrane proteins, roughly spherical in shape, polypeptide folds back on itself, enzymes

Fibrous Proteins

much or most of polypeptide chain is approximately parallel to a single axis

often mechanically strong

usually insoluble in water

structural role

Alpha Keratin

helical rod segments capped with non-helical N and C termini made of coiled coil of 2 alpha helices, left handed twist, 3.5 residues per turn, primary structure of rods has 7-residue repeats

• intra and inter-strand ionic interactions stabilize coiled coil

• coiled coils are frequently seen in the dimerization domains of transcription factors

Silk Fibroin

main protein in silk, antiparallel beta sheet structure with small side chains of ala and gly allowing close packing

• stabilized by H bonding within sheets, LDFs between sheets, hydrophobic interactions between ala sidechains

Collagen

made of 15-30% gly, pro, hydroxypro, and hydroxylys

strands crosslinked at N and C termini

many repeats of Gly - X - Y where X is pro and Y is 4hydroxypro - increases stability of collagen by increasing electrostatic interactions

gly occurs where chains cross by its NH H bonding to X carbonyl in adjacent strand

L-handed helix with 3 residues per turn

no intra-chain H bonds

R-handed superhelix

Globular Tertiary Structure

form wherever possible since big numbers of H bonds

helices and sheets often pack close together

backbone links between elements of secondary structure are usually short and direct

proteins fold to make the most stable structures, make H bonds, minimize solvent contact of hydrophobic residues

secondary elements themselves not usually active → must fold to become active

Globular Proteins

design principles:

• most polar residues face outside of protein + interact with solve

  • exceptions: membrane-spanning portion of membrane proteins, subunit interfaces (hydrophobic residues face interior and interact with each other)

• random coil is not random

• structures are not static (various elements and domains of protein move to different degrees)

• some parts are flexible and disordered

• empty space exists in form of small cavities

Structural Families

all alpha

all beta

a + b secondary structure elements separate in sequence, antiparallel beta strands

a/b mixed in the sequences, parallel strands in beta sheet

Motion in Globular Proteins

Repeated Structural Units in Proteins

motifs, small parts of proteins

• Rossman fold - nucleotide binding domain

domains, independently folded regions of structure that are functional when removed from larger protein

Rossman Fold

motif found in nucleotide binding domains

gly-x-gly-x-x-gly in ADP, NAD

gly-x-gly-x-x-ala in NADP

Other Structural Motifs

greek key

helix turn helix

beta turns

beta bulges

Domains

SH2 domains are found in proteins of the tyr kinase signaling cascade - binds phosphorylated tyr residues

Kringle Domains

around 80 amino acid looped domains with 3 disulfide bonds per domain

found in proteases of the blood clotting cascade and growth factors

2 families, one without 3 disulfides in ECM

Proteins with similar function…

may not have similar sequences but they will have similar structures

Quaternary Structure

interactions are specific and reproducible and increase stability of subunit

active site is at subunit interface

cooperativity of substrate improves binding efficiency and regulates activity

distal binding site for activators and enhancers increase sensitivity to such molecules

each chain in a homo oligomer is coded for by a different gene, if one is wrong other compensates

one gene has many subunits (genetic efficiency)

Protein Stability tertiary and quaternary Structure

electrostatic forces (ion-ion, close range forces, salt bridges)

van der waals (between permanent and induced dipoles, LDFs)

H bonds

hydrophobic interactions

What holds proteins together?

mostly the hydrophobic effect - the greasy stuff stays on the inside

• hydrophobic side chains get buried in layers like a cake

H bonds that are buried in the protein (30-40%)

buried salt bridges (smaller contribution)

Interior of the Protein

microenvironments affect properties of amino acids

charged side chains buried in hydrophobic pockets stay uncharged - increase pKa for (-) and decrease for (+)

buried salt bridge residues remain charged - decrease pKa for (-) and increase for (+)

Mutations

conservative mutations cause smaller changes except when at a critical location

mutations in surface residues cause smaller changes except if amino acid is part of interactions

nonconservative mutations cause more drastic changes except hydrophilic on outside

Methods for Determining Protein Structure

X-ray crystallography

NMR spectroscopy

CD

cryo electron microscopy

modelling

Protein Denaturation

native structure = biologically relevant 3D structure

denaturation = disrupting forces that hold native structure

• CD spec

• activity assays

• NMR spec

can be denatured by:

• heat or cold

• pH extremes

• organic solvents

• chaotropic agents: urea and guanidinium hydrochloride

Ribonuclease Refolding Experiment

ribonuclease has 8 cysteines linked via disulfide bonds

sequence alone determines native conformation when urea and 2-mercaptoethanol are removed slowly

Protein Folding

levinthal’s paradox: if a polypeptide searches all possible conformational space to find the lowest E state it will take a very long time

protein retains partially folded intermediates called nucleation condensation model

folding is hierarchical

hydrophobic collapse → secondary struc → tertiary struc → final conformation

protein disulfide isomerase: enzye reduces disulfides then helps reform new disulfide bonds

molecular chaperones: heat shock proteins, groEL, groES

GroEL and GroES - protein folding cages

each chamber has 7 subunits of groEL protein

cap is made of 7 subunits of groES protein

ATP binds groEL → misfolded portein binds to opening of chamber → chamber is capped up, ATP is hydrolyzed → ATP and a new substrate protein bind to other side → first protein and ADP are released

Alzheimer’s

normal folded peptide is in brain as helical bundle

one unfolding → others get beta sheet conformation → plaques in the brain

p53 Tumour Supressor

mutations in core of protein destabilize structure and make it unfold

mutants may be unable to bind to DNA/get fully unfolded

new target is small molecules that will stabilize the structure of the protein

Cystic Fibrosis

deletion of phe 508

destabilizes overall protein structure → misfolded protein degrades on ER

phe residues is important in stabilizing interdomain interactions via hydrophobic interactions