(2) protein structure, stability, & folding

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describe peptide group and their bonds

peptide group is planar and rigid

• C=O db prevents rotation

• peptide bond is stronger than esters

what kind of conformation do most peptides assume and why

trans; it minimizes steric bulk

how are the side chains aligned in a polypeptide

they are opposite to each other

what is primary structure

linear sequence of aa in a polypeptide chain

what is secondary structure

local spatial arrangement of polypeptide’s backbone atoms without regard to conformation of its side chains (alpha helix, beta sheet, turns)

what is tertiary structure

3D structure of an entire polypeptide, including its side chains

what is quaternary structure

spatial arrangement of 2+ polypeptides/subunits

when you unfold a tertiary structure and quaternary structure, how many polypeptide chains do you get

• tertiary: 1 chain

• quaternary: 2 or more chains

what are three things secondary structures are known for

alpha-helix, beta-sheet, reverse turns (beta-bends)

what is an α-helix? describe its residues per turn and distance it rises per turn

• right handed spiral with side chains extended outward

• helix has 3.6 residues (aa) per turn

• distance helix rises per turn: 5.4 A

what is the average length of alpha helices

12 residues (3 helical turns, and a length of 18 A)

what stabilizes the helix?

• H-bonds within the backbone

describe H-bonds and what the optimum distance is for them

• peptide C=O bond of the nth residue points along the helix axis twd peptide N-H grp of the (n+4)th residue (so 1st and 5th aa)

• optimum distance for H-bonds: 2.8 A

what kinds of amino acids would destabilize the helix

• proline; missing H-bond donor

• steric repulsion (Ile, Tyr)

• two amino acids with the same charge next to each other (electrostatic repulsion)

• positive charge aa at the positive N terminus

• negative charge aa at negative C terminus

what is a β sheet

• contains 2-22 polypeptide strands (avg 6)

• H-bonds between strands

• each strand could have up to 15 residues (avg 6)

in what direction does β sheet go? describe how you would differentiate antiparallel/parallel

• N terminus to C terminus

• antiparallel strands run in opposite directions; read one strand from N to C, then check the other strand!

what kind of β sheet strands have stronger interactions, why?

antiparallel because their hydrogen bonds are straight and shorter

what is the minimum strands needed for a stable antiparallel vs parallel β sheet

antiparallel = 2 strands

parallel = at least 5 strands

what is the function of a reverse turn (β bend)

to connect adjacent strands/secondary structures in a protein by making a sharp turn (connecting C-terminus to N-terminus)

which two amino acids are most common in reverse turns and why

• glycine: small no side chain

• proline: creates a natural kink in chain

which types of β sheet strands require longer loops to connect adjacent strands and why

parallel sheets require longer crossover loops because the C-terminus and N-terminus are farther apart (end of one strand is far from the start of the next strand)

what are motifs? name these/is C parallel or antiparallel

• combinations of secondary structures (supersecondary structure) present in tertiary structures

• (left to right), βαβ, β hairpin, αα, greek key

• antiparallel because the loop is shorter

what is tertiary structure

association of secondary structure units within a single polypeptide chain to give a 3D structure

what are 4 ways protein structures determined by? describe each

1. X-ray crystallography: needs crystals, highest resolution

2. Cryo-EM: no crystals; best for large complexes & multiple conformations

3. NMR spec: solution state, small proteins, shows dynamics & flexibility

4. AlphaFold: prediction, not experiment, good starting model, weak on ligand-bound pockets

what can most proteins be classified as?

α, β, α/β

what 2 factors binds retinol to the retinol-binding proteins

1. hydrophobic effect (entropic drive)

   1. retinol is fatty and lipophilic, it sits in protein’s hydrophobic pocket, displacing ordered water molecule; releasing trapped water increases entropy, driving strong binding

2. H-bonding

   1. polar OH grp at end of retinol STICKS out of pocket, forming H bonds with water and polar residue on protein exterior

3. These drive the shape of protein

why can’t retinol (vitamin A) leave the liver by itself

• it is vv lipophilic (fatty) and insoluble in water, os it can’t move freely through blood or leave liver without being packaged inside a transport protein

what structural feature of the β barrel allows it to transport hydrophobic mc

it has hydrophobic interior pocket that’s shielded from surrounding water, housing nonpolar molecules while its outer surface interacts favorably with aq envir

what are domains? how many resides or average diameter?

• domains: (only in tertiary) a globular cluster of secondary/supersecondary structures usually found in polypeptides with over 200 residues

  • consist of 40-200 residues with avg diameter of 25A

when you unfold a two domain protein (domain), how many strands do you get

1 strand

what are quaternary structure

non covalent association of 2 or more polypeptide chains/subunits to form a native protein structure

when you unfold quaternary structure of hemoglobin, how many chains and how manyy different chains do you get? what is this called

• 4 different chains

  • hetero-tetramer

what is the only covalent bond that can occur in quaternary structure

disulfide bond (2 Cys)

Transthyretin (TTR) is a homo-tetramer. when you unfold it, how many different chains do you get

4 of the SAME polypeptide chain

how many total polypeptide chains make up the fully assembled TTR-RBP(retinol binding protein) complex

6 total chains (4 TTR chains, 2 RBP chains)

what happens if a mutation prevents TTR from binding to RBP

RBP can’t leave the liver, causing excess vitamin A (retinol) to build up in liver and resylt in night blindness

what are the 2 advantages of quaternary structure

1. defects can be repaired only by replacing the flawed subunit

2. provides structural basis for regulation of their activities (allosteric/cooperative binding)

what do fibrous proteins have and give 2 ex

• have repeating secondary structures

• eg: keratin, collagen

what is α keratin, what is it rich in and where is it found

• coiled coil secondary structure

• rich in Cys residues

• found in: outer epidermis, hair, horn, nails, feather

describe the science of a hair perm (3)

1. ammonium thioglycolate is a reducing agent that adds H to break the covalent disulfide bonds

2. hair is styled, free -SH groups shift and align in new position

3. hydrogen peroxide is an oxidizing agent that reforms new covalent disulfide bonds in new positions, locking new style in place

describe collagen, where is it found

• most abundant vertebrate protein, with strong insoluble fibers (triple helix structure)

• component of connective tissues eg bone, teeth, tendon, cartilage, fibrous matrices of skin and blood vessels

how are the side chains in collagen linked? what aa is it mostly composed of ?what is it devoid of

• covalently cross-linked by reaction of histidine and activated lysine

• 30% gly, 15-20% Pro and Hyp (4-hyroxyprolyl)

• collagen is almost devoid of Cys residues

What reaction converts Proline (Pro) to Hydroxyproline (Hyp), and what enzyme/coenzyme performs it

adding OH grp via Prolyl hydroxylase, which requires Ascorbic acid (Vitamin C) as a coenzyme.

why is Hydroxyproline (Hyp) important for collagen structural integrity?

it makes H-bonds with other collagen fibers to hold structure together

how does vitamin c deficiency cause scurvy

low vitamin C → low prolyl hydroxylase activity → low Hyp → less H-bonds → weak collagen fibers

how are enzymes specific on substrates (5)

• geometric and electronic complementarity bt enzyme & substrate

• noncovalent forces (reversible binding)

• hydrophobic grps

• H-bonds

• lock/key & induced fit

what is the average MW of an aa

110 daltons

what is average MW of a protein

(# of amino acids) * 110

where are the nonpolar residues mostly located

mostly in the interior of a protein to avoid the aqueous solvent

where are the charged polar residues usually located

on the surface of a protein in contact with the aqueous solvent

where are the uncharged polar residues usually located

usually on the protein surface but also occur in the interior of the mc (when buried in protein, these aa’s are H-bonded to other grps)

what has the greatest influence on protein stability and structure, why?

the hydrophobic effect, dueto increase in entropy of water mc

what makes only minor contributions to protein stability

H bonds and ionic interactions
- the change in energy between the bonds made in unfolded vs native state is so little
- forming ion pairs reduces entropy (less movement)

what proteins have disulfide bonds

• extracellular proteins

• rare in intracellular bonds because the cytoplasm is a reducing environment

how do metal ions contribute to protein stability

they form coordinate covalent bonds with certain residues, stabilizing motifs

what is a zinc finger and what is their function

• 25-60 residues arranged around one or two Zn2+ ions that are tetrahedrally coordinated by the side chains of Cys and His

• finger-like shape that fits into binding pockets of DNA, RNA & proteins

what are five ways proteins can be denatured by

• heat (past 100C)

• acid/base pH changes that affect protein charge & H-bonds

• detergents like sodium dodecyl sulfate that affect hydrophobic interactions

• reducing agents that break disulfide bonds

  • eg dithiothreitol, beta-mercaptoethanol

• chaotropic agents that increase solubility of NP substances in water & disrupt H-bonds & hydrophobic intrxn

  • eg guanidinium ion, urea

what does polyphenol oxidase do in fruits

oxidates phenolics in fruits into quinones when exposed to oxygen when its cut

what are three ways to reduce the browning of fruit through inactivation of polyphenol oxidase

• cooking to denature enz

• reduce pH on fruit surface - add lemon juice

• putting fruit in water or vacuum pack to reduce available oxygen

how does urea & beta-mercaptoethanol denature proteins

• urea is denaturant that disrupt H-bonding & hydrophobic interaction

• beta-mercap is reductant that cleaves disulfide bond

why can many denatured proteins be renatured

as longas the primary structure is intact, the 3d structure will be the same

what is hydrophobic collapse

rapid shrinking and folding of protein into native conformation, driven by hydrophobic effect

what are molecular chaperones

proteins that bind to partially folded polypeptides to help it fold to final native conformation

• prevents improper hydrophobic associations

what are ATPases

enzymes that catalyze hydrolysis of ATP to ADP = P_i

• eg heat shock protein 90 Hsp90

why are molecular chaperones also ATPases

energy released from ATP is used to bind, manipulate, and release client proteins

what are heat shock proteins (Hsp)

important chaperones that recover heat-dentaured proteins and prevent misfolding under stressful conditions

• increased temp = inc hsp synthesis

why is Hsp90 a target for cancer therapy

hsp90 stabilizes proteins required for tumor growth