BIOC503 - Protein Strcuture

native fold

specific 3D conformation of a protein, making it able to perform a specific biological function

• has a large number of favorable interactions w/in the protein to stabilize it

hydrophobic effect

release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy

hydrogen bonds

interaction of N-H and C=O of the peptide bond leads to local regular structures such as alpha helices and beta sheets

London dispersion forces

medium-range weak attraction between all atoms contributes significantly to the stability in the interior of the protein

Electrostatic interactions

• long range strong interactions between permanently charged groups

• salt-bridges, especially those buried in the hydrophobic environment, strongly stabilize the protein

primary structure

the exact amino acid sequence of the protein and the peptide bonds

peptide bond

bond between two amino acids

• resonance hybrid

• less reactive due to resonance

• rigid

• nearly planar

• large dipole moment in trans configuration

dihedral angles

__ are made of 3 angles: phi, psi, and omega

• determine, with the identity of R groups, the secondary structures of the protein

phi

angle around the alpha carbon —amide nitrogen bond

psi

angle around the alpha carbon—carbonyl carbon bond

impossible

rotation around peptide bond is ___ due to resonance structure (double bond characteristic)

steric crowding

some dihedral angles combinations are unfavorable because of ___ of backbone atoms with other atoms in backbone or sidechains

favorable

some dihedral angles combinations are ___ because they increase the chance of H bonding along the backbone

Ramachandran plot

graph showing the distribution of dihedral angles that are found in a protein

• shows common secondary structure elements

• reveals regions with unusual backbone structure

right-handed alpha helix

found in the bottom left quadrant of Ramachandran plot

left-handed alpha helix

found in upper right quadrant of Ramachandran plot

beta sheets, collagen triple helix

found in upper left quadrant of Ramachandran plot

secondary structure

local spatial arrangement of the polypeptide backbone

• determined by H bonding

• alpha helix and beta sheet are common arrangements

• random coil found when irregularly arranged

random coil

irregular arrangement of the polypeptide chain on the secondary level

alpha helix

• stabilized by H bonds between backbone amides of n and n+4 residues

• right-handed or left-handed

• 3.6 residues per turn

• peptide bond roughly parallel with helical axis

• Side chains point out and are roughly perpendicular with helical axis.

• outer diameter (with side chains) fits perfectly in grooves of dsDNA

• inner diameter too small for anything to fit inside

• Full of small hydrophobic residues like Alanine and leucine

• has a large macroscopic dipole moment

proline, glycine

___ & ___ are not found in alpha helix because they are blocking rotation and too flexible respectively

similar

all peptide bonds in alpha helix have a ___ orientation

positive, negative

__ residues often occur near the positive end/N-terminus of helix, while __ residues occur near the negative/C-terminus of helix

Beta sheets

• stabilized by H bonds between backbone amides in different stands aka adjacent segments that may not be nearby

• pleated-like structure due to planarity of peptide bond and tetrahedral structure of alpha carbon

• side chain alternate between up & down directions

• multi-Beta-stand interactions are called sheets

• can be parallel or antiparallel

parallel beta sheet

B-stands oriented in the same direction

• carbonyl oxygen does NOT line up with hydrogen of next strand

• H bonds are bent, so weaker

• 6.5 A when 2 amino acids are bound

antiparallel beta sheet

strands are in the opposite directions

• carbonyl carbon and hydrogen of next strand lineup

• H bonds are linear, thus stronger

• 7 A when 2 amino acids are bound

Beta turn

• occurs frequently whenever B-strands in B-sheets change direction

• 180 degrees turn

• 4 amino acids

• stabilized by H bonds between carbonyl oxygen to amide proton 3 residues down sequence

• proline in position 2 or glycine in position 3

Type I

___ beta turn has proline in position 2

Type II

__ B-turns have glycine in position 3

trans

most peptide bonds involving proline are in the __ configuration

cis

6% of peptide bonds involving proline are in __ configuration

proline isomerases

enzymes catalyzing isomerization of proline

Circular Dichroism analysis

aka CD analysis

• measures molar absorption difference of left- and right-circularly polarized light

• CD signals depend on chain conformation

• useful at monitoring conversion of protein folding

• pretty niche and not used as much anymore

280

the __ nm wavelength is where you would see absorbance from tyrosine and tryptophan

tertiary structure

overall spatial arrangement of atoms in a protein (3D conformation, native fold)

• stabilized by numerous weak interactions between amino acid side chains

• largely hydrophobic and polar interactions stabilize it (maybe disulfide bonds)

• mainly either fibrous or globular

• interacting amino acids are not necessarily next to each other in primary sequence

fibrous proteins

• alpha-keratin (hair, nails, feathers)

• silk fibroin

• collagen (tendons, ligaments, bone matrix, connective tissues)

alpha-keratin

2 alpha helices form a 2-chain coiled coil, many of which make up the protofilament, many of which make up protofibril, a group of which make intermediate filaments.

AKA hair structure

curling/waving hair

made possible by reducing the disulfide bonds, which leads to the ___, and the disulfide bonds re-oxidize over time

collagen

• important component of connective tissues (tendons, cartilage, bones, cornea of the eye)

• each ___ chain is glycine and proline rich LEFT-HANDED HELIX

• 3 __ chains intertwine into a RIGHT-HANDED TRIPLE HELIX

• many triple helices form __ fibril whose crosslinks are covalent bonds between Lysine or hydroxylysine or histidine residues

4-hydroxyproline

• found in collagen

• forces proline ring into favorable pucker

• offer more H bonds between 3 strands of collagen

• made via posttranslational processing catalyzed by prolyl hydroxylase & requires alpha ketoglutarate, O2, and ascorbate (vitamin C)

silk fibroin

• main protein in spider and moth silk

• antiparallel B-sheets structure

• small side chains like glycine or alanine allow for very close packing

• stabilized by H bonding within sheets & London dispersion interactions between sheets

• crystalline parts of silk are ___ rich while the other part do not have as much and thus are more rubber-stretchy

motifs

specific arrangement of several secondary structure elements

stable geometrical arrangements of few secondary structures

• recurring structures in numerous proteins

• globular proteins are composed of different ___ folded together

• B-alpha-B loop

• B-barrel

• alpha-B barrel

intrinsically disordered proteins

segments of protein that lack definable structure on the tertiary level

• made of amino acids whose concentration forces less defined structure (lysine, arginine, glutamate, proline)

• these regions can conform to many different proteins, facilitating interaction with numerous different partner proteins

• usually at the C-terminus or N-terminus for adaptable regions

quaternary structure

formed by the assembly of individual polypeptides into a larger functional cluster

• aka structure made of many different polypeptide chains/proteins

• stabilized by H bonds, hydrophobic interactions, electrostatic interactions, Van der Waals, disulfide bonds

• determined by various methods

X-ray crystallography

1. purify protein

2. crystallize protein

3. collect diffraction data

4. calculate electron density

5. fit known amino acid residues to density

pros: no size limit &well established

cons: difficult for membrane proteins, cannot resolve/see hydrogens

biomolecular NMR

1. purify protein

2. dissolve protein

3. collect ___ data

4. assign ___ signals

5. calculate structure

pros: no need for crystallization & can see many hydrogens

cons: difficult for insoluble proteins & large proteins

cryo-EM

1. -Spot it onto a grid

2. -Put it in cryogenic conditions

3. -Put grid in electron microscope

4. -Collect images of individual proteins on grid and see their orientation

5. -Average across chosen particles based on images

6. -High resolution 3D map of protein is the result

pros: small sample of protein needed (little of it) and high resolution

cons: expensivedom

domain

large, generally independently folding part of a protein

• ___ often have distinct activities

• proteins can often be cleaved into separate active ___

denaturation

loss of structural integrity with accompanying loss of activity/function

Caused by:

• heat

• cold

• pH extremes

• organic solvent

• chaotropic agents (break bonds and unfold proteins)

ribonuclease refolding experiment

made by Chris Anfinsen

basically, broke ribonuclease disulfide bond using urea and 2-mercaptoethanol, removed the 2 agents, and the protein refolded properly

the primary sequence alone determines the native conformation

chaperones

__ proteins prevent misfolding and aggregation of unfolded peptides

AKA help other proteins fold by temporarily shielding hydrophobic groups

• can also refold proteins which unfolded because of stress

thermodynamically favorable

the direction toward the native structure is not random because it is the most _____

Levinthal paradox

protein folding thought to occur by randomly trying every conformation until lowest energy on ewas found