Amino acids and protein folding

Describe the traits of aliphatic amino acids (4)

1. hydrocarbon chains of variable lengths and branching

2. non-polar, hydrophobic

3. cluster together in protein structures, away from water

4. methionine contains an S

Name the aliphatic amino acids (5)

alanine ala a

valine val v

leucine leu L

isoleucine ile i

methionine met m

describe the traits of aromatic amino acids (5)

•Stable ring of atoms with shared, delocalised electrons

•Larger and variable hydrophobicity

•Tyrosine has a hydroxyl (OH) group → hydrogen bonds

•Nitrogen in tryptophan indole ring → hydrogen bonds

•π-stacking – aromatic rings stack on top of each other

name the aromatic amino acids (3)

phenylalanine, phe, f

tyrosine, tyr, Y

tryptophan, trp, W

Describe the traits of polar amino acids (3)

•Hydrophillic – prefer to interact with water outside the protein, or with each other through hydrogen bonds

•Histidine can also be considered polar

•Cysteine can form disulfide bonds

name the polar amino acids (5)

cysteine, cys, c

serine, ser, s

threonine, thr, T
asparagine, asn, n

glutamine, glm, q

describe the traits of charged amino acids (4)

•Ionisable sidechains – become charged in the cellular environment

•Basic amino acids are positively charged

•Acidic amino acids are negatively charged

•Histidine is not always/fully charged

name the basic amino acids (3)

lysine, lys, k

arginine, arg, r

histidine, his, H

name the acidic amino acids (2)

aspartic acid, asp, d

glutamic acid, glu, E

Beer-Lambert Law

A = ε c l

A = absorbance

ε = extinction coefficient

c = concentration

l - path length

calculating the extinction coefficient

5690(#Tryptophans) + 1280(#Tyrosines)

zwitterionic form definition

molecule where net charge is 0, but atleast one functional group has positive charge, and atleast one functional group has negative charge.

isoelectric point (pI)

pH at which particular molecule carries no charge

pH below pKa, weak acid is…

protonated

pKa below pH, weak acid is

dissociated

CORN rule spells CORN

L amino acid

CORN rule spells not corn

D amino acid

priority rule has priority groups highest to lowest clockwise

R amino acid

priority group has priority groups highest to lowest anticlockwise

S amino acid

the only natural amino acid that is R

L-cysteine

absolute nomenclature (r / s)

• uses relative ranking of the priority of various functional groups, based on molecular weight

• Label groups from 1 - 4, 1 having the highest MW

• flip so lowest priority group is pointing backwards

• priority groups 1 - 4 clockwise, R

• priority groups 1 - 4 anticlockwise, S

Nomenclature L or D

1. flip molecule horizontal so H is pointing towards you

2. Look down H-C bond

3. clockwise CORN - L amino acid

4. clockwise CONR - D amino acid

What’s more common: L or D amino acids?

L amino acids

Amino acid shape dictates ____ shape and protein ____ determines _____

1. protein

2. structure

3. function

Dextrorotatory / +

Amino acid rotates plane polarised light in CLOCKWISE direction

LEVOROTATORY / -

• amino acid rotates plane polarised light in COUNTERCLOCKWISE direction

Proteins are ______, so they are a series of amino acids linked together by ____ bonds, and so can be referred to as _____

1. polymers

2. peptide

3. polypeptides

peptide bonds are formed from the reaction of the _____ group of one amino acid with the ______ of another, in a ____ reaction

1. carboxyl

2. amino

3. Condensation

peptide bond stable (in absence of catalyst) in water for ____

~1000 years

residue

each amino acid unit in a polypeptide chain

why does a polypeptide chain have polarity

ends have opposite charges

polypeptides have regular repeating ____ or ______

backbone or main chain

varying region of polypeptide compromising ______

distinctive side chain

the polypeptide backbone is rich in hydrogen bonding potential because the…

• the carbonyl groups are hydrogen bond acceptors

• NH groups are hydrogen bond donors

most polypeptide chains contain _____ amino acid residues

50-2000

average mass of amino acid

mass of most proteins between ___

110 Da / 110g/mol

5500 Da and 220000 Da, or 5.5kDa and 22.5kDa

Polypeptides can form tightly packed _____ because peptide bonds are ____-

1. globular structures

2. peptide bonds are uncharged

peptide bonds have partial ____ character, so they are ____ with limited rotation

1. double bond

2. planar

what’s longer, a single bond or a double bond?

single bond

peptide bonds in proteins prefer the _____ configuration due to unfavourable ______ in the cis form

• trans

• steric clashes

sterich clash definition

when two atoms, not covalently bonded to each other, are impossibly close to each other

2 configurations, or ________ are possible for a peptide bond: cis or trans, explaining how the two _____ are positioned about the peptide bond

1. geometric isomers

2. alpha carbon atoms

Phi Φ

dihedral angle of rotation about the bond between nitrogen and alpha carbon atom

Psi ψ

dihedral angle rotation about the bond between an alha carbon atom and a carbonyl carbon

Ramachandran Plot

plot that assesses all possible conmbinations of phi and psi

patches of favoured angles define secondary structure

secondary structure

local spatial conformatino of the protein backbone, and its stabilisation through hydrogen bonding

alpha helices (6)

• rod-like structure

• tightly coiled backbone

• side chains arrayed around the outside

• rise of 5.4 Å per turn (1.5 Å per residue)

• strongly stabilised by hydrogen bonds, formed between i and i+4 residues

• 3.6 residues per helical turn (12 main chain atoms)

dihedral angles in an alpha helix

phi= -57 degrees

psi - -47 defrees

Why do alpha helices have an electronic dipole (dipole movement)

• N terminus - delta pos and stabilised by negative charged residues, can bind to negatively charged molecules eg phosphate

• C terminus - delta negative, stabilised by positive charged residue

alpha helices can present chemically _____ surfaces

• “distinct”

• helical wheels have angles of 100 degrees (360 / 3.6)

• polar / hydrophilic on one surface, hydrophobic on the other → amphiphilics

structure of b strands (2)

• almost fully extended

• side chains of adjacent amino acids point in opposite directions

dihedral angles of amino acids in beta strand

phi: -120

psi: +120

structure of b sheets (2)

• formed by linking to or more b strands by hydrogen bonds

• adjacent chains in a b sheet can run in opposite directions (antiparallel) or in the same direction (parallel)

hydrogen bonds for antiparallel beta sheets are between…

one amino acid’s NH and CO group of another amino acid on the adjacent strand

parallel beta sheet hydrogen bond

one amino acid on one strand with 2 amino acids on the adjacent strand

why don;t beta sheets have a dipole

dipoles on adjacent amino acids cancel out

beta sheets cannot be ____ because of the _______ between sidechains, so they ___ to minimise _______ of the side chains, but retain maximum ___—-

• planar

• steric clashes

• twist

• conformational energies

• hydrogen bonding

lower conformational energy…

higher stability

amino acids with longer side chains prefer…

• alpha helices

beta turns /hairpin bends/loops (3)

• loop regions in secondary structure, to fold back in on itself

• typically lie on the surface of proteins

• often participate in protein=protein interactions

Circular dichroism spectroscopy (3)

• utilises circularly polarised light

• chiral molecules absorb circularly polarised light differently

• useful for distinguishing protein secondary structure

Hydrogen bonding (4)

• electrostatic attraction between a lone pair of electrons on an electronegative atom and a hydrogen atom that is bonded to an electronegative atom

• between NH and CO on secondary structure

• part of the side chain reactions that drive the formation of tertiary structure

• neutralises polarity in the hydrophobic core of proteins

hydrophobic effect (5)

• hydrophobic regions of a protein cluster together, minimising interactions with water

• entropic, not a formal interaction

• water molecules form hydrogen bonds amongst themselves but cannot form favourable interactions with hydrophobic molecules

• A) ordered / low entropy, limited number of conformations for H2O molecules to maintain H bond network

• B) less ordered / higher entropy, decreases hydrophovic water interface, fewer ordered water molecules

Van der Waals interactions (4)

• between uncharged atoms held in proximity

• many weak interactions produce significant cumulative stability

• facilitates dense packing of protein hydrophobic cores

• random movement of electrons generates transient electron dipole, inducing dipole in nearby atom

ionic interactions (2)

• classic ionic bond: one atom donates an electron to fill the valence shell of another

• opp. charged amino acid sidechains held in proximity will form electrostatic attractions

pi interactions (6)

• non covalent interactions involving electron density of aromatic rings

• 6 p orbital overlap form delocalised electron cloud above and below plane

• partial negative charge in cloud

• partial positive at edge of rings

• pi stacking: opposite particle charges of edge and cloud in proximity cause different orientations

• cation pi interactions: positive charge can form favourable interaction with negative cloud of electron density

di sulfide bonds can link together 2 _____ residues

cysteine

unfolded polypeptides are ____, they move ____ to form ____ interactions and the most ____ conformation.

Once part of a folded structure has formed, it may ___ the likelihood of subsequent _______ interactions forming.

_____ to ____ energy conformation, often assisted by ____ ____ within cells

dynamic

randomly

stable

increase

favourable

cascade

lowest

chaperone proteins

The _____ effect of all interactions within a protein lead to a _____ _____ structure. Different interactions and bonds ____ eachother

• cumulative

• particular folded

• affect

β-mercaptoethanol

reducing agent - breaks sulfide bonds

8M Urea

chaotropic agent - disrupts non covalent interactions e.g. ionic

Why did this happen?

thermodynamics: decrease in free energy as scrambled ribonucleases are converted to stable, native confirmation

alpha keratins (4)

• quaternary structur

• main component in hair, claws, nails, hooves

• evolved for varying strength and rigidity

• formed from intertwined alpha helices, stabilised from disulfide bonds

quaternary structure

arrangement of protein subunits into 3D complexes

post translational modifications (PTMs)

• modulate protein function

• e.g. phosphorylation, lipidation, glycosylation, addition of proeins, proteolytic processing

escherichia coli as a producer of proteins in the laboratory (4)

• fast, cheap, easy to use

• can be manipulated to produce any protein by transformation of a plasmid

• can select for genes which have been successfully transformed due to genes for antibiotic resistance

• recombinant expression induced by IPTG

protein purification workflow with e coli (5)

• e coli cells produce desired protein

• cell lysis using chemical, homogenisation or sonication (high frequency sound waves) protocols

• chromatography - separate protein from interest from unwanted bacterial proteins in the lysate

• sds - page / gel electrophoresis

• protein assay

column chromatography 3 types and function

• size exclusion: separation depends on molecular size

• ion exchange - separation depends on molecular charge

• affinity chromatography - siparation depends on specific binding interaction

size exclusion chromatography (SEC)

• aka gel filtration

• separates proteins based on molecular size, with porous gel beads

ion exchange

• proteins separated based on difference in molecular charge

• wanted proteins bind to oppositely charged beads

• then removed using salt or pH gradients

affinity chromatography

• proteins separated based on specific binding interactions

• e.g. antibody affinity chromatography, Ni-NTA column

Checking protein purity using gel electrophoresis

• use SDS PAGE

• SDS - anionic detergent, denaturing proteins and binding to them, so that they have a negative charge

• migration towards anode separates proteins according to their size

protein assay (3)

• determining concentration of protein

• e.g. measure light absorbance at 280nm

• fluorescent dye / colour-metric