Protein folding and purification

Hydrophobic effect

• hydrophobic regions cluster together to minimise interactions with water - entropic effect

• water molecules for H bonds only with themselves, not with hydrophobic molecules

• adapted to reduce the size of the hydrophobic-water interface so fold into structures with nonpolar cores

Hydrogen bonding

• electrostatic attraction between lone pair of electrons on elec. neg. atom and H bonded to elec. neg. atom

• drive formation of 2* structure with NH and CO backbone

• drive formation of 3* structure with sidechains

Van der Waals interactions (ind. dip-dip)

• random movement generates transient electric dipole 

• many weak interactions produce sig. cum. stability 

• facilitates dense packing of protein hydrophobic cores

Ionic interactions

• oppositely charged a.a. sidechains held in proximity will form electrostatic interactions

Disulphide bonds

• very strong covalent bond

• not every sulphur forms disulphide bond, often many combinations but only one produces required structure

How can protein folding be aided?

• once part of folded structure has formed, may increase likelihood of subsequent favourable interactions (cascade of lowest energy conformation)

• chaperone proteins in cells increase likelihood of favourable interactions

• leads to native (completely folded) structure

What are 3 properties of peptide bonds?

• partial double bond character so planar with limited rotation

What is a residue?

Each amino acid unit in a polypeptide chain, sometimes what is left after a condensation reaction

What are the two terminus’ called in a polypeptide?

N (amino) and C (carboxyl)

From which terminus is the polypeptide named?

N to C

What is the charge of the N-terminus?

+ve

What is the charge of the C-terminus?

-ve

On average, how many amino acid residues are in each polypeptide chain?

50-2000 (any less it is just a peptide)

What is the average mass of an amino acid?

110 Da (gmol-1)

Which regions of the polypeptide are charged?

Ends, the backbone has no charge.

What is a Da?

1 Dalton → 1 g/mol

Pink as presence of glycine and proline makes beta turn more likely

Beta sheet as amino acid present have large R groups (amino acids such as W,F,Y)

Yellow as these amino acids have polar sidechains (other two are mostly hydrophobic)

Blue as both glycine and proline residues are present (they cannot both be incorporated into an alpha helix, especially proline)

What amino acid will not be found in an aplha helix?

Proline (will break them)

Name 6 sidechain interactions that produce teriary structures

1) Hydrophobic (entrpic effect)

2) Hydrogen bonding

3) Induced dip dip

4) Ionic

5) Disulphide

6) Aromatic stacking

What determines a native tertiary structure?

The primary sequence and a cumulative effect of all interactions

What can be added to a ribonuclease to denature it and determine its tertiary structure?

8M Urea and beta-mercaptoethanol

What happens as a denatured sequence becomes active (folded)?

Decrease in entropy (free energy)

What makes the quaternary structure of alpha keratin strong?

Many intertwined alpha helices, stabilised by disulphide bonds

What is the purpose of posttranslational modifications?

Tune activity of protein to be more specific to its purpose

Which amino acid lacks a chiral centre?

Glycine

D-Thr, L-Tyr

In a folded protein, charged amino acids tend to be on the…

Exposed on the outside of the protein

What type of interaction would be observed from glutamic acid and lysine sidechain?

Ionic interaction

Which interaction do we use beta-mercaptoethanol to disrupt?

Disulphide bonds

Name 3 types of column chromatograpy?

Size exclusion chromatography (based upon molecular size e.g. agarose gel)

Ion exchange (based on molecular charge think)

Affinity chromatography (based on specific binding interaction)

What is the step called in which you separate your protein of interest from unwanted bacterial proteins in the lysate?

Protein purification

How can protein purity be checked?

Gel electrophoresis (SDS-PAGE)

How can activity of proteins be assessed?

An assay (e.g. monitor activity of lactate dehydrogenase using light absorbtion)

Met → Phe or Val → Lys

Val → Lys as length, branching and charge are altered.

Met→ Phe is more likely to be incorporated as have similar hydrophobicity