Protein Folding & Biospectroscopy F14PFB Dr David Robinson Lecture 2

Protein Folding & Biospectroscopy

F14PFB

Dr David Robinson

Lecture 2

Principles of protein structure and function

• Function is derived from structure

• Structure is derived from amino acid sequence

• Different activities and shapes of proteins due to different amino acid sequences

A reminder…• Basic Amino Acid

Structure:– The side chain, R,

varies for each ofthe 20 amino acids

C

RR

C

H

NO

OHH

H

Aminogroup

Carboxylgroup

Side chain

The Peptide Bond

• Dehydration synthesis

• Repeating backbone: N–C –C –N–C –C

– Convention – start at amino terminus and proceed to carboxy terminus

O O

Levels of Protein StructureLevels of Protein Structure

- helix

Myoglobin

Hemoglobin

The folded protein structure is stabilized by a variety of weak chemical interaction, and in some cases covalent (disulfide) bonds between cysteine residues

R– CH2–S–S–CH2–R

Cys Cys

Disulfide bond:

Structural element Description

primary structure amino acid sequence of protein

secondary structure helices, sheets, turns/loops

super-secondary structure association of secondary structures

domain self-contained structural unit

tertiary structure folded structure of whole protein

• includes disulfide bonds

quaternary structure assembled complex (oligomer)

• homo-oligomeric (1 protein type)

• hetero-oligomeric (>1 type)

Protein structure:Protein structure: overview overview

Primary & Secondary StructurePrimary & Secondary Structure Primary structurePrimary structure = the linear sequence of

amino acids comprising a protein:AGVGTVPMTAYGNDIQYYGQVT…

Secondary structureSecondary structure• Regular patterns of hydrogen bonding in proteins

result in two patterns that emerge in nearly every protein structure known: the -helix and the-sheet

• The location of direction of these periodic, repeating structures is known as the secondary secondary structurestructure of the protein

The alpha helixThe alpha helix 60°

Properties of the alpha helixProperties of the alpha helix 60° Hydrogen bondsHydrogen bonds

between C=O ofresidue n, andNH of residuen+4

3.6 residues/turn 1.5 Å/residue rise 100°/residue turn

Properties of Properties of -helices-helices 4 – 40+ residues in length Often amphipathic or “dual-natured”

• Half hydrophobic and half hydrophilic• Mostly when surface-exposed

If we examine many -helices,we find trends…• Helix formers: Ala, Glu, Leu,

Met• Helix breakers: Pro, Gly, Tyr,

Ser

The beta strand (& sheet)The beta strand (& sheet) 135° +135°

Properties of beta sheetsProperties of beta sheets Formed of stretches of 5-10 residues in

extended conformation Pleated – each C a bit

above or below the previous Parallel/antiparallelParallel/antiparallel,

contiguous/non-contiguous

Parallel and anti-parallel Parallel and anti-parallel -sheets-sheets Anti-parallel is slightly energetically favoured

Anti-parallelAnti-parallel ParallelParallel

Turns and LoopsTurns and Loops Secondary structure elements are connected by

regions of turns and loops Turns – short regions

of non-, non-conformation

Loops – larger stretches with no secondary structure. Often disordered.• “Random coil”• Sequences vary much more than secondary

structure regions

Levels of Protein Levels of Protein StructureStructure

Secondary structure elements combine to form tertiary structure

Quaternary structure occurs in multienzyme complexes• Many proteins are

active only as homodimers, homotetramers, etc.

Protein Folding• Forming polypeptide chain requires energy

and information (template) – ie translation from RNA protein SEQUENCE

Protein Folding• Forming polypeptide sequence requires

energy and information (template)

• Forming native conformation requires NO ADDITIONAL energy or information

(SELF ASSEMBLY)

Protein folding

Amino acid sequence contains all information necessary for folding into a specific three-dimensional structure

Protein Folding

Proteins, in general, do NOT fold as they are synthesized on the ribosome

Folding of RNAse A in the test tubeFolding of RNAse A in the test tube

denaturation renaturation

Incubate proteinin guanidine

hydrochloride(GuHCl)or urea

100-folddilution of proteininto physiological

buffer

Anfinsen, CB (1973) Principles that govern the folding of protein chains. Science 181, 223-230.

- the amino acid sequence of a polypeptide is sufficient to specify its three-dimensional conformation

Thus: “protein folding is a spontaneous process that does not require the assistance of extraneous factors”

Protein Folding

Many proteins fold by Assisted Self Assembly

Correct assembly (native conformation) requires assistance

by CHAPERONES

Protein unfolding = DenaturationLoss of structure and function

– Heat– Extreme pH– Detergents– Urea

Protein unfolding = DenaturationWhy do these conditions cause loss of

structure and function?– Heat– Extreme pH– Detergents– Urea

Lysozyme

Lysozyme

Tertiary: complete three-dimensional

structure

Quaternary: arrangement of subunits

(in multisubunit protein)

Hemoglobin

Quaternary structure

• Held together by weak interactions between side (R/functional) groups as well as covalent disulfide bonds

Structure-function relationship• Function is derived from structure

• Structure is derived from sequence

Sickle-cell diseaseNormal red blood cells Sickle shaped red blood cells

Due to single amino acid change in haemoglobin

Sickle-cell disease

Sickle-cell disease

• Single specific amino acid change causes change in protein structure and solubility

• Results in change in cell shape

• Causes cells to clog blood vessels

Amino acids