Protein Folding & BiospectroscopyProtein Folding & Biospectroscopy

F14PFB

David Robinson

Mark Searle

Jon McMasterhttp://robinson.chem.nottingham.ac.uk/teaching

Module OverviewModule OverviewThe course will develop an understanding of protein

structure, stability, design and methods of structural analysis; understand the protein folding problem and experimental approaches to the analysis of protein folding kinetics and the application of site-directed mutagenesis. A range of experimental spectroscopic techniques will be introduced to probe protein structure and stability based on secondary structure and tertiary interactions and to probe the nature of the active site of metalloproteins using equilibrium and time-resolved spectroscopy.

Protein FoldingProtein Folding

1. Introduction

2. Protein Structure

3. Interactions

4. Protein Folding Models

5. Biomolecular Modelling

6. Bioinformatics

Handouts: http://robinson.chem.nottingham.ac.uk/teaching/F14PFB

3D Structure of Myoglobin3D Structure of Myoglobin

- first to be determined by x-ray crystallography

- revealed how the protein bound heme (loaded with oxygen) and gave the first detailed look at a protein structure

- now 10,000’s of protein structures are known

The many functions of proteinsThe many functions of proteins

Mechanoenzymes: myosin, actin Rhodopsin: allows vision Globins: transport oxygen Antibodies: immune system Enzymes: pepsin, renin, carboxypeptidase A Receptors: transmit messages through

membranes• And hundreds of thousands more…

Proteins are chains of amino acidsProteins are chains of amino acids Polymer – a molecule composed of repeating units

Amino acid compositionAmino acid composition 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 BondThe Peptide Bond

Dehydration synthesis Repeating backbone: N–C –C –N–C –C

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

O O

Peptidyl polymersPeptidyl polymers A few amino acids in a chain are called a

polypeptide. A protein is usually composed of 50 to 400+ amino acids.

Since part of the amino acid is lost during dehydration synthesis, we call the units of a protein amino acid residues.carbonylcarbonylcarboncarbon

amideamidenitrogennitrogen

Side chain propertiesSide chain properties Recall that the electronegativity of carbon is at

about the middle of the scale for light elements• Carbon does not make hydrogen bonds with water

easily – hydrophobic• O and N are generally more likely than C to h-bond

to water – hydrophilic We group the amino acids into three general

groups:• Hydrophobic• Charged (positive/basic & negative/acidic)• Polar

The Hydrophobic Amino AcidsThe Hydrophobic Amino Acids

Proline severelyProline severelylimits allowablelimits allowableconformations!conformations!

The Charged Amino AcidsThe Charged Amino Acids

The Polar Amino AcidsThe Polar Amino Acids

More Polar Amino AcidsMore Polar Amino Acids

And then there’s…And then there’s…

Amino acids

Planarity of the peptide bondPlanarity of the peptide bond

Phi () – the angle of rotation about the N-C bond.

Psi () – the angle of rotation about the C-C bond.

The planar bond angles and bond lengths are fixed.

Phi and psiPhi and psi = = 180° is

extended conformation

: C to N–H : C=O to C

C

C=O

N–H

The Ramachandran PlotThe Ramachandran Plot

G. N. Ramachandran – first calculations of sterically allowed regions of phi and psi

Note the structural importance of glycine

Observed(non-glycine)

Observed(glycine)Calculated

Four levels of protein structureFour levels of protein structurePrimary: amino acid sequence

Ser Val Tyr Cys

Four levels of protein structureFour levels of protein structure

Primary: amino acid sequence

Secondary: regular, repeated coiling

and folding of

polypeptide backbone

Four levels of protein structureFour levels of protein structure

Primary: amino acid sequence

Secondary: regular, repeated coiling and folding of polypeptide backbone

Tertiary: complete three-dimensional structure

Quaternary: arrangement of

subunits (in multisubunit

protein)

Secondary structureSecondary structure• Regular, repeated coiling and folding of

polypeptide backbone Due to hydrogen bonding Two patterns

(alpha) helix (beta) sheet

TertiaryTertiary

Complete three-dimensional structure Due to weak interactions between side (R)

groups as well as covalent disulfide bonds

Weak interactionsHydrogen bondsElectrostatic interactions (ionic bonds)Hydrophobic interactionsVan der Waals interactions

Tertiary Tertiary structure structure

formed through formed through side chain side chain

interactionsinteractions

TertiaryTertiary

Complete three-dimensional structure

Composed of:

• Motifs: specific combinations of secondary structural elements

• Domains: structurally independent units

MotifsMotifs

specific combinations of secondary structural elements

DomainsDomains

Structurally

independent units

Two different

binding domains

to bind two different

molecules

TertiaryTertiary

Complete three-dimensional structure

Native conformation: functional structure

Most stable conformation

TertiaryTertiaryFibrous Proteins = extended filaments

or

Globular proteins =

compact folded structure