Transcript protein - The Robinson Group – University of Nottingham

Protein Folding & Biospectroscopy
F14PFB
David Robinson
Mark Searle
Jon McMaster
http://robinson.chem.nottingham.ac.uk/teaching
Module Overview
The 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 Folding
1.
2.
3.
4.
5.
6.
Introduction
Protein Structure
Interactions
Protein Folding Models
Biomolecular Modelling
Bioinformatics
Handouts: http://robinson.chem.nottingham.ac.uk/teaching/F14PFB
3D 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 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 acids
 Polymer – a molecule composed of repeating units
Amino acid composition
 Basic Amino Acid
Structure:
• The side chain, R,
varies for each of
the 20 amino acids
Side chain
R
H
O
N C C
H
Amino
group
H
OH
Carboxyl
group
The Peptide Bond
 Dehydration synthesis
 Repeating backbone: N–C –C –N–C –C
O
O
• Convention – start at amino terminus and proceed
to carboxy terminus
Peptidyl 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.
carbonyl
carbon
amide
nitrogen
Side 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 Acids
Proline severely
limits allowable
conformations!
The Charged Amino Acids
The Polar Amino Acids
More Polar Amino Acids
And then there’s…
Amino acids
Planarity of the peptide bond
Psi () – the
angle of
rotation about
the C-C bond.
Phi () – the
angle of
rotation about
the N-C bond.
The planar bond angles and bond
lengths are fixed.
Phi and psi
  =  = 180° is
extended
conformation
  : C to N–H
  : C=O to C
C=O
C
N–H
The Ramachandran Plot
Observed
(non-glycine)
Calculated
Observed
(glycine)
 G. N. Ramachandran – first calculations of
sterically allowed regions of phi and psi
 Note the structural importance of glycine
Four levels of protein structure
Primary: amino acid sequence
Ser
Val
Tyr
Cys
Four levels of protein structure
Primary: amino acid sequence
Secondary: regular, repeated coiling
and folding of
polypeptide backbone
Four 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 structure
• Regular, repeated coiling and folding of
polypeptide backbone
 Due to hydrogen bonding
 Two patterns
•  (alpha) helix
•  (beta) sheet
Tertiary
 Complete three-dimensional structure
 Due to weak interactions between side (R)
groups as well as covalent disulfide bonds
Weak interactions
Hydrogen bonds
Electrostatic interactions (ionic bonds)
Hydrophobic interactions
Van der Waals interactions
Tertiary
structure
formed through
side chain
interactions
Tertiary
 Complete three-dimensional structure
 Composed of:
• Motifs: specific combinations of secondary
structural elements
• Domains: structurally independent units
Motifs
specific
combinations of
secondary
structural
elements
Domains
Structurally
independent units
Two different
binding domains
to bind two
different
molecules
Tertiary
 Complete three-dimensional structure
Native conformation: functional structure
Most stable conformation
Tertiary
Fibrous Proteins = extended filaments
or
Globular proteins =
compact folded structure