Transcript No Slide Title - The Robinson Group – University of Nottingham

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 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
Levels of Protein Structure
The folded protein structure is
stabilized by a variety of weak
chemical interaction,
and in some cases covalent
(disulfide) bonds between
cysteine residues
Disulfide bond:
R– CH2–S–S–CH2–R
Cys
Cys
- helix
Myoglobin
Hemoglobin
Protein structure: overview
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)
Primary & Secondary Structure
 Primary structure = the linear sequence of
amino acids comprising a protein:
AGVGTVPMTAYGNDIQYYGQVT…
 Secondary 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
structure of the protein
The alpha helix

 60°
Properties of the alpha helix
     60°
 Hydrogen bonds
between C=O of
residue n, and
NH of residue
n+4
 3.6 residues/turn
 1.5 Å/residue rise
 100°/residue turn
Properties of -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)
   135°
  +135°
Properties of beta sheets
 Formed of stretches of 5-10 residues in
extended conformation
 Pleated – each C a bit
above or below the previous
 Parallel/antiparallel,
contiguous/non-contiguous
Parallel and anti-parallel -sheets
 Anti-parallel is slightly energetically favoured
Anti-parallel
Parallel
Turns 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
Structure
 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 threedimensional structure
Protein Folding
Proteins, in general, do NOT fold as they
are synthesized on the ribosome
Folding of RNAse A in the test tube
denaturation
renaturation
Incubate protein
in guanidine
hydrochloride
(GuHCl)
or urea
100-fold
dilution of protein
into physiological
buffer
- 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”
Anfinsen, CB (1973) Principles that govern the folding of protein chains.
Science 181, 223-230.
Protein Folding
Many proteins fold by Assisted Self
Assembly
Correct assembly (native conformation)
requires assistance
by CHAPERONES
Protein unfolding = Denaturation
Loss of structure and function
–
–
–
–
Heat
Extreme pH
Detergents
Urea
Protein unfolding = Denaturation
Why 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 disease
Normal 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