Factors Determining Secondary and Tertiary Structure

1
Factors Determining Secondary and Tertiary
Structure

• Folding determined by amino acid sequence
• Danaturation unfold protein with temp., pH, or
  add urea, GuHCl
• Ribonuclease can be unfolded and refolded
  enzymatic activity restored
• Proteins naturally fold following synthesis, may
  need help to avoid misfolding or aggregation

2
Thermodynamics of folding

• Overall process must be favorable, -?G
• Entropy decreases during folding which causes
  ?G
• ?H must be negative to make up for, source is
  formation of non-covalent forces
• Charge-charge interactions
• salt bridges, such as K ?E, pH induced
  denaturation
• Internal hydrogen bonds
• Hydrogen bond donors form bonds with acceptors
• S and T , N, Q, H
• Van der Waals interactions Interior is tightly
  packed, allowing maximum contact between side
  chain atoms
• Protein has to give up H-bonds with water in
  random coil, thus the hydrogen bonds formed in
  folded structure help offset

3
The hydrophobic effect

• Hydrophobic amino acids become buried in the
  protein and are preventing interactions with
  water (less ordered cage like water structures
  around hydrophobic groups)
• Increase randomness and provides a negative ?S
• Hydrophobicity scales for amino acids
• Cooperativity of folding there are few
  intermediates in folding, thus the interactions
  that drive folding work in concert.

4
Introduction to Protein Structure Driving Forces
for Protein Folding
5
Role of Disulfide Bonds

• Bovine pancreatic trypsin inhibitor 3 disulfide
  bridges in 58 residues
• Mid-point of denaturation is highly dependent on
  disulfide bonds
• Bridges not necessary for folding, but stabilize
  folded structure
• S-S bonds are rare because of reducing conditions
  inside cells. Extracellular proteins are more
  likely to have.

6
Protein only exists in two conformations When
half-folded concentration is one-half folded,
one-half unfolded No table intermediates Cooperati
ve transition important partially folded
structures do not exist in the cell
7
(No Transcript)
8
Introduction to Protein Flexibility Example of
Protein Motion
Dihydrofolate reductase (DHFR)
Proteins are not rigid bodies. They are flexible
and have many types of motions from small
amplitude vibrations to large scale
conformational rearrangements to
folding/unfolding.
Animation from M.F. Thorpe
9
Introduction to Protein Flexibility Protein
Motion Often Facilitates Function
Animation from M.F. Thorpe
DHFR Mobile cleft
10
From Joan-Emma Shea
Time scales
Side-chain rotations
Loop closure
Protein aggregation
Protein folding
Folding of ?-hairpins
Helix formation
11
Introduction to Protein Folding Leventhals Paradox
Given a peptide sequence of 100 amino
acids. Question If all conformations are sampled
by brute force searching How long will it take
to find the native state?
Back of the envelope calculation Mean time to
find new conformation gt 10-15 seconds (1
Femtosecond) Estimated number of accessible
conformations gt (3)100 gt 1047 Total time to
search all conformations 1032 seconds. Problem
Estimated age of our Universe is ? 1018 seconds.
Conclusion Protein folding is biased. A protein
samples only a tiny fraction of phase space
(dihedral angle space, defining ?i, ?i, ?i
that is a-priori geometrically allowed.
12
Thermodynamic Hypothesis for Protein
Folding Christian Boehmer Anfinsen 1972 Nobel
prize in Chemistry
Note Coopers review paper was dedicated to
Chris Anfinsen
In 1972, Chris Anfinsen received the Nobel Prize
for demonstrating that the protein, ribonuclease,
spontaneously adopted its active conformation
from a completely unfolded state. Thus all
information specifying the native structure of a
protein resides in the sequence of its amino
acids.
Structural Biology Paradigm Sequence ? Structure
? Function
13
Modern View of Protein Folding Funnel-Shaped Free
Energy Landscape
Adopted from Ken Dills web site at UCSF
Free Energy
Ideal funnel
Folding Coordinate
14
Modern View of Protein Folding Funnel-Shaped Free
Energy Landscape
Adopted from Ken Dills web site at UCSF
Free Energy
Realistic funnel
Folding Coordinate
15
Dynamics of globular proteins

• Ribonuclease (1050 conformations possible) would
  take 1030 years to fold.
• Folding takes place through a series of
  intermediate states.
• Partially folded structures funneled by energy
  minimizations toward the final folded state.
• Many pathways all leading to folded state.
• Molten globule state compact structure that has
  not gone through hydrophobic packing.
• Trapped intermediates cells use mechanisms to
  eliminate

16
Protein Thermodynamic Stability Dependence on
Thermodynamic and Solvent Conditions and Sequence
Temperature induced denaturation R.M. Ballew, et
al., PNAS 93 5759 (1996)
Example of solvent induced denaturation G.
Pappenberger, Nature Structural Biology 8,
452-458 (2001)
Example of pH induced denaturation Spector and
Raleigh. JMB. 276 479-489, (1998)
17
Computational Biology Theoretical Modeling
Computational Methods Predictions
schematic
Develop models to facilitate computational
targeted-drug design predictions
Computer aided design of stable enzyme catalysts
that function in specified environmental
conditions and to obtain improved
protein-pharmacore interactions
18
(No Transcript)
19
(No Transcript)
20
(No Transcript)
21
(No Transcript)
22
Chaperonins

• Help proteins fold by preventing stable
  intermediates and aggregation
• Provide environment protected from solvent during
  folding process.
• Gro-El GroES Chaperonin, ring like structure
  forms a chamber.
• ATP-dependent conformational change in the
  interior hydrophobic to hydrophilic shift

23
Motions within proteins

• Three classes of motion
• vibrations and oscillations of individual atoms
  and groups 0.2 nm, 10-15-10-12, limits
  crystallography
• concerted motions of structural elements like
  alpha helices and groups of residues 0.2 -1 nm,
  10-12 10-8
• motions of whole domains, opening and closing of
  clefts 1 10 nm, gt10-8, protein-protein
  interactions catalytic activity

24
Protein folding an disease

• Prions conversion of PrPc to PrPsc off-pathway
  folded intermediate