Protein Dynamics:Measurement and Meaning

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Protein DynamicsMeasurement and Meaning
Reading Not much! The textbooks dont cover
dynamics. There is a nice review paper here but
its already at a higher level than expected for
the course. Well keep it simple and Ill try to
annotate the slides well!
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Proteins in motion.

• At all temperatures above 0 K, atoms and their
  constituents are in motion.
• Early protein NMR experiments detected
  ring-flipping motions of aromatic side chains,
  providing some of the first evidence that
  proteins are dynamic structures.
• Modern spectroscopic, time-resolved
  crystallographic, and computational studies have
  detected complex side chain and backbone thermal
  motions over time scales ranging from picoseconds
  to seconds.
• Chemical methods such as hydrogen exchange and
  disulfide trapping have probed thermal motions on
  longer time scales of microseconds to hours,
  revealing motional amplitudes as large as 1.5 nm
  on the millisecond time scale.
• Proteins in solution undergoes constant random
  thermal motions within a stable equilibrium
  structure. These motions involve displacements of
  individual atoms, bonds, functional groups, side
  chains, local regions of the backbone, secondary
  structure elements, and entire folded domains.
• Many proteins also undergo thermally driven
  transitions, called conformational changes,
  between two or more equilibrium structures.
• Both types of motions can play important
  functional roles.
• Random thermal motions act as a molecular
  lubricant during conformational changes, allowing
  the protein to sample conformational space.
• Random thermal motions and the average
  conformation can both change substantially when a
  protein is modified by substrate or ligand
  binding, docking to another macromolecule, or
  covalent modification (such as phosphorylation).
• Such changes often have important functional
  consequences for the tuning of binding affinities
  and the switching of regulatory proteins.

Joseph J. Falke Science 22 February 2002 Vol.
295. no. 5559, pp. 1480 - 1481
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Time Scale of Motions
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Dynamics of proteins

• Slower domain motions on the micro- to
  millisecond time scale (ms-ms) are biologically
  very important, because they are close to the
  time scales on which docking, protein folding,
  allosteric transitions, and product release take
  place and are therefore associated with
  functional processes.
• Three cases
• Tight ligand binding
• Allosteric Regulation
• Enzymatic Function

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Methods and Time-Scales

• Simulation molecular ps to ns
• NMR ns-ms
• Temperature Jump ps ms
• Stop-flow ms-s
• Flourescence miscroscopy min-hours

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The Role of Dynamics in the Life of a Protein

• .

Unfolded to folded transition weakens binding via
DS.
Unfolded upon binding increases affinity by
minimizing DS.
Functional regulation by shifting equilibrium.
Enzymatic function by shifting equilibrium. S
substrate, Pr product
Nature 438, 36-37 (3 November 2005)
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Timescale of some processes in proteins
Disulfide bridge rotations
Methyl group rotations
Aromatic group flipping Note the wide range of
rates.
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NMR has advantages

• Only method that can determine the dynamic
  parameters of an individual nucleus
• Can be tuned to detect dynamic behaviour on very
  different time scales

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Tight Binding to a Ligand
Proteins are generally tightly packed, except
around ligand-binding sites, which may be viewed
as packing deficiencies. These packing
deficiencies allow mobility of their perimeters,
which propagates through other parts of the
protein because they are so tightly
packed. Ligand binding repairs the packing
defect, leading to global protein rigidification
with concomitant entropy loss.
Ligand Binding Site
Protein
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An example of protein-ligand interactions SH2
domain binding to pY peptides
Y75
R36
pY
M
V
Natural ligand pY- Met/Val X Met, KI
100 nM pY phosphotyrosine
Kristensen et al, J. Mol. Biol, 299, 2000, pp.
771
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Backbone Relaxation Data
Ligand
- Ligand
More movement
ns-ms
More movement
ns
ms-ms
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Ligand Binding Reduces Dynamics at the active
site.
Ligand
- Ligand
slow
medium
fast
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Allostery Induced fit vs population shift
Allostery does not drive a system from one
conformation to another, but selects from a
number of available conformations.
Volkman et al, Science, 2001, 291, p. 2429
NtrC (receiver) inactive
NtrC (receiver) active
kinase
transcription
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Allostery in a single domain.
phosphoprotein
Activated mutant
wt
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Dynamics in Cooperative Binding
Ligand binding typically causes a loss of
entropy. If this occurs throughout the protein,
then there is no further loss of entropy when a
second ligand binds therefore automatically the
affinity will be increased. A general loss of
simply vibrational (fast motion) can create a few
kcal/mol of additional binding energy. DG -RT
lnKD 2 reduction in 400 aa protein ? 1.5
kcal/mol 4.5 increase in KD
Kern and Zuiderweg, TIBS, 2003, 13, p.748
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Cooperative Binding of O2 by Haemoglobin
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Take Home Lessons

• 3D Structures provide only static, incomplete
  information
• All processes are, by definition, dynamic!
• Dynamic processes span an enormous range of
  timescales
• Dynamics required for and may regulate molecular
  processes such as ligand binding, catalysis and
  allostery.
• Rigidification of backbone commonly seen ?
  enhanced specificity
• Sidechains
• Rigidification ? enhanced specificity
• Loosening ? less specificity
• Conformational change usually means a shift in
  the populations of pre-existing conformations.

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Sidechain Dynamics and Ligand BindingKay et al.,
Biochemistry, 1996, 35, p. 361
3
2
1
5
4
6
Phospholipase Cg SH2 domain and ligand
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Correlation of Sidechain Dynamics with Affinity
of Ligand Binding
Less movement
More movement
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How do we know that the 2 conformations are the
inactive and active ones?
Wt V115I D54E D86N D86N/A89T D86N/A89T/D86N/A89T15
I pNtrC
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Global Dynamics of CypA

• Cyclophilin A (CypA) is a proline cis-trans
  isomerase
• Binding of CspA inhibits CypA
• Study dynamics of free enzyme, enzyme-substrate
  and enzyme-inhibitor complexes using NMR.

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Global Dynamics of CypA
Nature 438, 117-121 (3 November 2005)
Free
Substrate Bound
Free
Substrate Bound
Methyls showing conformational exchange.
The same residues undergo conformational exchange
in both the free and working states.
Amides showing conformational exchange.
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Dynamic Exchange of 2 Populations
Free
Substrate Bound
By analysis of the NMR data it can be seen that
the protein is interconverting between two
conformations. Analysis of the chemical shifts
suggests that the two conformations in the free
state are the same as those in the substrate
bound state. The free state is undergoing
conformational exchange at about 1400 s-1. The
substrate bound state is undergoing
conformational exchange at about 2700 s-1. Using
the previous equation, the two conformers in the
free state are about 90/10. In the substrate
bound state they are about 50/50. The second
conformer is the one with bound substrate in the
trans (product) conformation. The substrate
catalysis rate is about 2500 s-1.
NMR Relaxation Dispersion data.
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Large Portions of CypA Undergo Correlated Motions.
Mutations within and outside of the active site
all effect the dynamic properties of the same set
of amino acids