Protein Dynamics as Probed by NMR Methods

1
Protein Dynamics as Probed byNMR Methods

• Karthik Raman
• Supercomputer Education Research Centre

2
Why study dynamics?

• Protein folding/unfolding
• Uncertainty in NMR and crystal structures
• Effect on NMR experiments
• Understanding of function
• Non-native states play role in protein transport,
  cellular processes
• Conformational Diseases?

3
Why NMR?

• Unique ability to characterise structure and
  dynamics of unfolded and partially folded protein
  states
• Ability to capture short-lived states
• Non-native protein states do not adopt unique 3D
  structures fluctuate rapidly over an ensemble
  of conformations
• Various NMR parameters have sensitivity to
  protein dynamics

4
Dynamics in Proteins

• Unfolding
• Tumbling
• Bond Librations
• Loop re-orientation

• SS flips
• Ring flips
• Side-chain rotation/ re-orientation
• Chemical Kinetics

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Parameters/Time-scales
Global unfolding
Local unfolding
bond librations
overall tumbling
Chemical kinetics
Slow loop reorientation
Fast loop reorientation
Side-chain rotation/re-orientation
SS flipping
Aromatic ring flips
HN exchange
T2, T1?
T1, T2, NOE
??
?J
Relaxation
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NMR Parameters for Protein Dynamics

• Number of signals per atom
• Line-widths
• Hydrogen Exchange
• Hetero-nuclear 15N, 13C
• Relaxation measurements
• T1 (spin-lattice relaxation time)
• T2 (spin-spin relaxation time)
• Hetero-nuclear NOE

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Chemical Shift and Line-width
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Number of Signals per atom

• Multiple signals indicate slow exchange between
  conformational states

Rate of exchange lt ?A - ?B

• Multiple states are difficult to detect from
  X-Ray Crystallography

9
Chemical Shift Time-scale
10
Hydrogen Exchange (HX)

• Constant exchange of backbone amide protons,
  polar side-chain NH/OH with solvent protons
• Exposed amide protons exchange faster
• HX can occur if
• hydrogen bond breaks (local fluctuations)
• or unfolding occurs

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Measuring HX

• Incubate protonated protein in D2O or labelled
  protein in H2O
• Proton occupancy of each amide hydrogen site is
  measured from intensity of NH peak in 1H-NMR
  spectrum
• Proton transfer affected by pH, T, neigbouring
  side-chains, isotopes calibrated effects

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Illustration of HX
The decrease of the NH NMR signal with time in
the process of the proton being exchanged with
deuteron, which is not detectable by NMR at the
1H NMR frequency
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HX

• Highly sensitive
• Can detect
• partially structured kinetic intermediates that
  are very short-lived
• intermediates that exist in small quantities as
  high-energy excited states in equilibrium with
  the native state
• HX with NMR can provide site-specific structural
  information about protein folding/unfolding,
  conformational dynamics

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Pulse labelling by HX
t 0
Exchange pulse with H2O at different times during
folding process
Deuterated protein
Quickly Lower pH to refold imprint of structure
at time of pulse
Exposed D exchange Protected D remain

• Useful to study folding pathways
• Structure of protein at time of pulse inferred
  from 2D 1H15N HSQC

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15N Spin Relaxation Measurements

• Probe of backbone dynamics
• Related to
• Longitudinal Relaxation Rate (R1)
• Transverse Relaxation Rate (R2)
• Steady-state NOE enhancement
• Sensitive to motions in psns time-scale
• R2 more sensitive to ns time-scale motions
• reflects contributions from slower (?s-ms)
  processes such as chemical exchange that cause
  line-broadening

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1H-15N NOE

• 1H 15N hetero-nuclear NOE most sensitive to
  high frequency motions of backbone
• Change in 15N intensity on perturbing equilibrium
  of directly bonded 1H in peptide bond
• Large and positive Low flexibility
• Negative Flexibility NH reorients in space
  independent of rest of protein
• Large unstructured region in human Bcl-xL
  (inhibitor of apoptosis) identified through NOEs
  (Muchmore et al, 1996)

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Structure of Calmodulin
15N1H NOEs indicate that the centre of the
connecting helix in holo calmodulin is floppy
(allowing molecule to wrap around recognition
helix of target protein)
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Correlation Function
Correlation function above is for the isotropic
diffusion of a rigid rotor Correlation time, ?c
time constant for exponential decay of function,
time the molecule takes to rotate 1
radian Short correlation times cause correlation
function to decay rapidly and vice-versa. ?c
depends primarily on molecular size and shape as
well as solvent viscosity, temperature, etc
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Spectral Density Function

• The spectral density function, J(?), is the
  Fourier transform of the correlation function
• Just as rapidly relaxing time domain signals
  give rise to broad lines, short correlation times
  have a broad spectral density function
• Goal in protein dynamics to know the spectral
  density function
• Peng and Wagner (1994) have described a method
  to measure the spectral density function at
  different NMR frequencies
• Many measured relaxation rates give rise to
  spectral densities

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Order Parameter (S2)

• Obtained from model-free analyses (Lipari-Szabo)
  no specific model for internal motion
• Measures magnitude of the angular fluctuation of
  a chemical bond vector such as the NH bond in a
  protein reflects flexibility of chain at these
  sites
• Depends on interactions responsible for relaxing
  nuclear spins in proteins
• Magnetic dipoledipole interactions
• Chemical Shift Anisotropy
• Electric Quadrupole
• S2 1, for a rigid sphere and S2 0 for a
  completely flexible molecule

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TIF in Methanococcus jannaschii
Colour-coded stereo view representation of the
values of the order parameters (S2) derived from
15N relaxation rate data for the backbone amide
nuclei. S2 lt 0.5 RED 0.5 lt S2 lt 0.7
ORANGE 0.7 lt S2 lt 0.8 GREEN S2 gt
0.8 BLUE Backbone amide nuclei with greatest
values of the S2 generally have most-restricted
motion on the ps-ns time scale. Residues 1,
8487, 101, and 102 are not included in the
figure, because their NH protons exchange too
rapidly with solvent for 15N relaxation rate data
to be obtained other residues for which no 15N
relaxation rate data was obtained, such as the
prolines and the isolated unassigned residues,
are labelled with the colour appropriate for the
adjacent residues
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TIF in Methanococcus jannaschii
Representation of rates with which backbone amide
protons exchange with deuterium, which are
derived from NMR data. Rex gt 10-1 s-1
RED 10-1 lt Rex lt 10-2 s-1 ORANGE 10-2 lt Rex
lt 10-4 s-1 GREEN Rex lt 10-4 s-1
BLUE Solvent exchange rates are normalized to
pH 7, assuming a 10-fold increase in exchange
rate for each increase of 1 pH unit. In general,
amide groups with the lowest solvent exchange
rates tend to be in the least flexible and least
solvent-exposed parts of the protein.
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Basic Pancreatic Trypsin Inhibitor (BPTI)

• NMR can be used to probe rate processes in
  proteins (Wüthrich, 2003)
• NMR spin relaxation
• NOE
• Aromatic ring flips, not indicated by X-ray
  crystal structure (temperature factors)

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Dynamics of Hen egg-white Lysozyme

• Comparison of NMR and X-Ray Crystallography
• Excellent agreement between X-ray diffraction and
  NMR data
• Haliloglu and Bahar (1999) have also proposed a
  model for predicting rotational dynamics of
  proteins based primarily on geometry

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Dynamics of Hen egg-white Lysozyme

Regions observed in 15N-H NMR relaxation to be
relatively disordered are shown in black and
indicated by the labels (2)(7). Residues
(shown in black) are 1619 (2), 4550 (3), 6770
(4), 8586 (5), 102106 (6), and 116117 (7).
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Probing Side-chain Dynamics

• Large variability of amplitude in side-chain
  dynamics, compared to backbone motions, which are
  largely uniform in most proteins
• Important in molecular recognition, protein
  stability
• T4 Lysozyme binding site is not accessible to
  ligands in static structures
• Probed using 15N Relaxation and exchange at
  backbone 15N sites to figure out residues that
  permit entry of ligand molecule

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Dynamics To Probe The OriginOf Structural
Uncertainty
Measurements show if high RMSD is due to high
flexibility (low S2)
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Other Methods for probing Protein Dynamics

• X-Ray Crystallography (B-factors) Thermal
  motions of atoms in crystalline state
• Site-Directed Spin Labelling (SDSL) followed by
  Electron Spin Resonance (ESR)

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References

• Chazin W (2004) Biochemistry 301 Principles of
  protein structure Lecture Notes,
  structbio.vanderbilt.edu
• Edison A S (2002) BCH 6745C Molecular Structure
  and Dynamics by NMR Lecture Notes, University of
  Florida
• Englander S W, Mayne L, Bai Y and Sosnick T R
  (1997) Hydrogen exchange The modern legacy of
  Linderstrøm-Lang Protein Science 611011109
• Haliloglu T and Bahar I (1999) Structure-Based
  Analysis of Protein Dynamics Comparison of
  Theoretical Results for Hen Lysozyme With X-Ray
  Diffraction and NMR Relaxation Data Proteins
  Structure, Function and Genetics 27654667
• Ishima R and Torchia D A (2000) Protein Dynamics
  from NMR Nature Structural Biology 7740743
• Juneja J and Udgaonkar J (2001) NMR studies of
  protein folding Current Science 84157172

30
References

• Kay L E (1998) Protein Dynamics from NMR Nature
  Structural Biology NMR Supplement 5513517
• Li W and Hoffman D W (2001) Structure and
  dynamics of translation initiation factor aIF-1A
  from the archaeon Methanococcus jannaschii
  determined by NMR spectroscopy Protein Science
  1024262438
• Muchmore S W, Sattler M, Liang H, Meadows R P,
  Harlan J E, Yoon H S, Nettesheim D, Chang B S,
  Thompson C B, Wong S-L, Ng S-C and Fesik S W
  (1996) X-ray and NMR structure of human Bcl-xL,
  an inhibitor of programmed cell death Nature
  381335341
• Mulder F A A, Skrynnikov N R, Hon B, Dahlquist F
  W and Kay L E (2001) Measurement of Slow (µs-ms)
  Time Scale Dynamics in Protein Side Chains by 15N
  Relaxation Dispersion NMR Spectroscopy
  Application to Asn and Gln Residues in a Cavity
  Mutant of T4 Lysozyme Journal of American
  Chemical Society 123967-975
• Peng and Wagner (1994) Investigation of Protein
  Motions via Relaxation Measurements Methods in
  Enzymology 239563596
• Wüthrich K (2003) NMR Studies of Structure and
  Function of Biological Macromolecules (Nobel
  Lecture) Angewandte Chemie 4233403363

31
Other Articles

• Hill R B, Bracken C, DeGrado W F and Palmer A G
  III (2000) Molecular Motions and Protein
  Folding Characterization of the Backbone
  Dynamics and Folding Equilibrium of ?2D Using 13C
  NMR Spin Relaxation Journal of American Chemical
  Society 1221161011619
• Daragan V A and Mayo K H (1998) A Simple
  Approach to Analyzing Protein Side-Chain Dynamics
  from 13C NMR Relaxation Data Journal of Magnetic
  Resonance 130329334
• Choy W-Y, Shortle D and Kay L E (2003) Side
  Chain Dynamics in Unfolded Protein States an NMR
  Based 2H Spin Relaxation Study of ?131? Journal
  of American Chemical Society 12517481758