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Protein Dynamics as Probed by NMR Methods


Constant exchange of backbone amide protons, polar side-chain NH/OH with solvent ... Proton occupancy of each amide hydrogen site is measured from intensity of NH ... – PowerPoint PPT presentation

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Title: Protein Dynamics as Probed by NMR Methods

Protein Dynamics as Probed byNMR Methods
  • Karthik Raman
  • Supercomputer Education Research Centre

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?

Why NMR?
  • Unique ability to characterise structure and
    dynamics of unfolded and partially folded protein
  • 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

Dynamics in Proteins
  • Unfolding
  • Tumbling
  • Bond Librations
  • Loop re-orientation
  • SS flips
  • Ring flips
  • Side-chain rotation/ re-orientation
  • Chemical Kinetics

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
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

Chemical Shift and Line-width
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

Chemical Shift Time-scale
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

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
  • Proton transfer affected by pH, T, neigbouring
    side-chains, isotopes calibrated effects

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
  • 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

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

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

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)

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)
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
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

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
  • 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

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
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.
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)

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

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).
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
  • 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

Dynamics To Probe The OriginOf Structural
Measurements show if high RMSD is due to high
flexibility (low S2)
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)

  • Chazin W (2004) Biochemistry 301 Principles of
    protein structure Lecture Notes,
  • Edison A S (2002) BCH 6745C Molecular Structure
    and Dynamics by NMR Lecture Notes, University of
  • 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

  • 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
  • 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
  • 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

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