1

COURSE#1022: Biochemical Applications of NMR Spectroscopy

http://www.bioc.aecom.yu.edu/labs/girvlab/nmr/course/
Heteronuclear Relaxation and
Macromolecular Structure and Dynamics
LAST UPDATE: 4/16/2010
2
References
Cavanagh, J ., W. J . Fairbrother, A. G. Palmer and N. J . Skelton (2007).
Protein NMR Spectroscopy: Principles and Practice, Academic Press.
Chapter 5 Relaxation and Dynamic Processes
Chapter 8 Experimental NMR Relaxation Measurements
Palmer, A. G. and F. Massi (2006). "Characterization of the dynamics
of biomacromoleculesusing rotating-frame spin relaxation NMR
spectroscopy." Chemical Reviews106(5): 1700-1719.
Igumenova, T. I., K. K. Frederick and A. J . Wand (2006).
"Characterization of the fast dynamics of protein amino acid side chains
using NMR relaxation in solution." Chemical Reviews106(5): 1672-
1699.
J arymowycz, V. A. and M. J . Stone (2006). "Fast time scale dynamics
of protein backbones: NMR relaxation methods, applications, and
functional consequences." Chemical Reviews106(5): 1624-1671.
Palmer, A. G. (2001). NMR probes of molecular dynamics: Overview
and comparison with other techniques. Annual Review of Biophysics
and Biomolecular Structure30: 129.
Palmer, A. G., C. D. Kroenkeand J . P. Loria(2001). Nuclear magnetic
resonance methods for quantifying microsecond-to-millisecond motions
in biological macromolecules. Nuclear Magnetic Resonance of
Biological Macromolecules, Pt B 339: 204.
Engelke, J . and H. Ruterjans(1999). Recent Developments in Studying
the Dynamics of Protein Structures from
15
N and
13
C Relaxation Time
Measurements. Biological Magnetic Resonance. N. R. Krishna and L. J .
Berliner. New York, Kluwer Academic/ Plenum Publishers. 17: 357-
418.
3
Fischer, M. W. F., A. Majumdar and E. R. P. Zuiderweg(1998).
Protein NMR relaxation: theory, applications and outlook. Progress in
Nuclear Magnetic Resonance Spectroscopy33(4): 207-272.
Daragan, V. A. and K. H. Mayo (1997). Motional Model Analyses of
Protein and Peptide Dynamics Using
13
C and
15
N NMR Relaxation.
Progress in Nuclear Magnetic Resonance Spectroscopy31: 63-105.
Nicholson, L. K., L. E. Kay and D. A. Torchia(1996). Protein
Dynamics as Studied by Solution NMR Techniques. NMR
Spectroscopy and Its Application to Biomedical Research. S. K. Sarkar.
Peng, J . W. and G. Wagner (1994). Investigation of protein motions
via relaxation measurements. Methods in Enzymology239: 563-96.
Wagner, G., S. Hybertsand J . W. Peng(1993). Study of Protein
Dynamics by NMR. NMR of Proteins. G. M. Cloreand A. M.
Gronenborn, CRC Press: 220-257.
Mini Reviews:
Akke, M. (2002). "NMR methods for characterizing microsecond to
millisecond dynamics in recognition and catalysis." Current Opinion in
Structural Biology12(5): 642-647.
Ishima, R. and D. A. Torchia(2000). Protein dynamics from NMR.
Nature Structural Biology7(9): 740-743.
Kay, L. E. (1998). Protein dynamics from NMR. Nature Structural
Biology5: 513-7.
Palmer, A. G., 3rd (1997). Probing molecular motion by NMR.
Current Opinion in Structural Biology7(5): 732-7.
4
Biomolecules are not static it is often Structure AND
Dynamics that determine Function:
rotational diffusion (
c
)
translational diffusion (D)
internal dynamics of backbone and sidechains(
i
)
degree of order for backbone and sidechains(S
2
)
conformational exchange (R
ex
)
interactions with other molecules (k
on
,k
off
)
Biomolecules are often not globular spheres:
anisotropy (D
xx
,D
yy
,D
zz
)
All of these parameters are accessible through NMR
measurements

c
S
2

i
R
ex
D
k
on
k
off
Types of Motion Involved in Dynamics
NMR relaxation measurements provide information on dynamics at a
wide range of time scales that is site specific:
5
time scale example experiment type
ns ps bond librations lab frame relaxation
reorientation of protein T
1
, T
2
motions of protein backbone
fast side chain rotations
us ms rapid conformational exchange lineshapeanalysis
rotating frame relax. (T
1
)
ms s interconversionof discrete magnetization exch.
conformations lineshapeanalysis
>s slow protein folding exchange rates
opening of 2
o
structures (H/D exchange)
Nuclei used to Report Protein/Nucleic Acid Dynamics Site
Specifically
1
H
15
N
13
C
2
H
31
P
Dynamics on Different Time Scales can be
Probed by Various NMR Experiments and Parameters
6
Structural Information from Relaxation
anisotropy of overall shape
distance information from cross-correlation relaxation
Thermodynamics from Relaxation
relationship to entropy and binding events
Function from Relaxation
binding sites for protein ligandinteractions
binding interfaces for protein proteininteractions
Importance of NMR relaxation measurements is underscored by
number of publications:
7
NMR Relaxation
Bloch equations introduce relaxation to account for return of
magnetization to equilibrium state:
excite
relax
treat relaxation as a first order process:
dM/dt =Mx B R(M-M
o
)
where
T
1
(longitudinal or spin-lattice relaxation time) is the time constant
used to describe rate at which M
z
component of magnetization returns to
equilibrium (the Boltzmandistribution) after perturbation.
T
2
(transverse or spin-spin relaxation time) is the time constant used
to describe rate at which M
xy
component of magnetization returns to
equilibrium (completely dephased, no coherence) after perturbation.
R =
1/T
2
0 0
0 1/T
2
0
0 0 1/T
1
8
so far, all we have is a time constant; is it possible to get a picture of
what is causing relaxation?
consider spontaneous emission of photon:
transition probability 1/
3
=10
-20
s
-1
for NMR
consider stimulated emission:
the excited state couples to the EMF inducing transitions this
phenomenon is observed in optical spectroscopy (eg. lasers) but
its effect is negligible in RF fields.
in a historic paper, Bloembergen, Purcell and Pound (Phys. Rev. 73,
679-712 (1948)) found that relaxation is related to molecular motion
they found that the NMR relaxation time varied as a function of
viscosity or temperature
they postulated that relaxation is caused by fluctuating fields
caused by molecular motion.
RF photon
NMR Relaxation, cont.
9
In other words, relaxation is dependent on motion of
molecule
Zeeman interaction is independent of molecular motion
therefore local fields must exist that are orientation
dependent and can causes relaxation:
fluctuating local fields create an oscillating field that induce
transitions between energy levels of spins
time dependence of interaction determines how efficiently
relaxation occurs
RF
source of local fields?
timescale of fluctuation?
NMR Relaxation, cont.
10
Some Relaxation Mechanisms
The relaxation of a nuclear spin is governed by the fluctuations of
local fields that result when molecules reorient in a strong external
magnetic field. Although a variety of interactions exist that can give rise
to a fluctuating local field, the dominant sources of local fields
experienced by
15
N and
13
C nuclei in biomoleculesare dipole-dipole
interactions andchemical shift anisotropy:
Magnetic Dipole-Dipole Interaction- the dipolar interaction is a
through-space coupling between two nuclear spins:

I
S
r
IS
The local field experienced by spin I is:
H
loc
=
S
h/r
3
IS
((3cos
2
1)/2)
11
Some Relaxation Mechanisms, cont.
Chemical Shift Anisotropy (CSA) - the chemical shift is due, in part,
to the distribution of electrons surrounding the nucleus and the local
magnetic field generated by these electrons as they precess under the
influence of the applied magnetic field. The effective field at the
nucleus is:
H
loc
= H
o
(1-)
where H
o
is the strength of the applied static magnetic field and is the
orientationallydependent component of the CSA tensor . The CSA
tensor determines how much the chemical shift varies with respect to
the orientation of the nucleus within the magnetic field the larger
the CSA, the larger the CSA relaxation.
12
Some Relaxation Mechanisms, cont.
In contrast to the case of the dipole-dipole interactions, the CSA
interaction constant depends on the strength of the static magnetic
field B
0
. As a consequence, the contribution of the CSA to the relaxation
rates increases with the increase of the static magnetic field strength.
Some CSA values of nuclei found in proteins:

13
C_CSA PROTEIN Cb ~32ppm

13
C_CSA PROTEIN Ca 46.5ppm

13
C_CSA PROTEIN CO 130ppm

15
N_CSA PROTEIN N -163ppm

1
H_CSA PROTEIN HN -8.9ppm
13
The additional broadening seen
in signals characterized by
exchange is given by the R
ex
term
14
Expressions for Relaxation Rates
The relaxation rate constants for dipolar, CSA and quadrupolar
interactions are linear combinations of spectral density functions, J ().
For example, one can derive the following equations for dipolar
relaxation of a heteronucleus(i.e.
15
N or
13
C) by a proton
R
1,N
= 1/T
1,N
= (d
2
/4)[J(
H
-
N
) + 3J(
N
) + 6J(
H
+
N
)]
R
2,N
= 1/T
2,N
= (d
2
/8)[4J(0) + J(
H
-
N
) + 3J(
N
) + 6J(
H
) +
6J(
H
+
N
)]
NOE
15N{1H}
= 1 + (d
2
/4)(
H
/
N
) [6J(
H
+
N
) - J(
H
-
N
)] x T
1,N
where d =(
H

N
(h/8)/r
HN
3
)
The J () terms are spectral density terms that tell us what frequency of
motions are going to contribute to relaxation. They have the form
J() =
c
/(1+
2

c
2
)
and allow the motional characteristics of the system (the correlation
time
c
) to be expressed in terms of the power available for relaxation
at a given frequency :

J()
10
7
10
8
10
9
10
10

c
=10
7

c
=10
8

c
=10
9
NOTE: maximum at J (=10
8
) term occurs
when
c
=10
-8
; motions most efficient for
inducing relaxation are
c
=1/
15
15
N Dipolar Relaxation Time as a Function of
Correlation Time
16
The Heteronuclear nOe as a
Function of Correlation Time
e.g.
15
N {
1
H}
Without NOE
S = A S = A
0 0
=1 =1
S = S =A A
0 0
+NOE= +NOE= - -4 4
NOE NOE
max max



2 2

5 5
17
Measurement of Relaxation Rates
spin lattice relaxation (T
1
) is measured using an inversion recovery
sequence:
180

=I
o
(1-2exp(-/T
1
))
18
Measurement of Relaxation Rates
spin-spin relaxation (T
2
) is measured using a spin echo sequence
(removes effect of field inhomogeneity):
90 180
I

=I
o
exp(-/T
2
)
I

19
Measurement of Relaxation Rates, cont.
The inversion-recovery sequence and spin-echo sequence
can be incorporated into a 2D
1
H-
15
N HSQC pulse sequence
in order to measure
15
N T
1
and T
2
for each crosspeakin the
HSQC:
Experimental techniques for
15
N (a) R
1
, (b) R
2
, and (c) {
1
H}
15
N NOE
spin relaxation measurements using two-dimensional, proton-detected
pulse sequences. R
1
and R
2
intensity decay curves are recorded by
varying the relaxation period T in a series of two dimensional
experiments. The NOE is measured by recording one spectrum with
saturation of
1
H magnetization and one spectrum without saturation.
20
Measurement of Relaxation Rates, cont.
Example 2D
1
H-
15
N spectra recorded with the pulse sequence used to
measure
15
N T
1
and corresponding decay curves:
21
Sample Output from
15
N Relaxation
Meaurements
22
Model Free analysis of relaxation based on Lipari, G. and A. Szabo
Model-Free Approach to the Interpretation of Nuclear Magnetic
Resonance Relaxation in Macromolecules. 1. Theory and Range of
Validity. J ournal of the American Chemical Society104: 4546 (1982).
Internal dynamics characterized by:
spatial restriction of motion of bond vector, S
2
S
2
=1 highly restricted
S
2
=0 no restriction
internal correlation time,
e
R
ex
, exchange contribution to T
2
Data Analysis
23
The spectral density terms in the relaxation equations are modified
with terms representing internal dynamics and spatial restriction of
bond vector:
The T
1
, T
2
and NOE can then be described in terms of the order
parameter (S
2
) and thecorrelation times (
m
,
e
). Analysis of relaxation
data using software package (eg. Model-Free or DASHA) allows the
dynamical parameters to be calculated:
measure:
15
N T
1
15
N T
2
15
N{
1
H} NOE
calculate
relaxation
data for a
given
m
recalculate
by varying
values of S
2
,

e
and R
ex
Compare
measured
vs. calc.
value
Data Analysis, cont.
Lipari-Szabo Model-Free Formulism
where:
m
is the overall motion of the protein

e
is the
1
H-
15
N internal motion
S
2
is the spatial restriction of internal motion (order parameter)

-1
=
e
-1
+
m
-1
If the internal motion is very rapid,
e
approaches zero.
If the internal motion is not present, S
2
approaches one.
24
Sample Output from
15
N Relaxation Analysis
Example: Using Dynamics to Probe the
Origin of Structural Uncertainty
15
N relaxation measurements
show if high RMSD is due to
high flexibility (low S
2
) or lack
of structural restraints (few
nOes)
Strong correlation
Weak correlation

26
Ribbon diagram of calbindinD9k (PDB file 2BCA).
The N-terminal (left) and C-terminal (right) EF
hand motifs are orange and green, respectively,
with the calcium-binding loops within these motifs
red and blue, respectively. Atoms involved in
calcium coordination are shown in stick
representation.
- CalbindinD9k is a small protein that contains a pair of calcium-binding
EF-hand motifs and is involved in the intracellular buffering of calcium
ions.
- observed that Ca
2+
-binding to site I reduces the mobility of both Ca
2+
-
binding motifs (i.e. site I and II) compared with the apostate
- Hypothesized that the long-range structural and dynamic changes
induced by binding of the first Ca
2+
ion lowers the free energy cost for
subsequent structural reorganization during the second binding step
consistent with observed cooperativityof calcium binding.
Akke, M., N. J . Skelton, J . Kordel, A. G. Palmer and W. J . Chazin(1993). "Effects of Ion Binding on
the Backbone Dynamics of CalbindinD9k Studied determined by 15N NMR Relaxation."
Biochemistry32: 9832-9844.
27
Example: Determining Domain Orientation in
Macromolecules Using
15
N Relaxation
Dependence of the observed
15
N T
1
/T
2
ratio on the angle between the NH
bond vectors and the unique axis of the
diffusion tensor
Tjandra, N., D. S. Garrett, et al. (1997). "Defining long range order in NMR structure
determination from the dependence of heteronuclear relaxation times on rotational diffusion
anisotropy." Nature Structural Biology4(6): 443-9.
The observed
15
N T
1
/T
2
can be used as a
restraint during structure refinement:
28
Variation in the experimentally determined backbone
15
N R
2
/R
1
ratio
versus protein sequence. Vertical bars represent the data for the unligated
(white) and ligated(black) SH(32) and for the free SH3 and SH2 domains
(hatching). Horizontal bars on the top indicate the location of the
individual domains in the Abl SH(32) dual domain sequence
Differences in the average levels of R
2
/R
1
ratio in the SH3 and SH2 parts
of the free dual domain construct, although small, indicate somedegree of
interdomainflexibility in SH(32). No significant difference was observed
for the two domains in the SH(32)/ligand complex, consistent with
restriction in the interdomainflexibility expected upon binding of the
consolidated ligand.
Fushman, D., R. Xu, et al. (1999). "Direct determination of changes of interdomainorientation on
ligation: Use of the orientational dependence of
15
N NMR relaxation in Abl SH(32)." Biochemistry
38(32): 10225-10230.
Using a SH3SH2 (SH32) segment from the human Abelson tyrosine
kinase, the relative orientation of the domains could be defined using
15
N
T
1
/T
2
for an unbound form and a form bound to a consolidated ligand.
29
Red indicates chemical shift changes
observed upon ligandbinding
Orientation dependence of relaxation data allows
positioning of domains with respect to each other.
30
Example: NMR Relaxation for Characterizing
Microsecond to Millisecond Dynamics in Catalysis
Eisenmesser, E. Z., D. A. Bosco, et al. (2002). "Enzyme dynamics during catalysis."
Science295(5559): 1520-1523.
Standard
15
N T
2
measurements were made for each residue in a peptidyl-
prolyl cis/trans isomerase, cyclophilinA, as a function of substrate
concentration (a prolyl-containing peptide) which allows characterization
of enzyme dynamics during catalysis.
Three-state model used for this study, cyclophilinA free (E), cyclophilin
A bound to substrate (ES
cis
) and cyclophilinA bound to product (ES
trans
):
31
Chemical shift changes of the amide signals in CypA upon titration with
the substrate Suc-Ala-Phe-Pro-Phe-4-NA. (A) At a constant CypA
concentration of 0.43 mM, spectra were recorded at 0 mM (blue), 0.38 mM
(orange), 1.01 mM (green), and 2.86 mM (red) substrate. The signal of
R55 is progressively shifting upon addition of increasing amounts of
substrate, indicating fast conformational exchange during catalysis. The
observed chemical shifts are population-weighted averages of E and ES,
and thus shift towards the position of the ES complex with increasing
amounts of substrate. In contrast, the signal of V139 is not affected by
catalysis. (B) The chemical shift differences between free CypA and in the
presence of 2.86 mM substrate were mapped onto the structure (1RMH)
with the use of a continuous color scale.
32
Differential build-up of backbone
15
N R
2
relaxation rates allows the
behavior of residues to be associated solely with binding (for example
K82) or with binding and isomerization(for example R55):
33
The catalytically essential arginine, R55, undergoes
conformational exchange on a timescale that corresponds well to
that of the catalyzed isomerizationreaction, strongly suggesting
that the processes are correlated.
34
Example: Changes in Side Chain Dynamics Upon
Formation of a ProteinPeptide Complex Using
2
H
Relaxation of Methyl Groups
Lee, A. L., S. A. Kinnear, et al. (2000). "Redistribution and loss of side chain entropy
upon formation of a calmodulin-peptide complex." Nature Structural Biology7(1):
72-77.
A detailed study of the complex between calcium saturated calmodulin
and a peptide model of the calmodulin-binding domain of smooth muscle
myosin light chain kinasedescribed the role of conformational entropy
changes involving side-chain motions. The backbone of calmodulinwas
found to be nearly unaffected by binding, whereas the dynamics of side
chains are significantly perturbed with an overall loss of psns time scale
mobility.
35
Using
2
H spin relaxation methods, the degree of spatial restriction of a
given methyl group was assessed via the themodel-free generalized
order parameter, S
2
. Values of the order parameter can range from 0 to
1, corresponding to isotropic disorder and a fixed orientation in the
molecular frame, respectively. For each generalized order parameter, a
corresponding effective internal correlation time (t
e
) was obtained.
36
Comparison of the total entropic cost of binding (estimated at 146 kJ
mol
-1
using relation between order parameter, S
2
, and entropy ) with the
total free energy change of complex formation (250 kJ mol
-1
) implies
considerable entropy/enthalpy compensation. The favorable enthalpic
contributions are provided by the extensive buried hydrophobic surface
that characterizes the calmodulinpeptide complex. Interestingly, despite
this global rigidification, some conserved methionineside-chains,
important for peptide recognition, exhibit significant increasesin psns
time-scale motion upon binding, reflecting a re-distribution of
conformational entropy at the protein surface.
37
Example: Characterizing Exchange Between the
Ground State and Excited State of a Protein Using
Rotating Frame
15
N and
13
C Relaxation Measurements
Mulder, F. A. A., A. Mittermaier, et al. (2001). "Studying excited states of
proteins by NMR spectroscopy." Nature Structural Biology8(11): 932-
935.
X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the
cavity is stericallyinaccessible to ligand, yet the protein is able to bind
substituted benzenes rapidly. Mulder et al. used relaxation dispersion
(rotating frame relaxation) NMR techniques to kinetically and
thermodynamically characterize a transition between a highly
populated (97%, 25 C) ground state conformation and an excited state
that is 2.0 kcal mol
1
higher in free energy. The residues involved
cluster about the cavity, providing evidence that the excited state
facilitates ligandentry.
38
Other Topics
- Field dependence
- protein unfolding
- pressure dependence
- temperature dependence