Publications by the Zuiderweg Group
at the University of Michigan (1991-2011)
Red: NMR spin-physics-related
Blue: Structural biology related
Green: Dynamics related
Bold:
hopefully of some impact
Ahmad A, Bhattacharya A, McDonald RA,
Cordes M, Ellington B, Bertelsen EB, Zuiderweg ER. Heat shock protein 70 kDa
chaperone/DnaJ cochaperone complex employs an unusual dynamic interface. Proc Natl Acad Sci U S A., 108;
18966-18971 (2011)
The Hsp70/DnaJ/NEF system assists in
intra-cellular protein (re)folding. Using solution NMR, we obtained the first
three-dimensional structure for a 75 kDa Hsp70-DnaJ complex in the ADP state,
loaded with substrate peptide. We establish that the J-domain (residues 1-70)
binds with its positively charged helix II to a negatively charged loop in the
Hsp70 nucleotide-binding domain. The complex shows an unusual “tethered” binding mode which is
stoichiometric and saturable, but which has a dynamic interface. The complex
represents part of a triple complex of Hsp70 and DnaJ both bound to substrate
protein. Mutagenesis data indicates that the interface is also of relevance for
the interaction of Hsp70 and DnaJ in the ATP state. The solution complex is
completely different from a crystal structure of a disulfide-linked complex of
homologous proteins (Jiang et al., 2007, Mol. Cell. 28:422-433).
Zuiderweg
E.R.P, Rousaki, A.
Gradient-enhanced TROSY described with Cartesian product operators Concepts in Magnetic
Resonance, 38A, 280-288 (2011)
TROSY, Transverse Relaxation
Optimized Spectroscopy, was developed more than a decade ago. Since that time,
the 15N-1H HSQC-TROSY experiment has become the standard
“fingerprint” correlation spectrum for proteins of high molecular weight. In
addition, its implementation in protein triple resonance experiments has pushed
the boundaries of NMR assignment up to about 100 kDa, making NMR a highly
relevant technique in structural biology.
TROSY exploits the dipole-CSA cross-correlated relaxation properties of
the NH system and selects for the narrowest of the HSQC J-correlation quartet
in both dimensions. The original publications and reviews of TROSY use shift
operators and/or single transition product operators to describe the TROSY coherence pathways selections. In this
review we offer a familiar Cartesian product operator approach to
comprehensively describe all of the events in the modern TROSY pulse sequence
such as multiplet selection, gradient coherence selection, gradient quadrature
and sensitivity enhancement.
Rousaki A, Miyata Y, Jinwal UK, Dickey
CA, Gestwicki JE, Zuiderweg ER. Allosteric drugs: the interaction of antitumor
compound MKT-077 with human Hsp70 chaperones. J Mol Biol., 411, 614-632. (2011)
The Hsp70 chaperones (Heat shock protein 70
kDa) are key to
cellular protein homeostatis. However, they also have the ability to inhibit
tumor apoptosis, and contribute to aberrant accumulation of hyperphosphorylated tau in
neuronal cells affected by tauopathies, including Alzheimer’s disease. Hence,
Hsp70 are increasingly been identified as targets for therapeutic intervention
in these widely abundant diseases.
Hsp70 proteins are allosteric machines and offer besides classical
active site targets, also opportunities to target the mechanism of allostery. In this work, it is demonstrated that the action of the
potent anti-cancer compound MKT-077, is through differential interaction with
the Hsp70 allosteric states. MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-
oxothiazolidin-2-ylidenemethyl] pyridinium chloride) is therefore an
“allosteric drug”. Using NMR spectroscopy, the compound’s binding site on human
HSPA8 (Hsc70) is identified. The binding pose is obtained from NMR-restrained
docking calculations, subsequently scored by molecular dynamics-based energy
and solvation computations.
Suggestions for improvement of the compound’s properties are made on the
basis of the binding location and pose.
Bagai, I., Ragsdale,
S.W. and Zuiderweg, E.R.P. Pseudo-4D triple resonance experiments
to resolve HN overlap in the
backbone assignment of unfolded proteins., J Biomol NMR.
49:69-74 (2011)
The solution NMR resonance
assignment of the protein backbone is most commonly carried out using triple
resonance experiments that involve 15N and 1HN
resonances. The assignment becomes
problematic when there is resonance overlap of 15N -1HN
cross peaks. For such residues,
one cannot unambiguously link the ”left” side of the NH root to the “right”
side, and the residues associated with such overlapping HN resonances remain
often unassigned. Here we present a solution to this problem: a hybrid (4d, 3d)
reduced-dimensionality HN(CO)CA(CON)CA sequence. In this experiment, the Ca(i) resonance
is modulated with the frequency of the Ca(i-1) resonance, which helps in resolving the
ambiguity involved in connecting the Ca(i) and Ca(i-1) resonances for overlapping NH roots. The experiment has limited sensitivity,
and is only suited for small or unfolded proteins. In a companion experiment, (4d, 3d) reduced-dimensionality HNCO(N)CA, the Ca(i) resonance is modulated with the frequency of the CO(i-1)
resonance, hence resolving the ambiguity existent in pairing up the Ca(i) and CO(i-1)
resonances for overlapping NH roots.
Crippen, G.M., Rousaki, A, Revington,
M., Zhang, Y. and Zuiderweg, E.R.P.SAGA: Rapid automatic mainchain NMR
assignment for large proteins J.
Biomol. NMR 46 281-298. (2010 )
Here we describe a new algorithm for automatically
determining the mainchain sequential assignment of NMR spectra for proteins.
Using only the customary triple resonance experiments, assignments can be
quickly found for not only small proteins having rather complete data, but also
for large proteins, even when only half the residues can be assigned. The
result of the calculation is not the single best assignment according to some
criterion, but rather a large number of satisfactory assignments that are
summarized in such a way as to help the user identify portions of the sequence
that are assigned with confidence, vs. other portions where the assignment has
some correlated alternatives. Thus very imperfect initial data can be used to
suggest future experiments.
Bhattacharya, A., Revington, M., and Zuiderweg, E.R.P. Measurement and interpretation of 15N-1H residual dipolar couplings in larger proteins J. Magn. Reson. 203 11–28, (2010)
A decade ago, Dr. L.E. Kay and
co-workers described an ingenious HNCO-based triple resonance experiment from
which several protein backbone RDCs can be measured simultaneously [1]. They implemented a J-scaling technique in the 15N dimension
of the 3D experiment to obtain the NH RDCs. We have used this idea to carry out J-scaling in a 2D 15N-1H
TROSY experiment and have found it to be an excellent method to obtain NH RDCs
for larger proteins upto 70 kDa, far superior to commonly used HSQC
in-phase/anti-phase and HSQC/TROSY comparisons. Here, this method, dubbed
“RDC-TROSY” is discussed in detail and the limits of its utility are assessed
by simulations. Prominent in the latter analysis is the evaluation of the
effect of amide proton flips on the “RDC-TROSY” linewidths. The details of the
technical and computational implementations of these methods for the
determination of domain-orientations in 45-60 KDa Hsp70 chaperone protein
constructs are described.
Weaver DS, Zuiderweg ER
Protein proton-proton dynamics from amide proton spin flip rates. J Biomol NMR.
2009 45:99-119. Residue-specific amide proton
spin-flip rates K were measured for peptide-free and peptide-bound calmodulin.
K approximates the sum of NOE build-up rates between the amide proton and all
other protons. This work outlines the theory of multi-proton relaxation, cross
relaxation and cross correlation, and how to approximate it with a simple model
based on a variable number of equidistant protons. This model is used to
extract the sums of K-rates from the experimental data. Error in K is estimated
using bootstrap methodology. We define a parameter Q as the ratio of
experimental K-rates to theoretical K-rates, where the theoretical K-rates are
computed from atomic coordinates. Q is 1 in the case of no local motion, but
decreases to values as low as 0.5 with increasing domination of sidechain
protons of the same residue to the amide proton flips. This establishes Q as a
monotonous measure of local dynamics of the proton network surrounding the
amide protons. The method is applied to the study of proton dynamics in Ca(2+)-saturated
calmodulin, both free in solution and bound to smMLCK peptide. The mean Q is
0.81 +/- 0.02 for free calmodulin and 0.88 +/- 0.02 for peptide-bound
calmodulin. This novel methodology thus reveals the presence of significant
interproton disorder in this protein, while the increase in Q indicates
rigidification of the proton network upon peptide binding, confirming the known
high entropic cost of this process.
Bhattacharya A, Kurochkin AV, Yip GN,
Zhang Y, Bertelsen EB, Zuiderweg ER. Allostery in Hsp70 chaperones is
transduced by subdomain rotations. J Mol Biol. 2009 388:475-90
Hsp70s (heat shock protein 70
kDa) are central to protein folding, refolding, and trafficking in organisms
ranging from archaea to Homo sapiens under both normal and stressed cellular
conditions. Hsp70s are comprised of a nucleotide-binding domain (NBD) and a
substrate-binding domain (SBD). The nucleotide binding site in the NBD and the
substrate binding site in the SBD are allosterically linked: ADP binding
promotes substrate binding, while ATP binding promotes substrate release.
Hsp70s have been linked to inhibition of apoptosis (i.e., cancer) and diseases
associated with protein misfolding such as Alzheimer's, Parkinson's, and
Huntington's. It has long been a goal to characterize the nature of allosteric
coupling in these proteins. However, earlier studies of the isolated NBD could
not show any difference in overall conformation between the ATP state and the
ADP state. Hence the question: How is the state of the nucleotide communicated
between NBD and SBD? Here we report a solution NMR study of the 44-kDa NBD of
Hsp70 from Thermus thermophilus in the ADP and AMPPNP states. Using the
solution NMR methods of residual dipolar coupling analysis, we determine that
significant rotations occur for different subdomains of the NBD upon exchange
of nucleotide. These rotations modulate access to the nucleotide binding cleft
in the absence of a nucleotide exchange factor. Moreover, the rotations cause a
change in the accessibility of a hydrophobic surface cleft remote from the
nucleotide binding site, which previously has been identified as essential to
allosteric communication between NBD and SBD. We propose that it is this change
in the NBD surface cleft that constitutes the allosteric signal that can be
recognized by the SBD.
Bertelsen EB, Chang L, Gestwicki JE,
Zuiderweg ER. Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone
complexed with ADP and substrate. Proc Natl Acad Sci U S A. 2009;106:8471-6.
DnaK is the canonical Hsp70 molecular chaperone protein from
Escherichia coli. Like other Hsp70s, DnaK comprises two main domains: a 44-kDa
N-terminal nucleotide-binding domain (NBD) that contains ATPase activity, and a
25-kDa substrate-binding domain (SBD) that harbors the substrate-binding site.
Here, we report an experimental structure for wild-type, full-length DnaK,
complexed with the peptide NRLLLTG and with ADP. It was obtained in aqueous
solution by using NMR residual dipolar coupling and spin labeling methods and
is based on available crystal structures for the isolated NBD and SBD. By using
dynamics methods, we determine that the NBD and SBD are loosely linked and can
move in cones of +/-35 degrees with respect to each other. The linker region
between the domains is a dynamic random coil. Nevertheless, an average
structure can be defined. This structure places the SBD in close proximity of
subdomain IA of the NBD and suggests that the SBD collides with the NBD at this
area to establish allosteric communication.
Weaver,
D.S and Zuiderweg, E.R.P. hz/k: a TROSY NMR
experiment measuring longitudinal relaxation interference. J Chem
Phys,128 155103 (14 pg) (2008)
NMR
spin relaxation experiments provide a powerful tool for the measurement of
global and local biomolecular rotational dynamics at sub-nanosecond time
scales. Technical limitations
restrict most spin relaxation studies to biomolecules weighing less than 10
kDa, considerably smaller than the average protein molecular weight of 30
kDa. In particular, experiments
measuring hz, the
longitudinal 1HN-15N dipole-dipole (DD)/15N
chemical shift anisotropy (CSA) cross-correlated relaxation rate, are among
those least suitable for use with larger biosystems. This is unfortunate
because these experiments yield valuable insight into the variability of the 15N
CSA tensor over the polypeptide backbone, and this knowledge is critical to the
correct interpretation of most 15N-NMR backbone relaxation
experiments, including R2 and R1. In order to remedy this
situation, we present a new 1HN-15N TROSY
experiment measuring hz suitable for applications
with larger proteins (up to at least 30 kDa). The presented experiment also yields k, the
site-specific rate of longitudinal 1HN-1H’ dipole-dipole
cross-relaxation. We describe the hz/k
experiment’s performance in protonated human ubiquitin at 30.0 °C and
in protonated calcium-saturated calmodulin/peptide complex at 20.0 °C,
and demonstrate preliminary experimental results for a deuterated E. coli DnaK
ATPase domain construct at 34 °C.
Jordan, D.M., Mills, K.M., Andricioaei, I., Bhattacharya, A., Palmo, K. and Zuiderweg, ERP. Parameterization of peptide 13C carbonyl chemical shielding anisotropy in molecular dynamics simulations. Chemphyschem. 8, 1375-1385 (2007).
NMR chemical shielding
anisotropy (CSA) relaxation is an important tool in the study of dynamical
processes in proteins and nucleic acids in solution. Here we investigate how
dynamical variations in local geometry affect the chemical shielding anisotropy
relaxation of the carbonyl nucleus, using the following protocol. (i) Using
Density Functional Theory, the carbonyl 13C’ CSA is computed for 103
conformations of the model peptide group N-methylacetamide (NMA) (ii) The variations
in computed 13C’ CSA parameters were fitted against quadratic
hyper-surfaces containing cross terms between the variables. (iii) The
predictive quality of the CSA hyper-surfaces was validated by comparing the
predicted and de-novo calculated 13C’ CSAs for 20 molecular dynamics
snapshots. (iv) The CSA
fluctuations and their auto- and cross-correlation functions due to bond-length
and bond-angle distortions were predicted for a CHARMM molecular dynamics trajectory
of Ca++ saturated calmodulin and GB3 from the hyper-surfaces, as
well as for a MD simulation of an NMR trimer using a quantum mechanically
correct forcefield. We find that the fluctuations can be represented by a 0.93
scaling factor of the CSA tensor for both R1 and R2
relaxation for residues in helix, coil and sheet alike. This result is
important, as it establishes that 13C’ relaxation is a valid tool
for measurement of interesting dynamical events in proteins.
Wang,
T., Weaver, D.S., Cai, S., Zuiderweg, E.R.P. Quantifying Lipari-Szabo modelfree
parameters from 13CO NMR relaxation experiments. J Biomol NMR. 36, 79-102.
(2006)
It is proposed to obtain effective Lipari-Szabo order parameters and local
correlation times for relaxation vectors of protein 13CO nuclei by
carrying out a 13CO-R1 auto relaxation experiment, a
transverse 13CO CSA / 13CO-13Ca CSA /
dipolar cross correlation and a transverse 13CO CSA / 13CO-15N
CSA / dipolar cross correlation experiment. Given the global rotational correlation time from 15N
relaxation experiments, a new program COMFORD (CO-Modelfree Fitting Of
Relaxation Data) is presented to fit
the 13CO data to
an effective order parameter S2CO, an effective
local correlation time and the orientation of the CSA tensor with respect to
the molecular frame. It is shown that the effective S2CO
is least sensitive to rotational fluctuations about an imaginary Ca-Ca axis
and most sensitive to rotational fluctuations about an imaginary axis parallel
to the NH bond direction. As such, the S2CO information
is fully complementary to the 15N relaxation order parameter, which
is least sensitive to fluctuations about the NH axis and most sensitive to
fluctuations about the Ca-Ca axis.
The new paradigm is applied on data of Ca2+ saturated Calmodulin,
and on available literature data for Ubiquitin. Our data indicate that the S2CO order
parameters rapport on slower, and sometimes different, motions than the 15N
relaxation order parameters. The CO local correlation times correlate well with
the calmodulin’s secondary structure.
Wang, J, Wang, T, Zuiderweg, E.R.P. and Crippen, G. CASA: An
Efficient Automated Assignment of Protein Mainchain NMR Data using an Ordered
Tree Search Algorithm. J. Biomol NMR. 33, 261-279
(2005).
Rapid analysis of
protein structure, interaction, and dynamics requires fast and automated
assignments of 3D protein backbone triple-resonance NMR spectra. We introduce a
new depth-first ordered tree search method of automated assignment, CASA, which
uses hand-edited peak-pick lists of a flexible number of triple resonance
experiments. The computer program was tested on experimental data for
ubiquitin calmodulin and
GrpE, and also on 13 artificially
simulated peak lists for proteins up to 723 residues. Within a few minutes of
CPU time it generated assignments that correspond to the ones reported in the
literature. The program was then tested on the proteins analyzed by other
methods using their tolerances, and it could generate good assignments in all
relevant cases. The robustness was further tested under various situations.
Yip, G.N.B. and Zuiderweg, E.R.P. Duty-Cycle Heating
Compensation in NMR Relaxation Experiments, J. Magnetic
Resonance, 176, 171-178 (2005).
To reliably measure NMR relaxation properties of macromolecules is a
prerequisite for precise experiments that identify subtle variations in
relaxation rates, as required for the determination of rotational diffusion
anisotropy, CSA tensor determination, advanced motional modeling or entropy
difference estimations. An underlying problem with current NMR relaxation
measurement protocols is maintaining constant sample temperature throughout the
execution of the relaxation series especially when rapid data acquisition is
required. Here, it is proposed to
use a combination of a heating compensation and a proton saturation sequence at the beginning of the NMR
relaxation pulse scheme. This simple extension allows reproducible, robust and
rapid acquisition of NMR spin relaxation data sets. The method is verified with
15N spin relaxation measurements for human ubiquitin.
Deep,
S., Im, S.C., Zuiderweg, E.R.P., and Waskell, L. Characterization and
Calculation of a cytochrome c-cytochrome b5 complex using NMR data Biochemistry
44, 10654-10668 (2005)
The
interaction between bovine cytochrome b5 (cyt b5) and horse-heart
cytochrome c
(cyt c) is
investigated by NMR spectroscopy.
Chemical shifts of cyt b5 backbone resonances and side chain methyl resonances
were monitored as a function of cyt c concentration. The shifts are small but
saturatable and indicate that the binding of cyt b5 with cyt c is in fast
exchange. An equilibrium
association constant of 6±3 x 104 M-1 was obtained with a lower limit
of 180 s-1 for the dissociation rate of the complex. To resolve
considerable ambiguities in the interpretation of the chemical shift mapping, 15N
relaxation experiments and cross-saturation experiments were used as
alternative methods to map the cyt b5-cyt c binding interface. Results from the three experiments
combined demonstrate that the conserved negatively charged region of cyt b5
surrounding the solvent exposed heme edge is involved in the interaction with
cyt c.
Revington, M., Zhang,
Yip, G.N.B., Kurochkin, A.V. and Zuiderweg, E.R.P. NMR investigations of
allosteric processes in a two-domain Thermus Thermophilus Hsp70 molecular
chaperone, J. Mol. Biol 349, 163-183 (2005)
Hsp70 chaperones are two-domain proteins which
assist in intra-cellular protein (re) folding processes in all species. The
protein folding activity of the substrate binding domain of the Hsp70’s
is regulated by nucleotide binding at the nucleotide binding domain through an
as yet undefined heterotropic allosteric mechanism. The available
structures of the isolated domains of Hsp70’s have given very limited
indications of nucleotide-induced conformational changes that could modulate
the affinity for substrate proteins. Here we present a multi-dimensional NMR
study of a prokaryotic Hsp70 homologue, Thermus thermophilus DnaK, using a 54
kDa construct containing both nucleotide binding domain and most of the
substrate binding domain. It is determined that the nucleotide
binding domain and substrate binding domain are closely associated in all
ligand states studied. Comparison of the assigned NMR spectra of the two-domain
construct with those of the previously studied isolated nucleotide binding
domain, allowed the identification of the nucleotide binding domain - substrate
binding domain interface. A global three-dimensional structure was obtained for
the two-domain construct on the basis of this information and of NMR residual
dipolar couplings measurements. This is the first experimental elucidation of
the relative positioning of the nucleotide binding domain and substrate binding
domain for any Hsp70 chaperone.
Comparisons of NMR data between various ligand states including
nucleotide free, ATP, ADP.Pi and ADP.Pi + peptide bound, identified residues
involved in the allosteric inter-domain communication. In particular, peptide
binding to the substrate binding domain was found to cause conformational
changes in the NBD extending to the nucleotide binding pocket. Detailed
analysis suggests that the inter-domain interface becomes tighter in the
(nucleotide binding domain ligation / substrate binding domain ligation) order
ATP/apo, ADP.Pi/apo ADP.Pi/peptide.
Wang,
T, King Frederick, K., Igumenova, T.I., Wand, A.J. and Zuiderweg, E.R.P.
Changes in Calmodulin backbone dynamics upon ligand binding revealed by
cross-correlated NMR relaxation measurements, J. Am.Chem.Soc. 127,
828-829 (2005)
Our findings indicate that the investigation
of protein backbone dynamics by NMR spectroscopy should be expanded to
routinely include dynamical information derived from 13CO-13Cα cross-correlated
relaxation experiments; this holds especially true if subtle changes in
dynamical properties, summed over many residues, are to be evaluated in terms
of change of conformational entropy.
Zhang, Y. and Zuiderweg, E.R.P. The Hsc70 chaperone
nucleotide binding domain in solution unveiled as a molecular machine that can
reorient its functional subdomains Proc Natl Acad Sci U S A. 2004,
101:10272-10277
The Hsc70 chaperone plays a crucial role in protein
(re-)folding and triage in the mammalian cytosol. Here we study, by NMR,
the 44 kDa nucleotide-binding domain of this molecule which regulates, by
binding either ADP or ATP in a cleft between two main lobes, the chaperoning
affinity of the attached substrate-binding domain. The nucleotide-binding
domain is also a center of interaction with co-chaperones that couple into the
allostery. By measuring residual dipolar couplings by NMR, we show that the
orientation of two lobes of the Hsc70 nucleotide-binding domain in solution
deviate upto 10 degrees from their positions in 14 superimposing X-ray
structures. Additional orientational differences of subdomains within the
lobes, unveil the Hsc70 nucleotide binding domain in solution as a flexible
molecular machine that can adjust the relative positions of all of its 4
subdomains. Since the residues interacting with the nucleotide emanate from all
four subdomains, adjustments in subdomain orientation should affect the
nucleotide chemistry and vice-versa. Our data suggests the hypothesis that
co-chaperone or substrate-domain binding perturbs the relative subdomain
orientations, thereby functionally and allosterically coupling to the
nucleotide state of the nucleotide-binding domain.
Revington, M. and
Zuiderweg, E.R.P. NMR study of
nucleotide-induced changes in the nucleotide binding domain of Thermus
Thermophilus Hsp70 chaperone DnaK: implications for the allosteric mechanism J
Biol Chem. 2004 279, 33958-33967
We present an NMR investigation of the
nucleotide dependent conformational properties of a 44 kDa nucleotide binding
domain (NBD) of an Hsp70 protein. Conformational changes
driven by ATP binding and hydrolysis in the N-terminal NBD are believed to
allosterically regulate substrate affinity in the C-terminal substrate binding
domain. Several crystal structures of Hsc70 NBD's in different
nucleotide states have, however, not shown significant structural
differences. We have previously reported the NMR assignments of the
backbone resonances of the NBD of the bacterial Hsp 70 homologue Thermus
thermophilus DnaK in the ADP bound state. In this study we
show, by assigning the NBD with the ATP/transition state analogue, ADP-AlFx,
bound, that it closely mimics the ATP-bound state. Chemical shift
difference mapping of the two nucleotide states identified differences in
a cluster of residues at the interface between subdomains 1A and
1B. Further analysis of the spectra revealed that the ATP state
exhibited a single conformation while the ADP state was in slow conformational
exchange between a form similar to the ATP state and another state unique to
the ADP bound form. A model is proposed of the allosteric mechanism based
on the nucleotide state altering the balance of a dynamic equilibrium between
the open and closed states.
Revington, M. &
Zuiderweg, E.R.P. TROSY-driven NMR backbone assignments of the 381-residue
nucleotide-binding domain of the Thermus Thermophilus DnaK molecular chaperone.
J. Biomol. NMR. 30, 113-4 (2004)
Backbone assignments were made at 55 0C, pH 7.4
for 333 of the 362 non-proline residues in the native sequence (92%) using 3D
HNCA-TROSY, HNCOCA-TROSY, HNCOCA-TROSY, HNCO TROSY, HNCACB-TROSY at 800 MHz
using perdeuterated protein. The spectra collected at pH 6.0 helped in
the assignment of 10 surface residues whose connectivities were too weak at pH
7.4. Examination of the HNCA and HNCACB spectra indicated
that D44 had undergone an aspartate to isoaspartate isomerization.
Yip, G. and Zuiderweg, E.R.P. A phase cycle
scheme that significantly suppresses offset-dependent artifacts in the R2-CPMG
15N relaxation experiment.” J.Magn. Reson. 171, 25-36 (2004)
It has been known for some time that in the
practical limit of finite pulse widths, which becomes acute when using
cryogenic probes, systematic errors in the apparent R2 relaxation behavior
occur for spins far off resonance from the RF carrier. Inaccurate measurement
of R2 rates propagates into quantitative models such as model-free relaxation
analysis, rotational diffusion tensor analysis and relaxation dispersion. The
root of the problem stems from evolution of the magnetization vectors out of
the XY-plane, both during the pulses as well as between the pulses. These
deviations vary as a function of pulse length, number of applied CPMG pulses,
and CPMG inter-pulse delay. Herein, we analyze these effects in detail
with experimentation, numerical simulations and analytical equations. Our work
suggests a surprisingly simple change in the phase progression of the CPMG
pulses, which leads to a remarkable improvement in performance. First, the
applicability range of the CPMG experiment is increased by a factor of two in
spectral width; secondly, the dynamical/kinetic processes that can be assessed
are significantly extended towards the slower time scale; finally, the
robustness of the relaxation dispersion experiments is greatly improved.
Kern, D. and Zuiderweg, E.R.P. The role of dynamics in allosteric
regulation, Current Opinion in Structural Biology, 13, 748-757 (2003)
The biomolecular conformational changes often associated
with allostery are, by definition, dynamic processes. Recent publications have
disclosed the role of pre-existing equilibria of conformational substates in
this process. In addition, the role of dynamics as an entropic carrier of free
energy of allostery has been investigated. Recent work thus shows that dynamics
is pivotal to allostery, and that it constitutes much more than just the move
from the ‘T’-state to the ‘R’-state. Emerging computational studies have
described the actual pathways of allosteric change.
Shao, W., Im, S.-C., Zuiderweg, E.R.P.* and
Waskell, L.* Mapping the Binding Interface of the Cytochrome b5- Cytochrome c
Complex by NMR Biochemistry 42, 14774-14784 (2003)
The interaction between bovine cytochrome b5
(cyt b5) and horse-heart cytochrome c (cyt c) is investigated by NMR
spectroscopy. Chemical shifts of cyt b5 backbone resonances and side
chain methyl resonances were monitored as a function of cyt c concentration.
The shifts are small but saturatable and indicate that the binding of cyt b5
with cyt c is in fast exchange. An equilibrium association constant of
6±3 x 104 M-1 was obtained with a lower limit of 180 s-1 for the dissociation
rate of the complex. To resolve considerable ambiguities in the interpretation
of the chemical shift mapping, 15N relaxation experiments and cross-saturation
experiments were used as alternative methods to map the cyt b5-cyt c binding
interface. Results from the three experiments combined demonstrate that
the conserved negatively charged region of cyt b5 surrounding the solvent
exposed heme edge is involved in the interaction with cyt c.
Stevens,S.Y., Cai, S., Pellecchia, M., Zuiderweg, E.R.P. The solution structure
of the bacterial HSP70 Chaperone protein domain DnaK(393-507) in complex with
the peptide NRLLLTG. Protein Science 12, 2588-2596 (2003)
The Hsp70 family of molecular chaperones
participates in a number of cellular processes, including binding to nascent
polypeptide chains and assistance in protein (re)folding and degradation. We
present the solution structure of the substrate binding domain (residues
393–507) of the Escherichia coli Hsp70, DnaK, that is bound to the peptide
NRLLLTG and compare it to the crystal structure of DnaK(389–607) bound to the
same peptide. The construct discussed here does not contain the -helical
domain that characterizes earlier published peptide-bound structures of the
Hsp70s. It is established that removing the -helical domain in its
entirety does not affect the primary interactions or structure of the DnaK(393–507)
in complex with the peptide NRLLLTG. In particular, the arch that protects the
substrate-binding cleft is also formed in th absence of the helical lid.
15N-relaxation measurements show that the peptide-bound form of DnaK(393– 507)
is relatively rigid. As compared to the peptide-free state, the peptide-bound
state of the domain shows distinct, widespread, and contiguous differences in
structure extending toward areas previously defined as important to the
allosteric regulation of the Hsp70 chaperones.
Cai, S., Stevens, S.Y., Budor,
A.P. and Zuiderweg, E.R.P. Solvent interaction of
a Hsp70 chaperone substrate-binding domain investigated with Water-NOE NMR
experiments Biochemistry 42, 11100-11108 (2003)
The interaction of solvent of the substrate binding
domain of the bacterial heat shock 70 chaperone protein DnaK was studied in its
apo form and with bound hydrophobic substrate peptide, using refined nuclear
magnetic resonance experiments. Distinct differences between the two states of
the protein were observed. According to our data, the apo form interacts more
extensively with solvent than the peptide-bound form. Significantly, the open
hydrophobic substrate binding cleft of DnaK in the apo form is found to contain
several molecules of water which are displaced by the binding of the
hydrophobic substrate, the peptide NRLLLTG. The solvent in the hydrophobic
cleft has a residence time longer than 400 ps. It is predicted that the
displacement of this trapped water must contribute to the binding free energy
of the natural hydrophobic substrates of this class of protein-folding
chaperone proteins.
Wang, T., Cai, S. and
Zuiderweg, E.R.P. Temperature dependence of
anisotropic protein backbone dynamics J. Am.
Chem. Soc. 125, 8639-8643 (2003).
The measurement of 15N NMR spin relaxation,
which reports the 15N-1H vector reorientational dynamics, is a widely
used experimental method to assess the motion of the protein backbone. Here, we
investigate whether the 15N-1H vector motions are representative of the overall
backbone motions, by analyzing the temperature dependence of the 15N-1H and
13CO-13CR reorientational dynamics for the small proteins binase and ubiquitin.
The latter dynamics were measured using NMR cross-correlated relaxation
experiments. The data show that, on average, the 15N-1H order parameters
decrease only by 2.5% between 5 and 30 °C. In contrast, the 13CO-13CR order
parameters decrease by 10% over the same temperature trajectory. This strongly
indicates that there are polypeptide-backbone motions activated at room
temperature that are not sensed by the 15N-1H vector. Our findings are at
variance with the common crank-shaft model for protein backbone dynamics, which
predicts the opposite behavior. This study suggests that investigation of the
15N relaxation alone would lead to underestimation of the dynamics of the
protein backbone and the entropy contained therein.
Khandelwal, P., Keliikuli, K., Smith, C.L., Saper,
M.A and Zuiderweg, E.R.P. Solution structure and phosphopeptide
binding to the N-terminal domain of Yersinia YopH, Comparison with a Crystal
Structure . Biochemistry, 41, 11425-11437 (2002).
Virulence of pathogenic bacteria of the genus
Yersinia requires the injection of six effector proteins into the cytoplasm of
host cells. The amino-terminal domain of one of these effectors, the tyrosine
phosphatase YopH, is essential for translocation of YopH, as well as for
targeting it to phosphotyrosinecontaining substrates of the type pYxxP. We
report the high-resolution solution structure of the N-terminal domain
(residues 1-129) from the Yersinia pseudotuberculosis YopH (YopH-NT) in complex
with N-acetyl-DEpYDDPF-NH2, a peptide derived from an in vivo protein
substrate. In contrast to the domainswapped dimer observed in a crystal
structure of the same protein (Smith, C. L., Khandelwal, P., Keliikuli, K.,
Zuiderweg, E. R. P., and Saper, M. A. (2001) Mol. Microbiol. 42, 967-979),
YopH-NT is monomeric in solution. The peptide binding site is located on a
â-hairpin that becomes the crossover point in the dimer structure. The binding
site has several characteristics that are reminiscent of SH2 domains, which
also bind to pYxxP sequences.
Pang, A, Buck, M., and Zuiderweg, E.R.P. Backbone Dynamics of the
Ribonuclease Binase Active Site Area using Multinuclear (15N and 13CO) NMR
Relaxation and Computational Molecular Dynamics, Biochemistry, 41,
2655-2666 (2002)
The nano-pico second backbone dynamics of the
ribonuclease binase, homologous to barnase, is investigated with 15N, 13C NMR
relaxation at 11.74 and 18.78 T and with a 1.1 ns molecular dynamics
simulation. The data are compared with the temperature factors reported for the
X-ray structure of this enzyme. The molecular dynamics and X-ray data
correspond well and predict motions in the loops 56- 61 and 99-104 that contain
residues that specifically recognize substrate and are catalytic (His101),
respectively. In contrast, the 15N relaxation data indicate that these loops
are mostly ordered at the nanopico second time scale. Nano-pico second motions
in the recognition loop 56-61 are evident from 13CO-13CR cross relaxation data,
but the mobility of the catalytic loop 99-104 is not detected by 13CO cross
relaxation either. From the results of this and previous work [Wang, L., Pang,
Y., Holder, T., Brender, J. R., Kurochkin, A., and Zuiderweg, E. R. P. (2001)
Proc. Natl. Acad. Sci. U.S.A., 98, 7684-7689], the
following dynamical characterization of the active site area of binase emerges:
a beta sheet, rigid at all probed time scales, supports the catalytic residue
Glu 72. Both substrate-encapsulating loops are mobile on both fast and slow
time scales, but the fast motions of the loop which contains the other
catalytic residue, His 101, as predicted by B-factors and computational
molecular dynamics is not detected by NMR relaxation. This work strongly argues
for the use of several measures in the study of protein dynamics.
Zuiderweg, E.R.P. Mapping of protein-protein
interactions in solution by NMR spectroscopy, Biochemistry, 41, 1-7
(2002).
Stevens, S.Y., Sanker, S., Kent, C. and
Zuiderweg, E.R.P. Delineation of the allosteric mechanism for a
cytidylyltransferase exhibiting negative cooperativity, Nature Structural
Biology 8, 947-952 (2001)
The dimeric enzyme CTP:glycerol-3-phosphate
cytidylyltransferase (GCT) displays strong negative cooperativity between the
first and second binding of its substrate, CTP. Using NMR to study the
allosteric mechanism of this enzyme, we observe widespread chemical shift
changes for the individual CTP binding steps. Mapping these changes onto the
molecular structure allowed the formulation of a detailed model of allosteric
conformational change. Upon the second step of ligand binding, NMR experiments
indicate an extensive loss of conformational exchange broadening of the
backbone resonances of GCT. This suggests that a fraction of the free energy of
negative cooperativity is entropic in origin.
Hall, D.A., Vander Kooi, C.W., Stasik, C.N.,
Stevens, S.Y., Zuiderweg, E.R.P., and Matthews, R.G., Mapping the Interactions
Between Flavodoxin and Its Physiological Partners Flavodoxin Reductase and
Cobalamin-dependent Methionine Synthase, Proc Natl Acad Sci U S A. 98,
9521-9526. (2001)
Flavodoxins are electron-transfer proteins that
contain the prosthetic group flavin mononucleotide. In Escherichia coli,
flavodoxin is reduced by the FAD-containing protein NADPH:ferredoxin
(flavodoxin) oxidoreductase; flavodoxins serve as electron donors in the
reductive activation of anaerobic ribonucleotide reductase, biotin synthase,
pyruvate formate lyase, and cobalamin-dependent methionine synthase. In
addition, domains homologous to flavodoxin are components of the multidomain
flavoproteins cytochrome P450 reductase, nitric oxide synthase, and methionine
synthase reductase. Although three-dimensional structures are known for many of
these proteins and domains, very little is known about the structural aspects
of their interactions. We address this issue by using NMR chemical shift
mapping to identify the surfaces on flavodoxin that bind flavodoxin reductase
and methionine synthase. We find that these physiological partners bind to
unique overlapping sites on flavodoxin, precluding the formation of ternary
complexes. We infer that the flavodoxin-like domains of the cytochrome P450
reductase family form mutually exclusive complexes with their electron-donating
and -accepting partners, complexes that require conformational changes for
interconversion.
Khandelwal, P, Keliikuli, K., Saper, M.A. and Zuiderweg, E.R.P. 1H,
15N and 13C assignments of the N-terminal domain of Yersinia outer protein H,
J. Biomol. NMR, 21, 69-70 (2001)
Wang, L., Pang, Y., Holder, T., Brender,
J.R., Kurochkin, A, Zuiderweg, E.R.P. Functional Dynamics in the active site of
the ribonuclease Binase, Proc. Natl. Acad. Sci. USA, 2001, 98, 7684-7689 (2001)
Binase, a member of a family of microbial guanyl-specific
ribonucleases, catalyzes the endonucleotic cleavage of single-stranded RNA. It
shares 82% amino acid identity with the well-studied protein barnase. We used
NMR spectroscopy to study the millisecond dynamics of this small enzyme, using
several methods including the measurement of residual dipolar couplings in
solution. Our data show that the active site of binase is flanked by loops that
are flexible at the 300-ms time scale. One of the catalytic residues, His-101,
is located on such a flexible loop. In contrast, the other catalytic residue,
Glu-72, is located on a b-sheet, and is static. The residues Phe-55, part of
the guanine base recognition site, and Tyr-102, stabilizing the base, are the
most dynamic. Our findings suggest that binase possesses an active site that
has a well-defined bottom, but which has sides that are flexible to facilitate
substrate accessyegress, and to deliver one of the catalytic residues. The
motion in these loops does not change on complexation with the inhibitor
d(CGAG) and compares well with the maximum kcat (1,500 s21) of these
ribonucleases. This observation indicates that the NMR-measured loop motions
reflect the opening necessary for product release, which is apparently rate
limiting for the overall turnover.
Pellecchia, M. Vander Kooi, C.W. , Keliikuli,
K., and Zuiderweg, E.R.P. Magnetization Transfer via Residual Dipolar
Couplings: Application to Proton-Proton Correlations in Partially Aligned
Proteins, J. Magn. Reson. 143, 435-439 (2000)
A novel three-dimensional NMR experiment is
reported that allows the observation of correlations between amide and other
protons via residual dipolar couplings in partially oriented proteins. The
experiment is designed to permit quantitative measurement of the magnitude of
proton–proton residual dipolar couplings in larger molecules and at higher
degree of alignments. The observed couplings contain data valuable for protein resonance
assignment, local protein structure refinement, and determination of
low-resolution protein folds.
Pang, Y. and Zuiderweg, E.RP.
Determination of Protein Backbone 13CO Chemical Shift Anisotropy Tensors in
Solution, J.Am. Chem.Soc. 122, 4841-4842 ( 2000) We obtain complete parametrization of peptide backbone 13CO
chemical shift anisotropy (CSA) tensors for a protein in solution, by measuring
the interference (cross correlation) between the 13CO CSA transverse
relaxation and three different dipole−dipole relaxations, 13CO−13Cα,
13CO−15N, and 13CO−1HN for all
sites.
Wang, L. Kurochkin, A.V. and
Zuiderweg, E.R.P. An iterative fitting procedure for the determination of
longitudinal NMR cross-correlation rates. J. Magn. Reson., 144,
175-185 (2000) We present a method to measure
N-15-H-1 dipolar/N-15 CSA longitudinal cross-correlation rates in protonated
proteins. The method depends on the measurement of four observables: the
cumulative proton-proton cross relaxation rates, the N-15 R-1 relaxation rate,
the multiexponential decay of 2N(Z)H(Z)(N) spin-order, and multiexponential
buildup of 2N(Z)H(Z)(N) spin-order. The N-15-H-1 dipolar/N-15 CSA longitudinal
cross-correlation rate is extracted from these measurements by an iterative
fitting procedure to the solution of differential equations describing the
coupled relaxation dynamics of the z-magnetization of the N-15 nucleus, the
two-spin-order 2N(Z)H(Z)(N), and a two-spin-order term 2N(Z)H(Z)(Q) describing
the interaction with remote protons. The method is applied to the microbial
ribonuclease binase. The method can also extract longitudinal cross-correlation
rates for those amide protons that are involved in rapid solvent exchange. The
experiment that serves for extracting proton-proton cross-relaxation rates is a
modification of 3D N-15-resolved NOESY-HSQC. The experiment restores the
solvent magnetization to its equilibrium state during data detection for all
phase cycling steps and all values of NOE mixing times and is also recommended
for use in standard applications.
Pellecchia, M., Stevens, S.Y., Vander Kooi,
C.W., Montgomery, D.H., Feng, E.H., Gierasch, L.M., and Zuiderweg,
E.R.P. Structural insights into substrate binding by the
molecular chaperone DnaK. Nature Structural Biology,7, 298- 303 (2000)
How substrate affinity is modulated by nucleotide binding
remains a fundamental, unanswered question in the study of 70 kDa heat shock
protein (Hsp70) molecular chaperones. We find here that the Escherichia
coliHsp70, DnaK, lacking the entire a-helical domain, DnaK(1–507),
retains the ability to support l phage replication in vivoand to pass
information from the nucleotide binding domain to the substrate binding domain,
and vice versa, in vitro. We determined the NMR solution structure of the corresponding
substrate binding domain, DnaK(393–507), without substrate, and assessed the
impact of substrate binding. Without bound substrate, loopL3,4 and strand b3
are in significantly different conformations than observed in previous
structures of the bound DnaK substrate binding domain, leading to occlusion of
the substrate binding site. Upon substrate binding, the b-domain shifts towards
the structure seen in earlier X-ray and NMR structures. Taken together, our
results suggest that conformational changes in the b-domain itself contribute
to the mechanism by which nucleotide binding modulates substrate binding
affinity.
Vander Kooi, C.W., Kupce, E., Zuiderweg, E.R.P.,
and Pellecchia, M.. Line Narrowing in Spectra of Proteins Dissolved in a Dilute
Liquid Crystalline Phase by Band-Selective Adiabatic Decoupling: Application to
1HN_15N Residual Dipolar Coupling Measurements, J. Biomol. NMR. 15, 335-338
(1999)
Residual heteronuclear dipolar couplings
obtained from partially oriented protein samples can provide unique NMR
constraints for protein structure determination. However, partial orientation
of protein samples also causes severe 1H line broadening resulting from
residual 1H-1H dipolar couplings. In this communication we show that
band-selective 1H homonuclear decoupling during data acquisition is an
efficient way to suppress residual 1H-1H dipolar couplings, resulting in
spectra that are still amenable to solution NMR analysis, even with high
degrees of alignment. As an example, we present a novel experiment with
improved sensitivity for the measurement of onebond 1HN-15N residual dipolar
couplings in a protein sample dissolved in magnetically aligned liquid
crystalline bicelles.
Pellecchia, M, Pang, Y, Wang, L.,
Kurochkin, A.V., Anil Kumar and Zuiderweg, E.R.P. Quantitative Measurement of
Cross-Correlations Between 15N and 13CO Chemical Shift Anisotropy Relaxation
Mechanisms by Multiple Quantum NMR, J. Am. Chem. Soc. 121, 9165-9170 (1999)
This paper describes an experiment that allows the
quantitative measurement of the CSA−CSA cross-correlation between backbone 15N
and 13CO nuclei in uniformly enriched proteins. The CSA−CSA
cross-correlation is obtained from the cross-peak intensity ratios of the double-
and the zero-quantum components observed with a modified triple-quantum 2D
CT-HNCO experiment. In addition, the 1HN−15N/1HN−13CO
dipole−dipole cross-correlation was measured without relying on resolved scalar
couplings using a complementary quantum 2D CT-HNCO experiment. The
cross-correlation rates measured for the protein binase (12.3 kDa) were
obtained with high precision but show a surprisingly large range of values.
Calculations show that this range is at least partially caused by dynamical processes.
The potential use of this information to characterize internal anisotropic
motion is discussed.
Morshauser, R.C. and Zuiderweg, E.R.P.
High Resolution Four-Dimensional HMQC-NOESY-HSQC Spectroscopy, J. Mag.
Reson,139, 232-239, 1999
Practical optimization of the 4D [H-1, C-13, C-13,
H-1] HMQC-NOESY-HSQC experiment in terms of distribution of resolution over the
indirect dimensions is analyzed in detail. Recommendations for an optimal
experiment are based on computer simulations assessing the effective resolution
of the experiment, defined as the percentage of all possible NOE cross peaks
that can be assigned unambiguously on the basis of the spectral data alone.
Using actual C-13-H-1 spectra of an 1.8-kDa chaperone protein, the analysis
shows that experiments with the best effective resolution are also among the
most sensitive ones. When combined with an efficient aliasing scheme that
reduces indirect spectral space 124-fold, a 4D experiment that yields
unambiguous assignments for 41% of all possible NOE cross peaks can be recorded
in 28 h. A high-resolution experiment, which can be recorded in 8 days, yields
61% unambiguous assignments and can be analyzed more easily using standard NMR
display software. The predictions are verified with experimental 4D spectra
from which 1850 NOEs (914 long-range) were extracted for the 1.8-kDa chaperone
protein.
Morshauser, R.C., Hu, W., Wang, H., Pang, Y.,
Flynn, G.C. and Zuiderweg, E.R.P. High resolution solution structure of the 18
kda substrate binding domain of the mammalian chaperone protein hsc70. J. Mol.
Biol, 289, 1387-1403 (1999) The
three-dimensional structure for the substrate-binding domain of the mammalian
chaperone protein Hsc70 of the 70 kDa heat shock class (HSP70) is presented. This
domain includes residues 383–540 (18 kDa) and is necessary for the binding of
the chaperone with substrate proteins and peptides. The high-resolution NMR solution structure is based on 4150 experimental distance
constraints leading to an average root-mean-square precision of 0.38 Å for the
backbone atoms and 0.76 Å for all atoms in the beta-sandwich sub-domain. The
protein is observed to bind residue Leu539 in its hydrophobic substrate-binding
groove by intramolecular interaction. The position of a helical latch differs
dramatically from what is observed in the crystal and solution structures of
the homologous prokaryotic chaperone DnaK. In the Hsc70 structure, the helix lies in a hydrophobic
groove and is anchored by a buried salt-bridge. Residues involved in this salt-bridge
appear to be important for the allosteric functioning of the protein. A
mechanism for interdomain allosteric modulation of substrate-binding is
proposed. It involves large-scale movements of the helical domain, redefining
the location of the hinge area that enables such motions.
Pang, Y., Wang, L., Pellecchia, M., Kurochkin,
A.V. and Zuiderweg, E.R.P. Evidence for extensive anisotropic local motions in
a small enzyme using a new method to determine NMR cross-correlated relaxation
rates in the absence of resolved scalar coupling, J. Biomol. 14, 297-306 (1999)
Transverse (CO)-C-13-(HN)-H-1
(dipole-dipole)/(CO)-C-13 (CSA) cross-correlated relaxation rates were measured
for the (CO)-C-13 resonances of the protein ribonuclease Binase from Bacillus
intermedius (12.3 kDa). This was carried out with a novel E.COSY-type
triple-resonance experiment, which allows the measurement of cross-correlated
transverse relaxation rate from multiplet effects in the absence of resolved
scalar coupling. The (CO)-C-13-(HN)-H-1 (dipole-dipole)/(CO)-C-13 (CSA)
cross-correlated relaxation rates were determined with an average precision of
+/- 5% and cover a range of values between -1.5 and +0.6 Hz. The average (-0.44
Hz) is to be compared with the computed value of -0.83 Hz for this interaction.
Mechanisms that potentially can cause the average to be smaller than the
theoretical value and the unexpected large spread in observed values are
discussed. It is suggested that large contributions to the variations are due
to large amplitude local anisotropic motions.
Fischer, M.W.F., Majumdar, A. and Zuiderweg, E.R.P.
Protein NMR relaxation: theory, applications and outlook, Progress in NMR
Spectrosc, 33, 207-272 (1998) Review of relaxation
theory including cross correlations
Fischer, M.W.F., Lei Zeng, L., Majumdar, A. and
Zuiderweg, E.R.P., Characterizing Semi-Local Motions in Proteins by NMR
Relaxation Studies, Proc. Natl. Acad. Sci, USA, 95, 8016-8019 (1998)
The understanding of protein function is incomplete without
the study of protein dynamics. NMR spectroscopy is valuable for probing
nanosecond and picosecond dynamics via relaxation studies. The use of 15N
relaxation to study backbone dynamics has become virtually standard. Here, we
propose to measure the relaxation of additional nuclei on each peptide plane
allowing for the observation of anisotropic local motions. This allows the
nature of local motions to be characterized in proteins. As an example,
semilocal rotational motion was detected for part of a helix of the protein
Escherichia coli flavodoxin.
Pang, Y., Zeng, L., Kurochkin,
A.V. and Zuiderweg, E.R.P. High-Resolution Detection of Five Frequencies
in a Single 3D Spectrum: HNHCACO - a Bi-directional Coherence Transfer
Experiment. J. Biomol. NMR, 11, 185-190 (1998)
Wang, H., Pang, Y, Kurochkin, A.V., Hu, W., Flynn,
G.C, and Zuiderweg, E.R.P. The solution structure of the 21 kDa
chaperone protein DnaK substrate binding domain: a preview of
chaperone – protein interaction. Biochemistry 37 , 7929-7940 (1998)
The solution
structure of the 21 kDa substrate-binding domain of the Escherichia coli
Hsp70-chaperone protein DnaK (DnaK 386-561) has been determined to a precision
of 1.00 Angstrom (backbone of the beta-domain) from 1075 experimental
restraints obtained from multinuclear, multidimensional NMR experiments. The
domain is observed to bind to its own C-terminus and offers a preview of the
interaction of this chaperone with other proteins. The bound protein region is
tightly held at a single amino acid position (a leucyl residue) that is buried
in a deep pocket lined with conserved hydrophobic residues. A second
hydrophobic binding site was identified using paramagnetically labeled
peptides. It is located in a region close to the N-terminus of the domain and
may constitute the allosteric region that links substrate-binding affinity with
nucleotide binding in the Hsp70 chaperones.
Fischer, M. W.F., Zeng, L., Pang, Y., Hu, W.,
Majumdar, A. and Zuiderweg, E.R.P.Experimental characterization of models
for backbone pico-second dynamics in proteins. Quantification of NMR
auto- and cross correlation relaxation mechanisms involving different nuclei of
the peptide plane. J. Am. Chem. Soc. 119, 12629-12642 (1997)
NMR relaxation parameters were measured
for the peptide-plane carbonyl and nitrogen nuclei for the protein E.
Coli Flavodoxin. A poor correlation between the general order
parameters of the C'-Ca vector (Zeng, L.; Fischer, M.W.F. ;
Zuiderweg, E.R.P. J. Biomol. NMR 1996, 7, 157-162 ) and the N-NH vector
was observed. We interpret this lack of correlation in this nearly spherical
protein as evidence of local or semi-local anisotropic
motion. A new experiment is introduced from which the cross
correlation between the carbonyl chemical shift anisotropy relaxation and
carbonyl-Ca dipole-dipole relaxation is obtained. We show theoretically
that the three relaxation measurements, reporting on the dynamics of the C'-Ca
vector, N-NH vector and CSA tensor components behave differently towards
under anisotropic motion. The cross-correlation order parameter formalism
for dipolar cross-correlation spectral densities, as introduced by
Daragan and Mayo (Daragan, V.A. ; Mayo, K.H., J. Magn. Reson B 1995, 107,
274-278), has been extended to include cross correlations between non-axial
chemical shift anisotropy and dipole-dipole relaxation. By analyzing our
experimental data with the theoretical models for anisotropic local
motion, dynamic models were obtained for the peptide planes of 32 residues
of E. Coli Flavodoxin.
Pang, Y, Zeng, L., Kurochkin, A.V. and Zuiderweg, E.R.P.
High-Resolution Detection of five frequencies in a single 3D spectrum: HNHCACO
- a Bi-directional coherence transfer experiment J. Biomol. NMR, 11, 185-190
(1998)
Cain, R.J., Glick, G.D. and Zuiderweg, E.R.P.
Extracting quantitative information from two- and three-dimensional NOE spectra
measured with short recycle delays. J. Magn. Reson. B 133, 252-255 (1996)
Cross
peaks in NOE spectra are important sources of information for NMR structure
determinations. The meaningful quantitation of NOE cross-peak volumes requires
the data to be recorded with a recycle delay between scans of at least five
times the longest 1H
nonselective longitudinal relaxation rate in the molecule. This assures thermal
equilibrium between scans and thus provides for a known boundary condition
for the differential relaxation
equations. Unfortunately, the use of such long recycle delays is rarely practical
when a series of 2D NOE spectra
with different mixing times is required. it certainly becomes prohibitively
time consuming when a series of 3D NOE data is to be collected for the
measurement of NOE buildup rates in larger molecules. Herein we present a
simple but robust protocol to alleviate this dilemma by allowing quantitative
cross-peak integrals to be extracted from a series of NOE spectra
collected a short recycle delay by
normalizing the series with a
single NOE spectrum recorded with a long recycle delay.
Fischer, M.W.F., Zeng, L. and Zuiderweg, E.R.P.
Use of 13C-13C NOE for the assignment of NMR lines of larger labeled proteins
at larger magnetic fields J. Am. Chem. Soc., 49, 12457-12458 (1996) It is demonstrated that it is possible to obtain good
3D (H)CCH-NOESY spectra using a 5 mm sample of a 1.2 mM solution of
a 21 kDa protein using a 600 MHz spectrometer.
Hu,
W. and Zuiderweg, E.R.P. Stereo-specific assignments of Val and Leu methyl
groups in a selectively 13C labeled 18 kDa polypeptide using 3D CT-(H)CCH-COSY
and 2D 1JCC edited heteronuclear correlation experiments, J. Magn. Reson.,
B113, 70-75 (1996)
The diastereotopic methyl groups of Val and Leu
were labeled selectively with 13C for a 18 kDa domain of the chaperone protein
Hsc-70, using the protocol described by D. Neri, T. Szyperski, G. Otting,
H. Senn, and K. Wüthrich (Biochemistry, 28, 7510-7516, 1989). For
the Hsc-70 domain, complete 1H and 13C stereo-specific assignments could not be
obtained using the recommended regular HSQC experiment due to severe
resonance overlap. Here, we propose to edit or resolve the 13C-1H
heteronuclear correlated spectrum in two or three dimensions using the
1JC-C coupling constant, thus facilitating the stereo-specific resonance
assignment.
Zuiderweg, E.R.P., Zeng, L., Brutscher, B. and
Morshauser, R.C. "Band-Selective Hetero- and Homo-nuclear Cross
Polarization Using Trains of Shaped Pulses" J. Biomol. NMR 8, 147-160
(1996)
The performance of solution cross polarization
using trains of shaped pulses on two channels is investigated by computer
simulation and experiment. It is determined that a Waltz modulation
pattern of Gaussian pulses of individual flip angles of 225 degrees, issued to
two coupled spins simultaneously, yields excellent coherence transfer with good
phasing behavior. Simulations and experimental verification were carried out
for both heteronuclear cross polarization between two restricted areas
(e.g. 1Ha-13Ca) and for homonuclear cross polarization between two spectral
regions (e.g. 13CO-13Ca). It is shown that shaped cross
polarization behaves as pure heteronuclear cross polarization when
the two r.f. fields are far apart, while it behaves in some aspects
analogous to homonuclear cross polarization when the two r.f. fields approach
eachother. The novel coherence transfer sequence, referred to as Cosine
modulated Shaped Waltz (CSW), was implemented in a 3D (H)C(CCO)NH
experiment using an 18 kDa isotopically labeled protein.
Zeng, L.,
Fischer, M.W.F. and Zuiderweg, E.R.P. Study of Protein Dynamics in Solution by
Measurement of 13Ca-13CO NOE and 13CO longitudinal relaxation. J. Biomol. NMR,
1996; 7, 157-162
13Ca -13CO homonuclear NOE and 13CO T1 relaxation are
measured for a 20 kDa protein using triple resonance pulse sequences. The
experiments are sufficiently sensitive to obtain statistically significant
differences in relaxation parameters over the molecule. The 13Ca -13CO cross
relaxation rate, obtained from these data, is directly proportional to an order
parameter describing local motion and largely independent of the local
correlation time. It is therefore a relatively straightforward observable
for the identification of local dynamics.
Van Doren, S.W., Kurochkin, A.V., Hu, W., Ye, Q.Z.,
Johnson, L.L., Hupe, D.J. and Zuiderweg, E.R.P. Solution structure of the
catalytic domain of human stromelysin complexed with a hydrophobic
inhibitor. Protein Science 1995; 4, 2487-2498
Stromelysin, a representative matrix
metalloproteinase and target of drug development efforts, plays a prominent
role in the pathological proteolysis associated with arthritis and secondarily
in that of cancer metastasis and invasion. To provide a structural template to
aid the development of therapeutic inhibitors, we have determined a
medium-resolution structure of a 20 kDa complex of human stromelysin's
catalytic domain with a hydrophobic peptidic inhibitor using multi-nuclear,
multi-dimensional nuclear magnetic resonance spectroscopy (NMR).
This domain of this zinc hydrolase contains a mixed b sheet comprising one
antiparallel strand and four parallel strands, three helices, and a
methionine-containing turn near the catalytic center. The ensemble of 20
structures was calculated using, on average, eight interresidue NOE restraints
per residue for the 166 residue protein fragment complexed with a four residue
substrate analog. The mean RMSD to the average structure for backbone
heavy atoms is 0.91 Å and for all heavy atoms is 1.42 Å. The structure
has good stereochemical properties, including its backbone torsion
angles. The b sheet and a helices of the catalytic domains of human
stromelysin (NMR model) and human fibroblast collagenase (X-ray
crystallographic model of Lovejoy et al., 1994b) superimpose well, having a
pairwise RMSD for backbone heavy atoms of 2.28 Å when three loop segments are
disregarded. The hydroxamate-substituted inhibitor binds across the
hydrophobic active site of stromelysin in an extended conformation. The
first hydrophobic side chain is deeply buried in the principal S1' subsite.
The second hydrophobic side chain is located on the opposite side of the
inhibitor backbone in the hydrophobic S2' surface subsite while a third
hydrophobic side chain (P3’) lies at the surface.
Sandusky, P., Wooten, E.W., Kurochkin, A.V.,
Mandecki, W. and Zuiderweg, E.R.P. Occurrence, Solution Structure and Stability
of DNA hairpins Stabilized by a CG/GA helix unit. Nucleic Acids Research,
1995; 23, 4717-4725
Morshauser, R.C., Wang, H., Flynn, G.C and Zuiderweg,
E.R.P. The peptide binding domain of the chaperone-protein Hsc70 has an unusual
secondary structure topology. Biochemistry Accelerated, 1995; 34,
6261-6266
Modern NMR methods were used to determine the
secondary structure topology of the 18 kDa peptide binding domain of the
chaperone protein Hsc70 in solution. This report constitutes the first
experimental conformational information on this important domain of the class
of Hsp70 proteins. The domain consists of two four-stranded anti-parallel beta
sheets and a single alpha helix. The topology does not resemble at all the
topology observed in the human leukocyte antigen (HLA) proteins of the major
histocompatibility complex. This is significant because such resemblance was
predicted on the basis of limited amino acid homology, secondary structure
prediction and related function. Moreover, the exact meander-type beta-sheet
topology identified in Hsc70 has to our best knowledge not been observed
in any other known protein structure.
Wang, H. and
Zuiderweg, E.R.P. HCCH-TOCSY spectroscopy of 13C-labeled proteins in H2O using
heteronuclear cross polarization and pulsed-field gradients J. Biomol. NMR
1995; 5, 207-211
A pulsed-field gradient enhanced, heteronuclear cross
polarization driven, 3D HCCH-TOCSY experiment is described, which in a single
scan can achieve nearly ideal solvent suppression for protein samples in H2O
solution. The 3D experiment can be transformed without additional
pre- or post-processing thus leaving solute resonances at the solvent-resonance
position undisturbed and easily identifiable. As the gradients are used
in combination with a 13C z-filter, only minimal relaxation losses are
encountered as compared to non-gradient versions.
Fischer, M.W.F., Majumdar, A., Dahlquist, F.W.
and Zuiderweg, E.R.P. 15N, 13C and 1H Assignments and Secondary Structure for
T4-lysozyme J. Mag. Res. 1995; B108, 143-154
Beckman, R.A. and Zuiderweg, E.R.P. Guidelines
for the use of oversampling in protein NMR J. Magn. Reson. 1995; , A 113,
223-231
Majumdar, A. and Zuiderweg, E.R.P.
Efficiencies of Double and triple-resonance J- cross polarization in
multidimensional NMR. J. Magn. Reson. 1995; A 113,
19-31. It
is generally observed that the performance of Double resonance
multiple-pulse-based J-Cross Polarization (DCP) is superior to pulsed-free-precession
(INEPT) based sequences for net-transfer of coherence between scalar
coupled spins. Here, effects of relaxation and radiofrequency field
inhomogeneity on transfer efficiency are analysed forboth methods. It is found
that relaxation differences are relatively small between INEPT and DCP. Rf
inhomogeneity effects were found to significantly favor DCP over
INEPT,contributing to the observed experimental differences in performance
between the two methods.The differences suggest that triple resonance cross
polarization (TCP) between three coupled spins should yieldbetter results
than analogous INEPT-based net coherence transfers. The
possibilities of TCP are theoretically analyzed by deriving the transfer
functions for this type ofexperiment. It is found that the TCP
transfer efficiency is low except in the case of equal scalar couplings. To
widen the applications of the potentially interesting TCP method, a scheme
involving a concatenation of triple and double resonance CP is introduced
(concatenated CP or CCP). It is theoretically derived that such a
sequence can be tuned to achieve complete in-phase transfer for all ratios of
scalar couplings. The transfer times in this scheme are shown to be somewhat
shorter than that required for optimally concatenated INEPT in-phase
transfers.The transfer efficiencies of CCP are verified with a 3D HACA(N)NH
experiment, in which the CA-N-(N)H
transfer is driven by the CCP scheme. The experiment was carried out with
labeled T4-lysozyme (19 kD). The CCP experiment has much higher
sensitivity than a version where the CA-N-(N)H transfer is driven by an INEPT
scheme.
Zuiderweg, E.R.P. Multi-dimensional
multi-nuclear high-resolution NMR of biomolecules in: Encyclopedia of
Analytical Science, Academic Press, London, 1995, pp 3568-3578.
Van Doren, S.W. and Zuiderweg, E.R.P.
Improvement in HSMQC-Type Double and Triple Resonance NMR experiments by using
full sweep (semi-) constant-time shift labeling. J. Magn. Reson. 1994; B 104,
193 - 198
Zuiderweg, E.R.P. and Majumdar, A. Modern
Multi-dimensional Protein NMR spectroscopy. Part 2. Trends in Analytical
Chemistry, 1994; 13, 73-81.
Zuiderweg, E.R.P. and Van Doren, S.R. Modern
Multi-dimensional Protein NMR spectroscopy. Part 1. Trends in Analytical
Chemistry, 1994; 13, 24-36.
Zuiderweg, E.R.P., Van Doren, S.R., Kurochkin,
A.V., Neubig, R.R. and Majumdar, A. Modern NMR spectroscopy of proteins and
peptides in solution and its relevance to drug design. Perspectives in Drug
Discovery and Design, 1993, 1, 391-417.
Van Doren, S.W., Kurochkin, A.V., Ye, Q.Z.,
Johnson, L.L., Hupe, D.J. and Zuiderweg, E.R.P. Assignments for the main chain
nuclear magnetic resonances and delineation of the secondary structure of
the catalytic domain of human stromelysin-1 as obtained from triple resonance
3D NMR experiments. Biochemistry 1993; 32, 13109-13122.
Majumdar, A. and Zuiderweg, E.R.P. Improved
13C-resolved HSQC-NOESY spectra in H2O, using pulsed field gradients. J.
Magnetic Resonance 1993; B 102, 242-244
Majumdar, A., Wang, H., Morshauser, R. and Zuiderweg,
E.R.P. Sensitivity improvement in 2D and 3D HCCH spectroscopy
using heteronuclear cross polarization J. Biomol. NMR 1993; 3,
387-397
A new method, which employs a sequence of heteronuclear-homonuclear-heteronuclear
Hartmann-Hahn (HEHOHEHAHA) cross-polarization steps for obtaining through-bond
H-C-C-H correlations in larger proteins (M, ~ 15 kDa), is presented. The method
has significantly higher sensitivity compared to INEPT- HOHAHA-INEPT-based
techniques. An additional feature of this experiment is that well-phaseable
spectra may be obtained with a minimal (4-step) phase cycle and, consequently,
experimental time can be utilized towards obtaining high resolution in indirect
dimensions. Results from 2D and 3D HEHOHEHAHA expenments on T4-lysozyme are
presented.
Van Doren, S.W. and Zuiderweg E.R.P.
An auxiliary RF channel with convenient phase control for NMR
spectrometers J. Magn. Reson.1993; A104, 222-225
Wang, H., Glick, G., and Zuiderweg, E.R.P.
A three-dimensional method for the separation of zero-quantum-coherence and NOE
in NOESY spectra. J. Magn. Reson. 1993; A102, 116-121 We demonstrate an experiment in which zero-quantum
J-correlation peaks in NOESY spectra are placed in a third dimension.