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 1HN15N/1HN13CO 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.

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