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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 2005, in press
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.

Hinton, A., Zuiderweg, E.R.P. and Ackerman, S.H. A Purified Subfragment of Yeast Atp11p Retains Full Molecular Chaperone Activity, J. Biol. Chem. 278, 34110-34113 (2003)
 
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.

Huber-Lang, M.S., Sarma, J.V., McGuire, S.R., Lu, K.T., Padgaonkar, V.A., Younkin, E.M., Guo, R.F., Weber, C.H., Zuiderweg, E.R.P., Zetoune, F.S., Ward, P.A. Structure -Function Relationships of Human C5a and C5aR  J. Immunol.  170, 6115-6124 (2003)

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.

Chung, D.A., Ramamoorthy, A., Zuiderweg , E.R.P., Neubig, R.R. NMR Structure of the Second Intracellular Loop of the a2A Adrenergic Receptor: Evidence for a Novel Cytoplasmic Helix Biochemistry, Biochemistry, 41, 3596-3604 (2002).

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).

Smith, C.L., Khandelwal, P., Keliikuli, K., Zuiderweg, E.R.P., and  Saper, M.A. Structure of the Type III Secretion and  Substrate-Binding Domain of the Yersinia YopH Phosphatase. Mol Microbiol. 42, 967-979. (2001)

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)


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.R.P. Determination of Protein Backbone 13CO Chemical Shift Anisotropy Tensors in Solution, J.Am. Chem.Soc. 122,  4841-4842 ( 2000)
 

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)
 

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, loop
L3,4 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)
 

Morshauser, R.C.  and Zuiderweg, E.R.P. High Resolution Four-Dimensional HMQC-NOESY-HSQC Spectroscopy, J. Mag. Reson,139, 232-239, 1999
 

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)
 

Stevens, S.Y., Hu, W., Gladysheva, T., Rosen, B.P., Zuiderweg, E.R.P.  and Lee, L. Secondary Structure and Fold Homology of the ArsC Protein  from the Escherichia coli Arsenic Resistance Plasmid R773, Biochemistry, 38, 10178-10186 (1999)
 

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)
 

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)
 

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)
 
 

Ziehler, W.A., Yang, Z., Kurochkin, A.V., Sandusky, P.O., Zuiderweg, E.R.P.and Engelke, D.R. Structural Analysis of the P10/11-P12 RNA Domain of Yeast Nuclear RNase P RNA and its Interaction with Magnesium, Biochemistry,  37, 3549-3557 (1998 )
 
 
 

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)
 

Park, Y-S., Gee, P., Sanker, S., Schurter, E.J., Zuiderweg, E.R.P. and Kent C. Identification of functional conserved residues of CTP:glycerol-3-phosphate-cytidylyltransferase: Role of histidines in the conserved HXGH in catalysis. J. Biol. Chem. 272, 15161-15166 (1997)

Buchler, N., Wang, H., Zuiderweg, E.R.P. and Goldstein, R.A. Protein Heteronuclear NMR Assignments using Mean-field Simulated Annealing, J. Magn. Reson. 125, 34-42 (1997)

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)

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.
 

Osborne, S.E., Cain, R.J., Goodwin, J.T., Stevens, S.Y., Wang, H., Zuiderweg, E.R.P. and Glick, G,D. Structure and Dynamics of disulfide cross-linked nucleic acids. In Biological Structure and Dynamics, Vol 2. Eds. Sarma, R.H. and Sarma, M.H. Adenine Press, Albany 1996, 217-231

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

Cain, R.J., Zuiderweg, E.R.P. and Glick, G.D. Solution structure of a DNA hairpin and its disulfide cross-linked analog.  Nucleic Acids Research,  1995; 12, 2153-2160

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

Wang, H., Zuiderweg, E.R.P. and Glick, G. The solution structure of a disulfide-stabilized DNA  hairpin. J.Am. Chem. Soc. 1995; 117, 2981-2991

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  

Wang, H., Osborne, S.E., Zuiderweg, E.R.P. and Glick, G. Three dimensional structure of a disulfide-stabilized non-ground-stateDNA hairpin. J. Am. Chem. Soc. 1994; 116, 5021-5022

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
 

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