Backbone dynamics of the oligomerization domain of p53 determined from 15N NMR relaxation measurements.

The backbone dynamics of the tetrameric p53 oligomerization domain (residues 319-360) have been investigated by two-dimensional inverse detected heteronuclear 1H-15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and heteronuclear NOEs were measured for 39 of 40 non-proline backbone NH vectors at both field strengths. The overall correlation time for the tetramer, calculated from the T1/T2 ratios, was found to be 14.8 ns at 35 degrees C. The correlation times and amplitudes of the internal motions were extracted from the relaxation data using the model-free formalism (Lipari G, Szabo A, 1982, J Am Chem Soc 104:4546-4559). The internal dynamics of the structural core of the p53 oligomerization domain are uniform and fairly rigid, with residues 327-354 exhibiting an average generalized order parameter (S2) of 0.88 +/- 0.08. The N- and C-termini exhibit substantial mobility and are unstructured in the solution structure of p53. Residues located at the N- and C-termini, in the beta-sheet, in the turn between the alpha-helix and beta-sheet, and at the C-terminal end of the alpha-helix display two distinct internal motions that are faster than the overall correlation time. Fast internal motions (< or = 20 ps) are within the extreme narrowing limit and are of uniform amplitude. The slower motions (0.6-2.2 ns) are outside the extreme narrowing limit and vary in amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

Backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from 15N NMR relaxation measurements.

The backbone dynamics of the uniformly 15N-labeled IIA domain of the glucose permease of Bacillus subtilis have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured, at a spectrometer proton frequency of 500 MHz, for 137 (91%) of the 151 protonated backbone nitrogens. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The T1 and T2 values for most residues were in the ranges 0.45-0.55 and 0.11-0.15 s, respectively; however, a small number of residues exhibited significantly slower relaxation. Similarly, (1H)-15N NOE values for most residues were in the range 0.72-0.80, but a few residues had much smaller positive NOEs and some exhibited negative NOEs. The molecular rotational correlation time was 6.24 +/- 0.01 ns; most residues had order parameters in the range 0.75-0.90 and tau e values of less than ca. 25 ps. Residues found to be more mobile than the average were concentrated in three areas: the N-terminal residues (1-13), which were observed to be highly disordered; the loop from P25 to D41, the apex of which is situated adjacent to the active site and may have a role in binding to other proteins; and the region from A146 to S149. All mobile residues occurred in regions close to termini, in loops, or in irregular secondary structure.     

The role of backbone conformational heat capacity in protein stability: temperature dependent dynamics of the B1 domain of Streptococcal protein G

The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15N NMR-derived order parameters. Longitudinal (R1) and transverse (R2) relaxation rates, transverse cross-relaxation rates (eta(xy)), and steady state [1H]-15N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50 degrees C for 89-100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R2/eta(xy) was used to identify nuclei for which conformational exchange makes a significant contribution to R2. Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter (S2) was used to calculate the contribution of each NH group to conformational heat capacity (Cp) and a characteristic temperature (T*), representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to approximately 0.8 kJ/mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be approximately 9 degrees C lower (78 degrees C rather than 87 degrees C). Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.     

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197].     

Analysis of the backbone dynamics of interleukin-1 beta using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy

The backbone dynamics of uniformly 15N-labeled interleukin-1 beta are investigated by using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. 15N T1, T2, and NOE data at a spectrometer frequency of 600 MHz are obtained for 90% of the backbone amide groups. The data provide evidence for motions on three time scales. All the residues exhibit very fast motions on a time scale of approximately less than 20-50 ps that can be characterized by a single-order parameter with an average value of 0.82 +/- 0.05. For a model comprising free diffusion within a cone, these residue-specific order parameters translate to an average cone semiangle of 20.7 +/- 3.3 degrees. Thirty-two residues also display motions on a time scale of 0.5-4 ns, slightly less than the overall rotational correlation time of the protein (8.3 ns). These additional motions must be invoked to account for the discrepancy between experiment and the simplest theoretical formulation in which the internal motions are described by only two parameters, a generalized order parameter and an effective correlation time [Lipari, G., & Szabo, A. (1982a) J. Am. Chem. Soc. 104, 4546-4559]. In particular, while the simple formulation can account for the 15N T1 and T2 data, it fails to account for the 15N-1H NOE data and yields calculated values for the NOEs that are either too small or negative, whereas the observed NOEs are positive. With the introduction of two internal motions that are faster than the rotational correlation time and differ in time scales by at least 1-2 orders of magnitude [Clore, G. M., Szabo, A., Bax, A., Kay, L. E., Driscoll, P. C., & Gronenborn, A. M. (1990) J. Am. Chem. Soc. 112, 4989-4991], all the relaxation data for these 32 residues can be fitted by two order parameters and an effective correlation time for the slower of the two internal motions. A simple model for these two motions is one in which the very fast motion involves axially symmetric diffusion within a cone, while the slower motion comprises jumps between two different orientations of the NH vector. For such a model the jump angle (excluding the C-terminal residue) ranges from 15 degrees to 69 degrees with a mean value of 28.6 +/- 14.0 degrees. Another 42 residues are characterized by some sort of motion on the 30-ns-10-ms time scale, which results in 15N line broadening due to chemical exchange between different conformational substates with distinct 15N chemical shifts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Guanine nucleotides differentially modulate backbone dynamics of the STAS domain of the SulP/SLC26 transport protein Rv1739c of Mycobacterium tuberculosis

Enzymatic catalysis and protein signaling are dynamic processes that involve local and/or global conformational changes occurring across a broad range of time scales. (1) H-(15) N relaxation NMR provides a comprehensive understanding of protein backbone dynamics both in the apo (unliganded) and ligand-bound conformations, enabling both fast and slow internal motions of individual amino acid residues to be observed. We recently reported the structure and nucleotide binding properties of the sulfate transporter and anti-sigma factor antagonist (STAS) domain of Rv1739c, a SulP anion transporter protein of Mycobacterium tuberculosis. In the present study, we report (1) H-(15) N NMR backbone dynamics measurements [longitudinal (T(1) ), transverse (T(2) ) and steady-state ({(1) H}-(15) N) heteronuclear NOE] of the Rv1739c STAS domain, in the absence and presence of saturating concentrations of GTP and GDP. Analysis of measured relaxation data and estimated dynamic parameters indicated distinct features differentiating the binding of GTP and GDP to Rv1739c STAS. The 9.55 ns overall rotational correlation time of Rv1739c STAS increased to 10.48 ns in the presence of GTP, and to 13.25 ns in the presence of GDP, indicating significant nucleotide-induced conformational changes. These conformational changes were accompanied by slow time scale (μs to ms) motions in discrete regions of the protein, as reflected by guanine nucleotide-induced changes in relaxation parameters. The observed nucleotide-specific alterations in the relaxation properties of individual STAS residues reflect an increased molecular anisotropy and/or the emergence of conformational equilibria governing functional properties of the STAS domain.     

(1)H/(15)N heteronuclear NMR spectroscopy shows four dynamic domains for phospholamban reconstituted in dodecylphosphocholine micelles

We report the backbone dynamics of monomeric phospholamban in dodecylphosphocholine micelles using (1)H/(15)N heteronuclear NMR spectroscopy. Phospholamban is a 52-amino acid membrane protein that regulates Ca-ATPase in cardiac muscle. Phospholamban comprises three structural domains: a transmembrane domain from residues 22 to 52, a connecting loop from 17 to 21, and a cytoplasmic domain from 1 to 16 that is organized in an "L"-shaped structure where the transmembrane and the cytoplasmic domain form an angle of approximately 80 degrees (Zamoon et al., 2003; Mascioni et al., 2002). T(1), T(2), and (1)H/(15)N nuclear Overhauser effect values measured for the amide backbone resonances were interpreted using the model-free approach of Lipari and Szabo. The results point to the existence of four dynamic domains, revealing the overall plasticity of the cytoplasmic helix, the flexible loop, and part of the transmembrane domain (residues 22-30). In addition, using Carr-Purcell-Meiboom-Gill-based experiments, we have characterized phospholamban dynamics in the micros-ms timescale. We found that the majority of the residues in the cytoplasmic domain, the flexible loop, and the first ten residues of the transmembrane domain undergo dynamics in the micros-ms range, whereas minimal dynamics were detected for the transmembrane domain. Hydrogen/deuterium exchange factors measured at different temperatures support the existence of slow motion in both the loop and the cytoplasmic helix. We propose that these dynamic properties are critical factors in the biomolecular recognition of phospholamban by Ca-ATPase and other interacting proteins such as protein kinase A and protein phosphatase 1.     

Structural and dynamics characterization of norovirus protease

Norovirus protease is an essential enzyme for proteolytic maturation of norovirus nonstructural proteins and has been implicated as a potential target for antiviral drug development. Although X‐ray structural studies of the protease give us wealth of structural information including interactions of the protease with its substrate and dimeric overall structure, the role of protein dynamics in the substrate recognition and the biological relevance of the protease dimer remain unclear. Here we determined the solution NMR structure of the 3C‐like protease from Norwalk virus (NV 3CLpro), a prototype strain of norovirus, and analyzed its backbone dynamics and hydrodynamic behavior in solution. ¹⁵N spin relaxation and analytical ultracentrifugation analyses demonstrate that NV 3CLpro is predominantly a monomer in solution. Solution structure of NV 3CLpro shows significant structural variation in C‐terminal domain compared with crystal structures and among lower energy structure ensembles. Also, ¹⁵N spin relaxation and Carr–Purcell–Meiboom–Gill (CPMG)‐based relaxation dispersion analyses reveal the dynamic properties of residues in the C‐terminal domain over a wide range of timescales. In particular, the long loop spanning residues T123–G133 show fast motion (ps‐ns), and the residues in the bII–cII region forming the large hydrophobic pocket (S2 site) undergo conformational exchanges on slower timescales (μs–ms), suggesting their important role in substrate recognition.     

Backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The extent of rapid (picosecond) backbone motions within the glucocorticoid receptor DNA-binding domain (GR DBD) has been investigated using proton-detected heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments containing the GR DBD. Sequence-specific 15N resonance assignments, based on two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69 backbone amides within the segment C440-A509 of the rat GR in a protein fragment containing a total of 82 residues (MW = 9200). Individual backbone 15N spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times (T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been measured at 11.74 T for a majority of the backbone amide nitrogens within the segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) characterizing internal motions in each backbone amide using an optimized value for the correlation time for isotropic rotational motions of the protein (tau R = 6.3 ns). Average S2 order parameters are found to be similar (approximately 0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection as well as quantitative analysis of the relaxation and NOE data suggests that the picosecond flexibility of the DBD backbone is limited and uniform over the entire protein, with the possible exception of residues S448-H451 of the first zinc domain and a few residues for which relaxation and NOE parameters were not obtained. in particular, we find no evidence for extensive rapid backbone motions within the second zinc domain. Our results therefore suggest that the second zinc domain is not disordered in the uncomplexed state of DBD, although the possibility of slowly exchanging (ordered) conformational states cannot be excluded in the present analysis.     

Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB

In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself.     

NMR characterization of structure, backbone dynamics, and glutathione binding of the human macrophage migration inhibitory factor (MIF).

Human macrophage migration inhibitory factor is a 114 amino acid protein that belongs to the family of immunologic cytokines. Assignments of 1H, 15N, and 13C resonances have enabled the determination of the secondary structure of the protein, which consists of two alpha-helices (residues 18-31 and 89-72) and a central four-stranded beta-sheet. In the beta-sheet, two parallel beta-sheets are connected in an antiparallel sense. From the total of three cysteines present in the primary structure of MIF, none was found to form disulfide bridges. 1H-15N heteronuclear T1, T2, and steady-state NOE measurements indicate that the backbone of MIF exists in a rigid structure of limited conformational flexibility (on the nanosecond to picosecond time scale). Several residues located in the loop regions and at the N termini of two helices exhibit internal motions on the 1-3 ns time scale. The capacity to bind glutathione was investigated by titration of a uniform 15N-labeled sample and led us to conclude that MIF has, at best, very low affinity for glutathione.     

Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl-enzyme intermediate

NMR spectroscopy was used to search for mechanistically significant differences in the local mobility of the main-chain amides of Bacillus circulans xylanase (BCX) in its native and catalytically competent covalent glycosyl-enzyme intermediate states. 15N T1, T2, and 15N[1H] NOE values were measured for approximately 120 out of 178 peptide groups in both the apo form of the protein and in BCX covalently modified at position Glu78 with a mechanism-based 2-deoxy-2-fluoro-beta-xylobioside inactivator. Employing the model-free formalism of Lipari and Szabo, the measured relaxation parameters were used to calculate a global correlation time (tau(m)) for the protein in each form (9.2 +/- 0.2 ns for apo-BCX; 9.8 +/- 0.3 ns for the modified protein), as well as individual order parameters for the main-chain NH bond vectors. Average values of the order parameters for the protein in the apo and complexed forms were S2 = 0.86 +/- 0.04 and S2 = 0.91 +/- 0.04, respectively. No correlation is observed between these order parameters and the secondary structure, solvent accessibility, or hydrogen bonding patterns of amides in either form of the protein. These results demonstrate that the backbone of BCX is well ordered in both states and that formation of the glycosyl-enzyme intermediate leads to little change, in any, in the dynamic properties of BCX on the time scales sampled by 15N-NMR relaxation measurements.     

Backbone dynamics of apocytochrome b5 in its native, partially folded state

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.     

Structural and dynamical changes of the bindin B18 peptide upon binding to lipid membranes. A solid-state NMR study

Structural and dynamical features of the B18 peptide from the sea urchin sperm binding protein were determined in the crystalline state and in zwitterionic lipid bilayers at a peptide:lipid molar ratio of 1:12 using solid-state NMR spectroscopy. The study was focused on three (13)C and (15)N uniformly labeled leucine residues, which were introduced into three different B18 peptides at positions evenly distributed along the B18 primary structure. Isotropic (13)C and (15)N chemical shift measurements showed that while B18 possesses a nonhelical and non-sheet-like structure in the crystalline state, the peptide adopts an oligomeric beta-sheet structure in the membrane in the presence of Zn(2+) ions at high peptide:lipid ratio. Torsion angle measurements for the three leucine sites supported these results, with phi torsion angles between -80 degrees and -90 degrees in the crystalline state and between -110 degrees and -120 degrees in the membrane-bound form. These phi torsion angles determined for membrane-bound B18 are consistent with a parallel beta-sheet secondary structure. Analysis of motionally averaged dipolar coupling measurements established an increase of the mobility in the leucine side chains upon binding to the membrane, whereas the backbone mobility remained essentially unchanged, except in the binding site of Zn(2+) ions. This difference in mobility was related to the H-bond network in the parallel beta-sheet structure, which involves the backbone and excludes the side chains of leucine residues. The parallel beta-sheet structure of B18 in the membrane in the presence of Zn(2+) appears to be an active state for the fusion of zwitterionic membranes in the presence of Zn(2+). A fluorescence fusion assay indicated that high B18 concentrations are required to induce fusion in these systems. Therefore, it was hypothesized that the oligomeric beta-sheet secondary structure revealed in the study represents an active state of the peptide in a membrane environment during fusion.     

15N NMR relaxation studies of free and ligand-bound human acidic fibroblast growth factor

15N NMR relaxation data have been used to characterize the backbone dynamics of the human acidic fibroblast growth factor (hFGF-1) in its free and sucrose octasulfate (SOS)-bound states. (15)N longitudinal (R(1)), transverse (R(2)) relaxation rates and (1H)-(15)N steady-state nuclear Overhauser effects were obtained at 500 and 600 MHz (at 25 degrees C) for all resolved backbone amide groups using (1)H- detected two-dimensional NMR experiments. Relaxation data were fit to the extended model free dynamics for each NH group. The overall correlation time (tau(m)) for the free and SOS-bound forms were estimated to be 10.4 +/- 1.07 and 11.1 +/- 1.35 ns, respectively. Titration experiments with SOS reveals that the ligand binds specifically to the C-terminal domain of the protein in a 1:1 ratio. Binding of SOS to hFGF-1 is found to induce a subtle conformational change in the protein. Significant conformational exchange (R(ex)) is observed for several residues in the free form of the protein. However, in the SOS-bound form only three residues exhibit significant R(ex) values, suggesting that the dynamics on the micro- to millisecond time scale in the free form is coupled to the cis-trans-proline isomerization. hFGF-1 is a rigid molecule with an average generalized parameter (S(2)) value of 0.89 +/- 0.03. Upon binding to SOS, there is a marked decrease in the overall flexibility (S(2) = 0.94 +/- 0.02) of the hFGF-1 molecule. However, the segment comprising residues 103-111 shows increased flexibility in the presence of SOS. Significant correlation is found between residues that show high flexibility and the putative receptor binding sites on the protein.     

Solution structure and dynamics of a designed monomeric variant of the lambda Cro repressor.

The solution structure of a monomeric variant of the lambda Cro repressor has been determined by multidimensional NMR. Cro K56[DGEVK] differs from wild-type Cro by the insertion of five amino acids at the center of the dimer interface. 1H and 15N resonances for 70 of the 71 residues have been assigned. Thirty-two structures were calculated by hybrid distance geometry/simulated annealing methods using 463 NOE-distance restraints, 26 hydrogen-bond, and 39 dihedral-angle restraints. The root-mean-square deviation (RMSD) from the average structure for atoms in residues 3-60 is 1.03 +/- 0.44 A for the peptide backbone and 1.6 +/- 0.73 A for all nonhydrogen atoms. The overall structure conforms very well to the original design. Although the five inserted residues form a beta hairpin as expected, this engineered turn as well as other turns in the structure are not well defined by the NMR data. Dynamics studies of backbone amides reveal T1/T2 ratios of residues in the alpha2-alpha3, beta2-beta3, and engineered turn that are reflective of chemical exchange or internal motion. The solution structure and dynamics are discussed in light of the conformational variation that has been observed in other Cro structures, and the importance of flexibility in DNA recognition.     

NMR structure and dynamics of the second transmembrane domain of the neuronal acetylcholine receptor beta 2 subunit

Structure and backbone dynamics of a selectively [(15)N]Leu-labeled 28-residue segment of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) beta(2) subunit were studied by (1)H and (15)N solution-state NMR in dodecylphosphocholine micelles. The TM2e structure was determined on the basis of the nuclear Overhauser effects (NOEs) and the hydrogen bond restraints, which were inferred from the presence of H(alpha)(i)-H(N)(i+3), H(alpha)(i)-H(beta)(i+3), and H(alpha)(i)-H(N)(i+4) NOE connectivity and from the slow amide hydrogen exchange with D(2)O. The TM2e structure of the nAChR beta(2) subunit contains a helical region between T4 and K22. Backbone dynamics were calculated using the model-free approach based on the (15)N relaxation rate constants, R(1) and R(2), and on the (15)N-[(1)H] NOE. The data acquired at 9.4 and 14.1 T and calculations using different dynamic models demonstrated no conformational exchange and internal motions on the nanosecond time scale. The global tumbling time of TM2e in micelles was 14.4 +/- 0.2 ns; the NOE values were greater than 0.63 at 9.4 T, and the order parameter, S(2), was 0.83-0.96 for all (15)N-labeled leucine residues, suggesting a restricted internal motion. This is the first report of NMR structure and backbone dynamics of the second transmembrane domain of the human nAChR beta(2) subunit in a membrane-mimetic environment, providing the basis for subsequent studies of subunit interactions in the transmembrane domain complex of the neuronal nAChR.     

Backbone dynamics of barstar: a (15)N NMR relaxation study

Backbone dynamics of uniformly (15)N-labeled barstar have been studied at 32 degrees C, pH 6.7, by using (15)N relaxation data obtained from proton-detected 2D (1)H-(15)N NMR spectroscopy. (15)N spin-lattice relaxation rate constants (R(1)), spin-spin relaxation rate constants (R(2)), and steady-state heteronuclear (1)H-(15)N NOEs have been determined for 69 of the 86 (excluding two prolines and the N-terminal residue) backbone amide (15)N at a magnetic field strength of 14.1 Tesla. The primary relaxation data have been analyzed by using the model-free formalism of molecular dynamics, using both isotropic and axially symmetric diffusion of the molecule, to determine the overall rotational correlation time (tau(m)), the generalized order parameter (S(2)), the effective correlation time for internal motions (tau(e)), and NH exchange broadening contributions (R(ex)) for each residue. As per the axially symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D( parallel)/D( perpendicular)) is 0.82 +/- 0.03. The two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these residues at three frequencies: 0, omega(H), and omega(N), where omega(H),(N) are proton and nitrogen Larmor frequencies. The value of tau(m) obtained from model-free analysis of the relaxation data is 5.2 ns. The reduced spectral density analysis, however, yields a value of 5.7 ns. The tau(m) determined here is different from that calculated previously from time-resolved fluorescence data (4.1 ns). The order parameter ranges from 0.68 to 0.98, with an average value of 0.85 +/- 0.02. A comparison of the order parameters with the X-ray B-factors for the backbone nitrogens of wild-type barstar does not show any considerable correlation. Model-free analysis of the relaxation data for seven residues required the inclusion of an exchange broadening term, the magnitude of which ranges from 2 to 9.1 s(-1), indicating the presence of conformational averaging motions only for a small subset of residues.