Backbone dynamics of the channel-forming antibiotic zervamicin IIB studied by 15N NMR relaxation

The backbone dynamics of the channel-forming peptide antibiotic zervamicin IIB (Zrv-IIB) in methanol were studied by 15N nuclear magnetic resonance relaxation measurements at 11.7, 14.1 and 18.8 T magnetic fields. The anisotropic overall rotation of the peptide was characterized based on 15N relaxation data and by hydrodynamic calculations. 'Model-free' analysis of the relaxation data showed that the peptide is fairly rigid on a sub-nanosecond time-scale. The residues from the polar side of Zrv-IIB helix are involved in micro-millisecond time-scale conformational exchange. The conformational exchange observed might indicate intramolecular processes or specific intermolecular interactions of potential relevance to Zrv-IIB ion channel formation.     

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.     

Backbone dynamics of the cytotoxic ribonuclease alpha-sarcin by 15N NMR relaxation methods

The cytotoxic ribonuclease -sarcin is a 150-residue protein that inactivates ribosomes by selectively cleaving a single phosphodiester bond in a strictly conserved rRNA loop. In order to gain insights on the molecular basis of its highly specific activity, we have previously determined its solution structure and studied its electrostatics properties. Here, we complement those studies by analysing the backbone dynamics of -sarcin through measurement of longitudinal relaxation rates R₁, off resonance rotating frame relaxation rates R₁, and the ¹⁵N¹HNOE of the backbone amide ¹⁵N nuclei at two different magnetic field strengths (11.7 and 17.6 T). The two sets of relaxation parameters have been analysed in terms of the reduced spectral density mapping formalism, as well as by the model-free approach. -Sarcin behaves as an axial symmetric rotor of the prolate type (D/D=1.16 ± 0.02) which tumbles with a correlation time _m of 7.54 ± 0.02 ns. The rotational diffusion properties have been also independently evaluated by hydrodynamic calculations and are in good agreement with the experimental results. The analysis of the internal dynamics reveals that -sarcin is composed of a rigid hydrophobic core and some exposed segments which undergo fast (ps to ns) internal motions. Slower motions in the s to ms time scale are less abundant and in some cases can be assigned to specific motional processes. All dynamic data are discussed in relation to the role of some particular residues of -sarcin in the process of recognition of its ribosomal target.     

Backbone dynamics of the human MIA protein studied by (15)N NMR relaxation: implications for extended interactions of SH3 domains

The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by (15)N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting beta-sheets, such as the RT-loop for example, display increased mobility on picosecond to nanosecond time scales. One of the most important dynamic features is the pronounced flexibility of the distal loop, comprising residues Asp 68 to Ala 75, where motions on time scales up to milliseconds occur. Further, significant exchange contributions are observed for residues of the canonical binding site of SH3 domains including the RT-loop, the n-Src loop, for the loop comprising residues 13 to 19, which we refer to as the"disulfide loop", in part for the distal loop, and the carboxyl terminus of human MIA. The functional importance of this dynamic behavior is discussed with respect to the biological activity of several point mutations of human MIA. The results of this study suggest that the MIA protein and the recently identified highly homologous fibrocyte-derived protein (FDP)/MIA-like (MIAL) constitute a new family of secreted proteins that adopt an SH3 domain-like fold in solution with expanded ligand interactions.     

Backbone dynamics of the calcium-signaling protein apo-S100B as determined by 15N NMR relaxation

Backbone dynamics of homodimeric apo-S100B were studied by (15)N nuclear magnetic resonance relaxation at 9.4 and 14.1 T. Longitudinal relaxation (T(1)), transverse relaxation (T(2)), and the (15)N-[(1)H] NOE were measured for 80 of 91 backbone amide groups. Internal motional parameters were determined from the relaxation data using the model-free formalism while accounting for diffusion anisotropy. Rotational diffusion of the symmetric homodimer has moderate but statistically significant prolate axial anisotropy (D( parallel)/D( perpendicular) = 1.15 +/- 0.02), a global correlation time of tau(m) = 7.80 +/- 0.03 ns, and a unique axis in the plane normal to the molecular symmetry axis. Of 29 residues at the dimer interface (helices 1 and 4), only one has measurable internal motion (Q71), and the order parameters of the remaining 28 were the highest in the protein (S(2) = 0.80 to 0.91). Order parameters in the typical EF hand calcium-binding loop (S(2) = 0.73 to 0.87) were slightly lower than in the pseudo-EF hand (S(2) = 0.75 to 0.89), and effective internal correlation times, tau(e), distinct from global tumbling, were detected in the calcium-binding loops. Helix 3, which undergoes a large, calcium-induced conformational change necessary for target-protein binding, does not show evidence of interchanging between the apo and Ca(2+)-bound orientations in the absence of calcium but has rapid motion in several residues throughout the helix (S(2) = 0.78 to 0.88; 10 < or = tau(e) < or = 30 ps). The lowest order parameters were found in the C-terminal tail (S(2) = 0.62 to 0.83). Large values for chemical exchange also occur in this loop and in regions nearby in space to the highly mobile C-terminal loop, consistent with exchange broadening effects observed.     

Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible.

The backbone dynamics of Ca(2+)-saturated recombinant Drosophila calmodulin has been studied by 15N longitudinal and transverse relaxation experiments, combined with 15N(1H) NOE measurements. Results indicate a high degree of mobility near the middle of the central helix of calmodulin, from residue K77 through S81, with order parameters (S2) in the 0.5-0.6 range. The anisotropy observed in the motion of the two globular calmodulin domains is much smaller than expected on the basis of hydrodynamic calculations for a rigid dumbbell type structure. This indicates that, for the purposes of 15N relaxation, the tumbling of the N-terminal (L4-K77) and C-terminal (E82-S147) lobes of calmodulin is effectively independent. A slightly shorter motional correlation time (tau c approximately 6.3 ns) is obtained for the C-terminal domain compared to the N-terminal domain (tau c approximately 7.1 ns), in agreement with the smaller size of the C-terminal domain. A high degree of mobility, with order parameters of approximately 0.5, is also observed in the loop that connects the first with the second EF-hand type calcium binding domain and in the loop connecting the third and fourth calcium binding domain.     

A novel view of domain flexibility in E. coli adenylate kinase based on structural mode-coupling (15)N NMR relaxation.

Adenylate kinase from Escherichia coli (AKeco), consisting of a single 23.6 kDa polypeptide chain folded into domains CORE, AMPbd and LID, catalyzes the reaction AMP+ATP-->2ADP. In the ligand-free enzyme the domains AMPbd and LID execute large-amplitude movements controlling substrate binding and product release during catalysis. Domain flexibility is investigated herein with the slowly relaxing local structure (SRLS) model for (15)N relaxation. SRLS accounts rigorously for coupling between the global and local N-H motions through a local ordering potential exerted by the protein structure at the N-H bond. The latter reorients with respect to its protein surroundings, which reorient on the slower time scale associated with the global protein tumbling. AKeco diffuses globally with correlation time tau(m)=15.1 ns, while locally two different dynamic cases prevail. The domain CORE features ordering about the equilibrium N-H bond orientation with order parameters, S(2), of 0.8-0.9 and local motional correlation times, tau, mainly between 5-130 ps. This represents a conventional rigid protein structure with rapid small-amplitude N-H fluctuations. The domains AMPbd and LID feature small parallel (Z(M)) ordering of S(2)=0.2-0.5 which can be reinterpreted as high perpendicular (Y(M)) ordering. M denotes the local ordering/local diffusion frame. Local motion about Z(M) is given by tau( parallel) approximately 5 ps and local motion of the effective Z(M) axis about Y(M) by tau( perpendicular)=6-11 ns. Z(M) is tilted at approximately 20 degrees from the N-H bond. The orientation of the Y(M) axis may be considered parallel to the C(alpha)(i-1)-C(alpha)(i) axis. The tau( perpendicular) mode reflects collective nanosecond peptide-plane motions, interpretable as domain motion. A powerful new model of protein flexibility/domain motion has been established. Conformational exchange (R(ex)) processes accompany the tau( perpendicular) mode. The SRLS analysis is compared with the conventional model-free analysis.     

Backbone dynamics of calcium-loaded calbindin D9k studied by two-dimensional proton-detected 15N NMR spectroscopy.

Backbone dynamics of calcium-loaded calbindin D9k have been investigated by two-dimensional proton-detected heteronuclear nuclear magnetic resonance spectroscopy, using a uniformly 15N enriched protein sample. Spin-lattice relaxation rate constants, spin-spin relaxation rate constants, and steady-state [1H]-15N nuclear Overhauser effects were determined for 71 of the 72 backbone amide 15N nuclei. The relaxation parameters were analyzed using a model-free formalism that incorporates the overall rotational correlation time of the molecule, and a generalized order parameter (S2) and an effective internal correlation time for each amide group. Calbindin D9k contains two helix-loop-helix motifs joined by a linker loop at one end of the protein and a beta-type interaction between the two calcium-binding loops at the other end. The amplitude of motions for the calcium-binding loops and the helices are similar, as judged from the average S2 values of 0.83 +/- 0.05 and 0.85 +/- 0.04, respectively. The linker region joining the two calcium-binding subdomains of the molecule has a significantly higher flexibility, as indicated by a substantially lower average S2 value of 0.59 +/- 0.23. For residues in the linker loop and at the C-terminus, the order parameter is further decomposed into separate order parameters for motional processes on two distinct time scales. The effective correlation times are significantly longer for helices I and IV than for helices II and III or for the calcium-binding loops. Residue by residue comparisons reveal correlations of the order parameters with both the crystallographic B-factors and amide proton exchange rates, despite vast differences in the time scales to which these properties are sensitive. The order parameters are also utilized to distinguish regions of the NMR-derived three-dimensional structure of calbindin D9k that are poorly defined due to inherently high flexibility, from poorly defined regions with average flexibility but a low density of structural constraints.     

Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study

Backbone dynamics of uniformly ¹⁵N-labeled free barnase and its complex with unlabelled barstar have been studied at 40°C, pH 6.6, using ¹⁵N relaxation data obtained from proton-detected 2D {¹H}-¹⁵N NMR spectroscopy. ¹⁵N spin-lattice relaxation rate constants (R₁), spin-spin relaxation rate constants (R₂), and steady-state heteronuclear {¹H}-¹⁵N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N-terminal residue) backbone amide ¹⁵N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times (_m) are 5.0 and 9.5 ns for the free and complexed barnase, respectively. The average order parameter is found to be 0.80 for free barnase and 0.86 for the complex. However, the changes are not uniform along the backbone and for about 5 residues near the binding interface there is actually a significant decrease in the order parameters on complex formation. These residues are not involved in the actual binding. For the residues where the order parameter increases, the magnitudes vary significantly. It is observed that the complex has much less internal mobility, compared to free barnase. From the changes in the order parameters, the entropic contribution of NH bond vector motion to the free energy of complex formation has been calculated. It is apparent that these motions cause significant unfavorable contributions and therefore must be compensated by many other favorable contributions to effect tight complex formation. The observed variations in the motion and their different locations with regard to the binding interface may have important implications for remote effects and regulation of the enzyme action.     

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)     

Protein dynamics studied by rotating frame 15N spin relaxation times

Summary Conformational rate processes in aqueous solutions of uniformly ¹⁵N-labeled pancreatic trypsin inhibitor (BPTI) at 36°C were investigated by measuring the rotating frame relaxation times of the backbone ¹⁵N spins as a function of the spin-lock power. Two different intramolecular exchange processes were identified. A first local rate process involved the residues Cys³⁸ and Arg³⁹, had a correlation time of about 1.3 ms, and was related to isomerization of the chirality of the disulfide bond Cys¹⁴-Cys³⁸. A second, faster motional mode was superimposed on the disulfide bond isomerization and was tentatively attributed to local segmental motions in the polypeptide sequence-Cys¹⁴-Ala¹⁵-Lys¹⁶-. The correlation time for the overall rotational tumbling of the protein was found to be 2 ns, using the assumption that relaxation is dominated by dipolar coupling and chemical shift anistropy modulated by isotropic molecular reorientation.Abbreviations BPTI basic pancreatic trypsin inhibitor - 2D two-dimensional - COSY 2D correlation spectroscopy - TOCSY 2D total correlation spectroscopy - RF radio frequency - CW continuous wave - TPPI time-proportional phase incrementation - CSA chemical shift anisotropy - T₁ longitudinal relaxation time - T₂ transverse relaxation time - T₁ relaxation time in the rotating frame , correlation time for overall rotational reorientation of the protein - _ex ^s , _ex ^f , correlation times for two conformational exchange processes (slow and fast).     

Backbone dynamics of the ribonuclease binase active site area using multinuclear ((15)N and (13)CO) NMR relaxation and computational molecular dynamics

The nano-pico second backbone dynamics of the ribonuclease binase, homologous to barnase, is investigated with (15)N, (13)C 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 (15)N relaxation data indicate that these loops are mostly ordered at the nano-pico second time scale. Nano-pico second motions in the recognition loop 56-61 are evident from (13)CO-(13)C cross relaxation data, but the mobility of the catalytic loop 99-104 is not detected by (13)CO 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.     

Escherichia coli adenylate kinase dynamics: comparison of elastic network model modes with mode-coupling (15)N-NMR relaxation data

The dynamics of adenylate kinase of Escherichia coli (AKeco) and its complex with the inhibitor AP(5)A, are characterized by correlating the theoretical results obtained with the Gaussian Network Model (GNM) and the anisotropic network model (ANM) with the order parameters and correlation times obtained with Slowly Relaxing Local Structure (SRLS) analysis of (15)N-NMR relaxation data. The AMPbd and LID domains of AKeco execute in solution large amplitude motions associated with the catalytic reaction Mg(+2)*ATP + AMP --> Mg(+2)*ADP + ADP. Two sets of correlation times and order parameters were determined by NMR/SRLS for AKeco, attributed to slow (nanoseconds) motions with correlation time tau( perpendicular) and low order parameters, and fast (picoseconds) motions with correlation time tau( parallel) and high order parameters. The structural connotation of these patterns is examined herein by subjecting AKeco and AKeco*AP(5)A to GNM analysis, which yields the dynamic spectrum in terms of slow and fast modes. The low/high NMR order parameters correlate with the slow/fast modes of the backbone elucidated with GNM. Likewise, tau( parallel) and tau( perpendicular) are associated with fast and slow GNM modes, respectively. Catalysis-related domain motion of AMPbd and LID in AKeco, occurring per NMR with correlation time tau( perpendicular), is associated with the first and second collective slow (global) GNM modes. The ANM-predicted deformations of the unliganded enzyme conform to the functional reconfiguration induced by ligand-binding, indicating the structural disposition (or potential) of the enzyme to bind its substrates. It is shown that NMR/SRLS and GNM/ANM analyses can be advantageously synthesized to provide insights into the molecular mechanisms that control biological function.     

Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease

This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments. Because T1 values and heteronuclear NOEs are sensitive to high-frequency motions (10(8)-10(12) s-1) while T2 values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. We have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+ and labeled uniformly with 15N. T1, T2, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid 1H-15N bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, 15N line widths suggest a possible correlation between secondary structure and motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.     

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.     

A comparison of 15N NMR relaxation measurements with a molecular dynamics simulation: Backbone dynamics of the glucocorticoid receptor DNA-binding domain

The rapid motions of the backbone of the DNA-binding domain of the glucocorticoid receptor (GR DBD) have been investigated using proton-detected heteronuclear NMR experiments on ¹⁵N-labeled protein at pH 6.0 and with a 200 psec molecular dynamics simulation of hydrated GR DBD. The experimental data were interpreted in terms of a generalized order parameter (S²) and an effective correlation time (τ_e) for the internal motion of each amide bond. A back calculation, using the same model, yielded the {¹H}-¹⁵N nuclear Overhauser effects (NOEs) and the ¹⁵N spin-lattice relaxation times (T₁) from the simulated data. The rapid motions of the backbone turned out to be rather limited and uniform throughout the protein, with a somewhat reduced mobility in the two major α-helical regions and a slightly enhanced flexibility for some residues in the first zinc coordinating region. The agreement between the experimental and simulated S²-values was as good as quantitative for most of the residues, except for some residues that were subject to a more large-scale, and in the simulation thus poorly sampled, motion. Examples of such motions that were found in the simulation include jumps of the amide bond of Ile-487 between the charged oxygens of the side chain of Asp-485 and less distinct large scale motions for some of the residues in the extended regions, that were shown to give rise to noisy and/or fast decaying internal reorientational correlation functions. For these residues large differences in the simulated and experimental τ_e-values were found, indicating that motions on different time scales were dominating in the experimental and simulated values. The lower (<0.7) experimental NOEs for these residues could not be reproduced in the simulation and were shown to be a consequence of the lower τ_e-values estimated in the simulation. By combining information from the simulation and the experiment a more complete picture of the motions for these residues can be obtained as is illustrated with an estimation of the jump angle and jump frequency for the amide bond of Ile-487. © 1993 Wiley-Liss, Inc.     

Backbone dynamics of the regulatory domain of calcium vector protein, studied by 15N relaxation at four fields, reveals unique mobility characteristics of the intermotif linker

CaVP is a calcium-binding protein from amphioxus. It has a modular composition with two domains, but only the two EF-hand motifs localized in the C-terminal domain are functional. We recently determined the solution structure of this regulatory half (C-CaVP) in the Ca(2+)-saturated form and characterized the stepwise ion binding. This paper reports the (15)N nuclear relaxation rates of the Ca(2+)-saturated C-CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral density function for the majority of the amide H(N)-N vectors. Fitting the spectral density values at eight frequencies by a model-free approach, we obtained the microdynamic parameters characterizing the global and internal movements of the polypeptide backbone. The two EF-hand motifs, including the ion binding loops, behave like compact structural units with restricted mobility as reflected in the quite uniform order parameter and short internal correlation time (< 20 nsec). Comparative analysis of the two Ca(2+) binding sites shows that site III, having a larger affinity for the metal ion, is generally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneously by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C-CaVP characterized here is discussed in relation with other well-characterized Ca(2+)-binding proteins. Supplemental material: See www.proteinscience.org     

The binding of ATP and AMP to Escherichia coli adenylate kinase investigated by 1H and 15N NMR spectroscopy

[N6 15N]ATP and [N6 15N]AMP, complexed with E.coli adenylate kinase (AKe), were observed with 15N isotope-filtered NMR pulse sequences and 1H[15N] heterocorrelated experiments to determine differences between binding sites based on chemical shifts and competition by substrate analogs. The chemical shifts of the N6 amino proton and nitrogen signals changed significantly after mixing with adenylate kinase. Differences in chemical shifts between the bound ATP and AMP signals are slight. The response of these shifts to further addition of other substrates or Mg2+ supports the view that the unchelated nucleotides can bind to both the sites, whereas the metal complexed species are restricted to the MgATP/MgADP binding site.