Comparison of backbone dynamics of oxidized and reduced putidaredoxin by 15N NMR relaxation measurements.

The backbone dynamics of uniformly 15N-labeled reduced and oxidized putidaredoxin (Pdx) have been studied by 2D 15N NMR relaxation measurements. 15N T1 and T2 values and 1H-15N NOEs have been measured for the diamagnetic region of the protein. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameters (S2), the effective correlation time for internal motions (tau e), and the 15N exchange broadening contributions (Rex) for each residue, as well as the overall correlation time (tau(m)). Order parameters for the reduced Pdx are generally higher than for the oxidized Pdx, and there is increased mobility on the microsecond to millisecond time scale for the oxidized Pdx, in comparison with the reduced Pdx. These results clearly indicate that the oxidized protein exhibits higher mobility than the reduced one, which is in agreement with the recently published redox-dependent dynamics studied by amide proton exchange. In addition, we observed very high T1/T2 ratios for residues 33 and 34, giving rise to a large Rex contribution. Residue 34 is believed to be involved in the binding of Pdx to cytochrome P450cam (CYP101). The differences in the backbone dynamics are discussed in relation to the oxidation states of Pdx, and their impact on electron transfer. The entropy change occurring on oxidation of reduced Pdx has been calculated from the order parameters of the two forms.     

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 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.     

Loop mobility in a four-helix-bundle protein: 15N NMR relaxation measurements on human interleukin-4.

15N NOE, T1, and T2 measurements have been carried out on uniformly 15N-labeled human interleukin-4. Analysis of the results in terms of order parameters (S2) shows that although the helical core of this four-helix-bundle protein exists as a well-defined structure with limited conformational flexibility (S2 congruent to 0.9), other regions of the molecule experience substantial fluctuations in the conformation of the main chain (S2 = 0.3-0.8). These regions include both the N- and C-termini and two of the loops joining the helices. The majority of these internal motions are fast compared with the overall rotational correlation time (tau R = 7.6 ns at 35 degrees C) and are localized in regions that are relatively ill-defined in the NMR structures previously determined for this protein [Smith, L. J., Redfield, C., Boyd, J., Lawrence, G. M. P., Edwards, R. G., Smith, R. A. G., & Dobson, C. M. (1992) J. Mol. Biol. 224, 899-904]. Other motions are on a slower time scale and appear to be associated with two of the three disulfide bonds and the beta-sheet region in the protein. The dynamic properties of interleukin-4 in solution have been compared with features of the X-ray structures of other four-helix-bundle proteins. The results suggest that the dynamic properties observed here may be general for this class of proteins and may be significant for the interpretation of both their structural and functional properties.     

Thermodynamic interpretation of protein dynamics from NMR relaxation measurements

Protein dynamics and thermodynamics can be characterized through measurements of relaxation rates of side chain (2)H and (13)C, and backbone (15)N nuclei using NMR spectroscopy. The rates reflect protein motions on timescales from picoseconds to milliseconds. Backbone and methyl side chain NMR relaxation measurements for several proteins are beginning to reveal the role of protein dynamics in protein stability and ligand binding.     

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.     

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.     

Accessibility of tobacco lipid transfer protein cavity revealed by 15N NMR relaxation studies and molecular dynamics simulations

Plant LTP1 are small helical proteins stabilized by four disulfide bridges and are characterized by the presence of an internal cavity, in which various hydrophobic ligands can be inserted. Recently, we have determined the solution structure of the recombinant tobacco LTP1_1. Unexpectedly, despite a global fold very similar to the structures already known for cereal seed LTP1, its binding properties are different: Tobacco LTP1_1 is able to bind only one monoacylated lipid, whereas cereal LTP1 can bind either one or two. The 3D structure of tobacco LTP1_1 revealed the presence of a hydrophobic cluster, not observed on cereal LTP1 structures, which may hinder one of the two entrances of the cavity defined for wheat LTP1. To better understand the mechanism of lipid entrance for tobacco LTP1_1 and to define the regions of the protein monitoring the accessibility of the cavity, we have complemented our structural data by the study of the internal dynamics of tobacco LTP1_1, using (15)N magnetic relaxation rate data and MD simulations at room and high temperatures. This work allowed us to define two regions of the protein experiencing the largest motions. These two regions delineate a portal that opens up during the simulation constituting a unique entrance of the hydrophobic cavity, in contrast with wheat LTP1 where two routes were detected. The hydrophobic interactions resulting from a few point mutations are strong enough to completely block the second portal so that the accessibility of the cavity is restricted to one entrance, explaining why this particular LTP1 binds only one lipid molecule.     

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.     

Redox-controlled backbone dynamics of human cytochrome c revealed by N NMR relaxation measurements

Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D ¹⁵N NMR relaxation experiments. ¹⁵N T₁ and T₂ values and ¹H-¹⁵N NOEs of uniformly ¹⁵N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S²), the effective correlation time for internal motion (τ_e), the ¹⁵N exchange broadening contributions (R_ex) for each residue, and the overall correlation time (τ_m) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S² value was increased from 0.88 ± 0.01 to 0.92 ± 0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S² values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system.     

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 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 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.     

Fast-time scale dynamics of outer membrane protein A by extended model-free analysis of NMR relaxation data

In order to better understand the dynamics of an integral membrane protein, backbone amide ¹⁵N NMR dynamics measurements of the β-barrel membrane protein OmpA have been performed at three magnetic fields. A total of nine relaxation data sets were globally analyzed using an extended model-free formalism. The diffusion tensor was found to be prolate axially symmetric with an axial ratio of 5.75, indicating a possible rotation of the protein within the micelle. The generalized order parameters gradually decreased from the mid-plane towards the two ends of the barrel, counteracting the dynamic gradient of the lipids in a matching bilayer, and were dramatically reduced in the extracellular loops. Large-scale internal motions on the ns time scale indicate that entire loops most likely undergo concerted (“sea anemone”-like) motions emanating from their anchoring points on the barrel. The case of OmpA in DPC micelles also illustrates inherent limitations of analyzing the data with even the most sophisticated current models of the model-free formalism. It is likely that conformational exchange processes on the ms-μs also play a role in describing the motions of some residues, but their analysis did not produce unique results that could be independently verified.     

Spectral densities of nitrogen nuclei in Escherichia coli ribonuclease HI obtained by 15N NMR relaxation and molecular dynamics

Summary Spectral densities of the ¹⁵N amide in Escherichia coli ribonuclease HI, obtained from NMR relaxation experiments, were compared with those calculated using a molecular dynamics (MD) simulation. All calculations and comparisons assumed that the auto-correlation function describing the internal motions of the molecule was independent of the auto-correlation function associated with overall rotational diffusion. Comparisons were limited to those residues for which the auto-correlation function of internal motions rapidly relaxed and reached a steady state within 205 ps. The results show the importance of frequency components as well as amplitudes of internal motions in order to obtain a meaningful comparison of MD simulations with NMR data.     

Characteristic domain motion in the ribosome recycling factor revealed by 15N NMR relaxation experiments and molecular dynamics simulations

The backbone dynamics of ribosome recycling factor (RRF) from Escherichia coli in water were characterized by (15)N NMR relaxation analysis and molecular dynamics (MD) simulation. RRF is composed of two domains connected by a joint region that consists of two peptide chains, such that the overall structure seems to mimic that of tRNA. MD trajectories indicated that the relative orientation of domains varies on the nanosecond time scale. We analyzed the observed (15)N T(1), T(2), and NOE using an extended model-free spectral density function in which the domain motions with a nanosecond time scale were considered. At 30 degrees C, the order parameters of slow motion () were determined to be approximately 0.9 for domain I and 0.7 for domain II, respectively. These values indicate that domain I is nearly fixed on the molecular diffusion frame, and domain II is wobbling in a cone for which the semi-angle is about 30 degrees.     

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).