Mapping of the spectral densities of N-H bond motions in eglin c using heteronuclear relaxation experiments.

A new strategy is used for studying the internal motions of proteins based on measurements of NMR relaxation parameters. The strategy yields values of the so-called spectral density functions J(omega) for N-H bond vectors. The spectral density functions are related to the distribution of frequencies contained in the rotational (overall and internal) motions of these NH bond vectors. No a priori model assumptions about the dynamics are required in this approach. The method involves measurements of six relaxation parameters consisting of 15N longitudinal relaxation rates, transverse relaxation rates of in-phase and antiphase coherence, the relaxation rates of heteronuclear 1H-15N two-spin order, the heteronuclear 1H-15N nuclear Overhauser effects, and longitudinal relaxation rates of the amide protons. The values of the spectral density functions at the five frequencies 0, omega N, omega H + omega N, omega H, and omega H - omega N are determined from the relaxation parameters using analytical relations derived previously [Peng & Wagner (1992) J. Magn. Reson. 98, 308-332]. Here, the method is applied to characterize the backbone dynamics of the 15N-enriched proteinase inhibitor eglin c, a protein of 70 residues. The values for J(0) and J(omega N = 50 MHz) vary significantly with the amino acid sequence, whereas the spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are typically much smaller and show no significant variation with the sequence. The collective behavior of the J(omega) values indicate greater internal motion for the proteinase binding loop residues and the first eight N-terminal residues. The additional internal motion in these regions is in the rate range below 450 MHz. The values of J(omega) are also compared with root mean square deviations (rmsds) of backbone atoms as obtained in NMR structure determinations. Low values of J(0) and J(omega N) are correlated with high rmsds. Spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are small and show no correlation with rmsds. A comparison with the spectral density functions obtained by fitting the experimental data to the functional dependence of the Lipari and Szabo formalism [Lipari & Szabo (1982a) J. Am. Chem. Soc. 104, 4546-4559] is made.     

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.     

Main chain and side chain dynamics of oxidized flavodoxin from Cyanobacterium anabaena.

Oxidized flavodoxin from Cyanobacterium anabaena PCC 7119 is used as a model system to investigate the fast internal dynamics of a flavin-bearing protein. Virtually complete backbone and side chain resonance NMR assignments of an oxidized flavodoxin point mutant (C55A) have been determined. Backbone and side chain dynamics in flavodoxin (C55A) were investigated using (15)N amide and deuterium methyl NMR relaxation methods. The squared generalized order parameters (S(NH)(2)) for backbone amide N-H bonds are found to be uniformly high ( approximately 0.923 over 109 residues in regular secondary structure), indicating considerable restriction of motion in the backbone of the protein. In contrast, methyl-bearing side chains are considerably heterogeneous in their amplitude of motion, as indicated by obtained symmetry axis squared generalized order parameters (S(axis)(2)). However, in comparison to nonprosthetic group-bearing proteins studied with these NMR relaxation methods, the side chains of oxidized flavodoxin are unusually rigid.     

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)     

Comparison of 15N- and 13C-determined parameters of mobility in melittin

Backbone and tryptophan side-chain mobilities in the 26-residue, cytolytic peptide melittin (MLT) were investigated by ¹⁵N and ¹³C NMR. Specifically, inverse-detected ¹⁵N T₁ and steady-state NOE measurements were made at 30 and 51 MHz on MLT at 22 °C enriched with ¹⁵N at six amide positions and in the Trp¹⁹ side chain. Both the disordered MLT monomer (1.2 mM peptide at pH 3.6 in neat water) and -helical MLT tetramer (4.0 mM peptide at pH 5.2 in 150 mM phosphate buffer) were examined. The relaxation data were analyzed in terms of the Lipari and Szabo model-free formalism with three parameters: _m, the correlation time for the overall rotation; S², a site-specific order parameter which is a measure of the amplitude of the internal motion; and _e, a local, effective correlation time of the internal motion. A comparison was made of motional parameters from the ¹⁵N measurements and from ¹³C measurements on MLT, the latter having been made here and previously [Kemple et al. (1997) Biochemistry, 36, 1678–1688]. _m and _e values were consistent from data on the two nuclei. In the MLT monomer, S² values for the backbone N-H and C-H vectors in the same residue were similar in value but in the tetramer the N-H order parameters were about 0.2 units larger than the C-H order parameters. The Trp side-chain N-H and C-H order parameters, and _e values were generally similar in both the monomer and tetramer. Implications of these results regarding the dynamics of MLT are examined.     

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)     

Backbone dynamics of a model membrane protein: 13C NMR spectroscopy of alanine methyl groups in detergent-solubilized M13 coat protein

The filamentous coliphage M13 possesses multiple copies of a 50-residue coat protein which is inserted into the inner membrane of Escherichia coli during infection. 13C nuclear magnetic resonance (NMR) spectroscopy has been used to probe the structure and dynamics of M13 coat protein solubilized in detergent micelles. A comparison of backbone dynamics within the hydrophobic core region and the hydrophilic terminal domains was obtained by biosynthetic incorporation of [3-13C]alanine. Alanine is distributed throughout the protein and accounts for 10 residues (i.e., 20% of the total). Similar 13C NMR spectra of the protein have been obtained in two anionic detergents, sodium deoxycholate and sodium dodecyl sulfate, although the structures and physical properties of these solubilizing agents are quite different. The N-terminal alanine residues, assigned by pH titration, and the penultimate residue, assigned by carboxypeptidase A digestion, give rise to analogous peaks in both detergent systems. The pKa of Ala-1 (approximately 8.8) and the relaxation parameters of individual carbon atoms (T1, T2, and the nuclear Overhauser enhancement) are also generally similar, suggesting a similarity in the overall protein structure. Relaxation data have been analyzed according to the model-free approach of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall correlation times were obtained by fitting the three experimental relaxation values for a given well-resolved single carbon atom to obtain a unique value for the generalized order parameter, S2, and the effective correlation time, tau e. The former parameter reflects the spatial restriction of motion, and the latter, the rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the ability of molecular dynamics force fields to recapitulate NMR derived protein side chain order parameters

Molecular dynamics (MD) simulations have become a central tool for investigating various biophysical questions with atomistic detail. While many different proxies are used to qualify MD force fields, most are based on largely structural parameters such as the root mean square deviation from experimental coordinates or nuclear magnetic resonance (NMR) chemical shifts and residual dipolar couplings. NMR derived Lipari–Szabo squared generalized order parameter (O²) values of amide N? H bond vectors of the polypeptide chain were also often employed for refinement and validation. However, with a few exceptions, side chain methyl symmetry axis order parameters have not been incorporated into experimental reference sets. Using a test set of five diverse proteins, the performance of several force fields implemented in the NAMDD simulation package was examined. It was found that simulations employing explicit water implemented using the TIP3 model generally performed significantly better than those using implicit water in reproducing experimental methyl symmetry axis O² values. Overall the CHARMM27 force field performs nominally better than two implementations of the Amber force field. It appeared that recent quantum mechanics modifications to side chain torsional angles of leucine and isoleucine in the Amber force field have significantly hindered proper motional modeling for these residues. There remained significant room for improvement as even the best correlations of experimental and simulated methyl group Lipari–Szabo generalized order parameters fall below an R² of 0.8.     

Backbone dynamics of proteins derived from carbonyl carbon relaxation times at 500, 600 and 800 MHz: Application to ribonuclease T1

The backbone dynamics of uniformly 13C/15N-enriched ribonuclease T1 have beeninvestigated using carbonyl carbon relaxation times recorded at three different spectrometerfrequencies. Pulse sequences for the determination of the longitudinal (T1) and transverse (T2)relaxation times are presented. The relaxation behaviour was analysed in terms of a multispinsystem. Although the chemical shift anisotropy relaxation mechanism dominates at highmagnetic field strength, the contributions of the dipole–dipole interactions and thecross-correlation between these two relaxation mechanisms have also been considered.Information about internal motions has been extracted from the relaxation data using themodel-free approach of Lipari and Szabo in order to determine order parameters (S2) andeffective internal correlation times (i). Using a relatively simple relation between themeasured relaxation rates and the spectral density function, an analytical expression for themicrodynamical parameters in dependence of T1 and T2 has been derived. The spectraldensity mapping technique has been applied in order to study the behaviour of the carbonylcarbon resonances in more detail.     

Backbone dynamics of ribonuclease T1 and its complex with 2′GMP studied by two-dimensional heteronuclear NMR spectroscopy

Summary The backbone dynamics of free ribonuclease T1 and its complex with the competitive inhibitor 2GMP have been studied by ¹⁵N longitudinal and transverse relaxation experiments, combined with {¹H, ¹⁵H} NOE measurements. The intensity decay of individual amide cross peaks in a series of (¹H, ¹⁵N)-HSQC spectra with appropriate relaxation periods (Kay, L.E. et al. (1989) Biochemistry, 28, 8972–8979; Kay, L.E. et al. (1992) J. Magn. Reson., 97, 359–375) was fitted to a single exponential by using a simplex algorithm in order to obtain ¹⁵N T₁ and T₂ relaxation times. These experimentally obtained values were analysed in terms of the model-free approach introduced by Lipari and Szabo (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559; 4559–4570). The microdyramical parameters accessible by this approach clearly indicate a correlation between the structural flexibility and the tertiary structure of ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The results obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.     

Backbone dynamics of (1–71)- and (1–36)bacterioopsin studied by two-dimensional 1H-15N NMR spectroscopy

Summary The backbone dynamics of uniformly ¹⁵N-labelled fragments (residues 1–71 and 1–36) of bacterioopsin, solubilized in two media (methanol-chloroform (1:1), 0.1 M ²HCO₂NH₄, or SDS micelles) have been investigated using 2D proton-detected heteronuclear ¹H-¹⁵N NMR spectroscopy at two spectrometer frequencies, 600 and 400 MHz. Contributions of the conformational exchange to the transverse relaxation rates of individual nitrogens were elucidated using a set of different rates of the CPMG spin-lock pulse train and were essentially suppressed by the high-frequency CPMG spin-lock. We found that most of the backbone amide groups of (1–71)bacterioopsin in SDS micelles are involved in the conformational exchange process over a rate range of 10³ to 10⁴ s^-1. This conformational exchange is supposed to be due to an interaction between two -helixes of (1–71)bacterioopsin, since the hydrolysis of the peptide bond in the loop region results in the disappearance of exchange line broadening. ¹⁵N relaxation rates and ¹H-¹⁵N NOE values were interpreted using the model-free approach of Lipari and Szabo [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559]. In addition to overall rotation of the molecule, the backbone N-H vectors of the peptides are involved in two types of internal motions: fast, on a time scale <20 ps, and intermediate, on a time scale close to 1 ns. The intermediate dynamics in the -helical stretches was mostly attributed to bending motions. A decrease in the order parameter of intermediate motions was also observed for residues next to Pro⁵⁰, indicating an anisotropy of the overall rotational diffusion of the molecule. Distinctly mobile regions are identified by a large decrease in the order parameter of intermediate motions and correspond to the N- and C-termini, and to a loop connecting the -helixes of (1–71)bacterioopsin. The internal dynamics of the -helixes on the millisecond and nanosecond time scales should be taken into account in the development of a model of the functioning bacteriorhodopsin.Abbreviations BO bacterioopsin - 2D two-dimensional - CPMG Carr-Purcell-Meiboom-Gill (Carr and Purcell, 1954) - SDS sodium dodecyl(²H₂₅) sulfate - R(S_x), R(S_z) ¹⁵N transverse and longitudinal relaxation rates, respectively     

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.     

Quantitative analysis of backbone motion in proteins using MAS solid-state NMR spectroscopy

We present a comprehensive analysis of protein dynamics for a micro-crystallin protein in the solid-state. Experimental data include ¹⁵N T ₁ relaxation times measured at two different magnetic fields as well as ¹H–¹⁵N dipole, ¹⁵N CSA cross correlated relaxation rates which are sensitive to the spectral density function J(0) and are thus a measure of T ₂ in the solid-state. In addition, global order parameters are included from a ¹H,¹⁵N dipolar recoupling experiment. The data are analyzed within the framework of the extended model-free Clore–Lipari–Szabo theory. We find slow motional correlation times in the range of 5 and 150 ns. Assuming a wobbling in a cone motion, the amplitude of motion of the respective amide moiety is on the order of 10° for the half-opening angle of the cone in most of the cases. The experiments are demonstrated using a perdeuterated sample of the chicken α-spectrin SH3 domain.     

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.     

Investigation of the backbone dynamics of the IgG-binding domain of streptococcal protein G by heteronuclear two-dimensional 1H-15N nuclear magnetic resonance spectroscopy.

The backbone dynamics of the immunoglobulin-binding domain (B1) of streptococcal protein G, uniformly labeled with 15N, have been investigated by two-dimensional inverse detected heteronuclear 1H-15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and nuclear Overhauser enhancement data were obtained for all 55 backbone NH vectors of the B1 domain at both field strengths. The overall correlation time obtained from an analysis of the T1/T2 ratios was 3.3 ns at 26 degrees C. Overall, the B1 domain is a relatively rigid protein, consistent with the fact that over 95% of the residues participate in secondary structure, comprising a four-stranded sheet arranged in a -1, +3x, -1 topology, on top of which lies a single helix. Residues in the turns and loops connecting the elements of secondary structure tend to exhibit a higher degree of mobility on the picosecond time scale, as manifested by lower values of the overall order parameter. A number of residues at the ends of the secondary structure elements display two distinct internal motions that are faster than the overall rotational correlation time: one is fast (< 20 ps) and lies in the extreme narrowing limit, whereas the other is one to two orders of magnitude slower (1-3 ns) and lies outside the extreme narrowing limit. The slower motion can be explained by large-amplitude (20-40 degrees) jumps in the N-H vectors between states with well-defined orientations that are stabilized by hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.