Study of molecular dynamics of bovine carbonic anhydrase B by 13C-NMR in two strongly polarizing magnetic fields. I. Intramolecular mobility of polypeptide chains of the native enzyme

The study of the dynamics of enzyme segmental movement is of considerable importance in the understanding of the physics of the catalytic function of these macromolecules, which cannot be adequately described without introduction of intramolecular mobility of their polypeptide chains. At present high resolution [13C]NMR is mostly used as an effective and selective method for the observation of spectral and relaxation parameters that are sensitive to structure, conformation and local motion. The molecular dynamics of bovine carbonic anhydrase B (carbonate hydrolase EC. 4.2.1.1) in the native form was studied. Measurements of the relaxation parameters (T1, T2 and NOE) of the alpha-carbons of the polypeptide chain in two high magnetic fields (4.7 and 11.7 T) were carried out. The model-free approach of Lipari and Szabo to the interpretation of these experimental data show a satisfactory agreement between theory and experiment for these carbon nuclei if an internal degree of motion such as libration or restricted diffusion in a cone with angular amplitude in the 10 degrees less than theta less than or equal to 20 degrees range and an effective correlation time tau e approximately equal to 6 to 7 x 10(-11) S in addition to the tau R = 3 x 10(-8) S reorientation correlation time of the whole molecular is introduced.     

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)     

Motion of aromatic side chains, picosecond fluorescence, and internal energy transfer in Escherichia coli thioredoxin studied by site-directed mutagenesis, time-resolved fluorescence spectroscopy, and molecular dynamics simulations

We have determined the picosecond fluorescence of the four aromatic amino acid residues (W28, W31, Y49, and Y70) in wild-type Escherichia coli thioredoxin (wt Trx) and a mutant Trx with W31 replaced by phenylalanine, Trx-W28-W31F. The internal motions of the four aromatic side chains were also analyzed. We examined the possibility of using internal energy transfer from tyrosine to tryptophan as a measure of long-range distances. The major features of the lifetime distribution of tryptophan fluorescence were unchanged in the W31F mutation, indicating that the environment of W28 is similar in both wt Trx and Trx-W28-W31F. However, the mutation of W31F changed the mobility of W28, situated close to the active-site disulfide/dithiol, but not the mobility of two tyrosines, Y49 and Y70, situated on the other side of the molecule. The mobility of the two tyrosine residues increased upon reduction of the active-site disulfide, indicating a looser structure with reduction. This increased motion could also be seen from molecular dynamics simulations. The change in energy transfer rates, as judged by tyrosine fluorescence lifetimes, was in agreement with energy transfer rates calculated from the molecular dynamics simulations. The anisotropy of tryptophan and tyrosine fluorescence could be separated in three parts: (I) overall rotation of the protein (10(-9)s), (II) internal mobility of side chains (10(-10)s), and (III) a very fast relaxation (10(-12)s). We can only experimentally detect this very fast relaxation when the internal motion is not present.     

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.     

31P NMR relaxation studies of the activation of the coenzyme phosphate of glycogen phosphorylase. The role of motion of the bound phosphate.

Spin-lattice and spin-spin relaxation rates (1/T1 and 1/T2) have been determined for the catalytically essential coenzyme phosphate at the active site of glycogen phosphorylase in both activated (R state) and inactive (T state) conformations of the enzyme. Dipolar contributions to 31P relaxation due to exchangeable protons on the phosphate group have been determined by measurement of relaxation rates at different concentrations of H2O and D2O, and field dependence studies have been performed to estimate the contribution of chemical shift anisotropy to the remaining 31P relaxation in D2O. At 109 MHz, dipolar relaxation from exchangeable protons was found to account for 50% of the spin-lattice relaxation for activated phosphorylase in 75% H2O, the remainder being due to chemical shift anisotropy. The spin-lattice relaxation rates in D2O for R-state glycogen phosphorylase are very similar to those measured for other proteins of very different size such as actin (Brauer, M., and B. D. Sykes, 1981, Biochemistry. 20:6767-6775), alkaline phosphatase (Coleman, J. E., I. D. Armitage, J. F. Chlebowski, J. D. Otvos, and A. J. M. S. Uiterkamp, 1979), and phosphoglucomutase (Rhyu, G. I., W. J. Ray, Jr., and J. L. Markley, 1984, Biochemistry. 23:252-260). In inactive (T state) phosphorylase the spin-lattice relaxation rates were almost an order of magnitude slower, while the spin-spin relaxation rates were essentially identical. These results have been analyzed by calculating the theoretically expected 31P relaxation rates in the presence of internal motions that are included in the relaxation calculation using the model-free approach of Lipari and Szabo (1982, J. Am. Chem. Soc. 104:4564-4559).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tryptophan sidechain dynamics in hydrophobic oligopeptides determined by use of 13C nuclear magnetic resonance spectroscopy.

Two oligopeptides, t-boc-LAWAL-OMe and t-boc-LALALW-OMe, were synthesized for the purpose of examining the sidechain dynamics of the tryptophan residue in hydrophobic environments by 13C nuclear magnetic resonance and fluorescence spectroscopy. In both peptides, the tryptophan sidechain was greater than 95% enriched with 13C at the C delta 1 position. Spin-lattice relaxation time (T1) and steady-state nuclear Overhauser effect (NOE) data were obtained at 50.3 and 75.4 MHz for both peptides in CD3OD, and at 75.4 MHz for t-boc-LALALW-OMe in lysolecithin-D2O micelles. We have adapted the model-free approach of G. Lipari and A. Szabo (1982, J. Am. Chem. Soc. 104:4546) to interpret the 13C-NMR data. Computer-generated curves based on experimental data obtained at a single frequency demonstrate relationships between an effective correlation time for tryptophan sidechain motion (tau e), a generalized order parameter (sigma) describing the extent of motional restriction, and an overall correlation time for the peptide (tau m). Assuming predominantly dipolar relaxation, least-squares fits of the dual frequency relaxation data provide values for these parameters for both peptides. The contribution of chemical shift anisotropy (CSA), however, is also explicitly assessed in the data analysis, and is shown to perturb the predicted sigma, tau e, and tau m values and to decrease chi(2) values observed in nonlinear least-squares analysis of the data. Because of uncertainty in the contribution of CSA to the relaxation of the indole ring 13C delta 1 atom, nonlinear least-squares analysis of the relaxation data were performed with and without inclusion of a CSA term in the appropriate relaxation equations. Neglecting CSA, an overall peptide correlation time of 0.69 ns is predicted for t-boc-LAWAL-OMe in CD3OD at 20 degrees C compared with 1.28 ns for t-boc-LALALW-OMe. Given these tau m values and taking into account the effect of measurement error in the T1 and NOE data, the internal dynamics of the tryptophan residue of t-boc-LAWAL-OMe in this isotropic environment are described by a range of tau e values from 70 to 112 ps and sigma values between 0.22 and 0.36. Similarly, for t-boc-LALALW-OMe, 68 less than or equal to tau e less than or equal to 93 ps and 0.09 less than or equal to sigma less than or equal to 0.17. The Ch-terminal position of the tryptophan residue in the hexapeptide may account for its lower order parameter.(ABSTRACT TRUNCATED AT 400 WORDS)     

Internal mobility of the ologonucleotide duplexes d(TCGCG)2 and d(CGCGCG)2 in aqueous solution from molecular dynamics simulations

Summary In this paper we present longitudinal relaxation times, order parameters and effective correlation times for the base and sugar carbons in both strands of the oligonucleotide duplexes d(TCGCG)₂ and d(CGCGCG)₂, as calculated from 400 ps molecular dynamics trajectories in aqueous solution. The model-free approach (Lipari and Szabo, 1982) was used to determine the amplitudes and time scales of the internal motion. Comparisons were made with NMR relaxation measurements (Borer et al., 1994). The order parameters could acceptably be reproduced, and the effective correlation times were found to be lower than the experimental estimates. Reasonable T₁ relaxation times were obtained in comparison with experiment for the nonterminal nucleosides. The T₁ relaxation times were found to depend mainly on the order parameters and overall rotational correlation time.Abbreviations MD molecular dynamics - CSA chemical shift anisotropy To whom correspondence should be addressed.     

Off-resonance rotating frame spin-lattice NMR relaxation studies of phosphorus metabolite rotational diffusion in bovine lens homogenates

The rotational diffusion behavior of phosphorus metabolites present in calf lens cortical and nuclear homogenates was investigated by the NMR technique of 31P off-resonance rotating frame spin-lattice relaxation as a means of assessing the occurrence and extent of phosphorus metabolite-lens protein interactions. 31P NMR spectra of calf lens homogenates were obtained at 10 and 18 degrees C (below and above the cold cataract phase transition temperature, respectively) at 7.05 T. Effective rotational correlation times (tau 0,eff) for the major phosphorus metabolites present in cortical and nuclear bovine calf lens homogenates were derived from nonlinear least-squares analysis of R vs omega e (spectral intensity ratio vs precessional frequency about the effective field) data with the assumption of isotropic reorientational motion. Intramolecular dipole-dipole (1H-31P, 31P-31P), chemical shift anisotropy (CSA), and solvent (water) translational intermolecular dipole-dipole (1H-31P) relaxation contributions were assumed in the analyses. In those cases where the limiting value of the spectral intensity ratio failed to reach unity at large offset frequency, a modified formalism incorporating chemical exchange mediated saturation transfer between two sites was used. Values of tau 0,eff for phosphorus metabolites present in the cortex varied from a low of ca. 2 ns [L-alpha-glycero-phosphocholine (GPC)] to a high of 12 ns (alpha-ATP) at 10 degrees C, whereas at 18 degrees C the range was from ca. 1 to 9 ns. For the nucleus the tau 0,eff values ranged from ca. 3 ns (GPC) to 41 ns (Pi) at 10 degrees C; at 18 degrees C the corresponding values ranged from 4 to 39 ns. For PME (phosphomonoester; in lens the predominant metabolite is L-alpha-glycerol phosphate) at 18 degrees C evidence was obtained for binding and subsequent exchange with solid like protein domains. The diversity in tau 0,eff values for lenticular phosphorus metabolites is suggestive of differential binding to more slowly tumbling macromolecular species, most likely lens crystallin proteins. Corresponding measurement of tau 0,eff values for the mobile protein fraction present in calf lens cortical and nuclear homogenates at 10 and 18 degrees C, by 13C off-resonance rotating frame spin-lattice relaxation, provided average macromolecular correlation times that were assumed to represent the bound metabolite state. A fast-exchange model (on the T1 time scale), between free and bound forms, was employed in the analysis of the metabolite R vs omega e curves to yield the     

NMR (13)C-relaxation study of base and sugar dynamics in GCAA RNA hairpin tetraloop

Intramolecular dynamics of a 14-mer RNA hairpin including GCAA tetraloop was investigated by (13)C NMR relaxation. R(1) and R(1rho) relaxation rates were measured for all protonated base carbons as well as for C1' carbons of ribose sugars at several magnetic field strengths. The data has been interpreted in the framework of modelfree analysis [G. Lipari and A. Szabo. J Am Chem Soc 104, 4546-4559 (1982); G. Lipari and A. Szabo. J Am Chem Soc 104, 4559-4570 (1982)] characterizing the internal dynamics of the molecule by order parameters and correlation times for fast motions on picosecond to nanosecond time scale and by contributions of the chemical exchange. The fast dynamics reveals a rather rigid stem and a significantly more flexible loop. The cytosine and the last adenine bases in the loop as well as all the loop sugars exhibit a significant contribution of conformational equilibrium on microsecond to millisecond time scale. The high R(1rho) values detected on both base and sugar moieties of the loop indicate coordinated motions in this region. A semiquantitative analysis of the conformational equilibrium suggests the exchange rates on the order of 10(4) s(-1). The results are in general agreement with dynamics studies of GAAA loops by NMR relaxation and fluorescent spectroscopy and support the data on the GCAA loop dynamics obtained by MD simulations.     

Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents

The dynamics in isotopic solvents of selectively 13C labeled synthetic melittin and three analogues have been investigated by using NMR and fluorescence techniques both separately and in combination. In conjunction with the "model-free" approach to interpretation of NMR relaxation data [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4570], the availability of steady-state fluorescence anisotropy and lifetime data augment T1, T2, and NOE data to provide quantitative information about fluorophore dynamics in these peptides. A method is presented for using combined fluorescence and NMR data to obtain technique- and model-independent values for parameters describing local motion of 13C-labeled fluorophores in peptides and proteins. The dynamics of melittin and melittin analogues are found to be consistent with structural characteristics inferred from CD, fluorescence, and NMR spectral information presented in the preceding paper (Weaver et al., 1989). In particular, the mobility of the random coil peptide monomers is shown to be quite similar, while side-chain as well as peptide backbone motion in the aggregated or oligomeric species differs markedly among the analogues. For melittin itself, experimentally determined overall rotational correlation times for the monomer and tetramer agree very well with values predicted on the basis of solvent-accessible protein surface area. The local dynamics of selectively 13C-labeled Trp-19 and Gly-12 residues of melittin are also found to be consistent with peptide structure. In random coil melittin monomer, a specific model for the motion indicates that the Trp side chain moves through an approximate angle of +/- 71 degrees about the beta-gamma bond with a correlation time of 159 +/- 24 ps. In melittin tetramer, the indole moiety is spatially more confined with a flip angle of +/- 37 degrees, yet demonstrates an increased rate of motion with a correlation time of 56 +/- 8 ps. The constrained mobility of the Trp-19 side chain is consistent with motional constraints inferred from the X-ray structure of melittin tetramer. These results show that protein side-chain motion, even of moieties as large as indole, can occur on the picosecond time scale and that these motions are reasonably similar to those inferred from molecular dynamics simulations.     

13C NMR relaxation times of hepatic glycogen in vitro and in vivo

The field dependence of relaxation times of the C-1 carbon of glycogen was studied in vitro by natural-abundance 13C NMR. T1 is strongly field dependent, while T2 does not change significantly with magnetic field. T1 and T2 were also measured for rat hepatic glycogen enriched with [1-13C]glucose in vivo at 4.7 T, and similar relaxation times were observed as those obtained in vitro at the same field. The in vitro values of T1 were 65 +/- 5 ms at 2.1 T, 142 +/- 10 ms at 4.7 T, and 300 +/- 10 ms at 8.4 T, while T2 values were 6.7 +/- 1 ms at 2.1 T, 9.4 +/- 1 ms at 4.7 T, and 9.5 +/- 1 ms at 8.4 T. Calculations based on the rigid-rotor nearest-neighbor model give qualitatively good agreement with the T1 field dependence with a best-fit correlation time of 6.4 X 10(-9) s, which is significantly smaller than tau M, the estimated overall correlation time for the glycogen molecule (ca. 10(-5) s). A more accurate fit of T1 data using a modified Lipari and Szabo approach indicates that internal fast motions dominate the T1 relaxation in glycogen. On the other hand, the T2 relaxation is dominated by the overall correlation time tau M while the internal motions are almost but not completely unrestricted.     

Real-time kinetics of the interaction between the two subunits,Escherichia coli thioredoxin and gene 5 protein of phage T7 DNA polymerase

T7 phage DNA polymerase is a tight 1:1 complex of the gene 5 protein (g5p) (80 kDa) of phage T7 and thioredoxin (12 kDa) from the Escherichia coli host. The holoenzyme is essential for the replication of the phage. We estimated the real-time kinetics and thermodynamics of the interaction of g5p with thioredoxin (wild type and mutants) using surface plasmon resonance. Thioredoxin was immobilized on a CM5 sensor chip through a six-carbon spacer (6-amino-n-hexanoic acid) using standard amine coupling. Reduced thioredoxin bound g5p but oxidized thioredoxin did not. The association and dissociation phases of the complex fit a two-exponential model with an apparent equilibrium dissociation constant (KD) of 2.2 nm for thioredoxin with 4.7 x 104.M-1.s-1 and 10.5 x 10-5.s-1 as the corresponding association (ka) and dissociation (kd) rate constants. The strong binding of g5p to thioredoxin is therefore due to fast association and very slow dissociation, a situation similar to antigen-antibody interactions. Thioredoxin mutants P34S, D26A, K57M, D26A/K57M, W31F, W31Y, K36A, K36E, and Y49F had KD values in the range of 1 to 8 nm, whereas mutant W28A had a KD of 12.5 nm. No detectable interaction was observed for mutants P40G, W31H, W31A, and C35A. The effect of temperature on KD and the changes in enthalpy (-DeltaH = 20.2 kcal.m-1) and entropy (TDeltaS =-8.4 kcal.m-1) upon formation of the complex suggested that the interaction is driven by an increase in enthalpy and opposed by a decrease in entropy.     

High-resolution field-cycling NMR studies of a DNA octamer as a probe of phosphodiester dynamics and comparison with computer simulation

Phosphorus-spin longitudinal relaxation rates of the DNA duplex octamer [d(GGAATTCC)](2) have been measured from 0.1 to 17.6 T by means of conventional and new field-cycling NMR methods. The high-resolution field-cycling method is identical to a conventional relaxation experiment, except that after preparation the sample is moved pneumatically from its usual position at the center of the high-resolution magnet upward to a lower field above its normal position and then returned to the center for readout after it has relaxed for the programmed relaxation delay at the low field. This is the first measurement of all longitudinal relaxation rates R(1) of a nuclear species in a macromolecule over virtually the entire accessible magnetic field range. For detailed analysis, three magnetic field regions can be delineated: (i) dipolar relaxation dominates at fields below 2 T, (ii) chemical shift anisotropy (CSA) relaxation is roughly constant from 2 to 6 T, and (iii) a square-law increasing dependence is seen at fields higher than approximately 6 T due to internal motion CSA relaxation. The analysis provides a rotational correlation time (tau(r) = 4.1 +/- 0.3 ns) for the duplex at both 1.5 and 0.25 mM concentrations (of duplex) at 22 degrees C. For comparison, extraction of tau(r) in the conventional way from the ratio of T(1)/T(2) at 14 T yields 3.2 ns. The tau(r) discrepancy disappears when we exclude the contribution of internal motion from the R(1) in the ratio. The low-field dipolar relaxation provides a weighted inverse sixth power sum of the distances from the phosphorus to the protons responsible for relaxation. This average is similar for all phosphates in the octamer and similar to that in previous B-DNA structures (its inverse sixth root is about 2.40 A for two different concentrations of octamer). The CSA relaxation at intermediate field provides an estimate of the order parameter squared, S(c)(2), for each phosphorus. S(c)(2) is about 0.7-1, clearly different for different phosphate linkages in the octamer duplex. The increasing R(1) at high fields reflects CSA relaxation due to internal motions, for which a correlation time, tau(hf), can be approximately extracted with the aid of additional measurements at 14.0 and 17.6 T. We conclude that tau(hf) values are relatively large, in the range of about 150 ps. Insight into the motions leading to this correlation time was gained by a 28 ns molecular dynamics simulation of the molecule. S(2) and tau(s) (corresponding to tau(hf)) predicted by this simulation were in good agreement with the experimental values from the field-cycling data. Both the effect of Mg(2+) on the dynamic parameters extracted from (31)P relaxation rates and the field dependence of relaxation rates for several protons of the octamer were measured. High-resolution field cycling opens up the possibility of monitoring residue-specific dipolar interactions and dynamics for the phosphorus nuclei of diverse oligonucleotides.     

Structure and dynamics of melittin in lysomyristoyl phosphatidylcholine micelles determined by nuclear magnetic resonance.

Mixed micelles of the 26-residue, lytic peptide melittin (MLT) and 1-myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) in aqueous solution at 25 degrees C were investigated by (13)C- and (31)P-NMR spectroscopy. (13)C alpha chemical shifts of isotopically labeled synthetic MLT revealed that MLT in the micelle is predominantly alpha-helical and that the peptide secondary structure is stable from pH 4 to pH 11. Although the helical transformation of MLT as determined from NMR is evident at lipid:peptide molar ratios as low as 1:2, tryptophan fluorescence measurements demonstrate that well-defined micellar complexes do not predominate until lipid:peptide ratios exceed 30:1. (31)P linewidth measurements indicate that the interaction between phosphate ions in solution and cationic groups on MLT is pH dependent, and that the phosphoryl group of MMPC senses a constant charge, most likely +2, on MLT from pH 4 to pH 10. (13)C-NMR relaxation data, analyzed using the model-free formalism, show that the peptide backbone of MLT is partially, but not completely, immobilized in the mixed micelles. Specifically, order parameters (S(2)) of C alpha-H vectors averaged 0.7 and were somewhat larger for residues in the N-terminal half of the molecule. The amino terminal glycine had essentially the same range of motion as the backbone carbons. Likewise, order parameters for the trp side chain were similar to those found for the peptide C alpha moieties, as was verified by trp fluorescence anisotropy decay data. In contrast, the motion of the lysine side chains was less restricted, the average S(2) values for the C epsilon-H vectors being 0.19, 0.30, and 0.44 for lys-7, 21, and 23, respectively, for MLT in the mixed micelles. Values of the effective correlation time of the local motion tau e were in the motional narrowing limit and usually longer for side-chain atoms than for those in the backbone. The dynamics were independent of pH from pH 4 to pH 9, but at pH 11 the correlation time for the rotational motion of the mixed micelles as a whole increased from 10 ns to 16 ns, and S(2) for the lys side chains increased. Overall it appears that the MLT helix lies near the surface of the micelle at low to neutral pH, but at higher pH its orientation changes, accompanied by deeper penetration of the lysine side chains into the micelle interior. It is apparent, however, that the MLT-lipid interaction is not dependent on deprotonation of any of the titratable cationic groups in the peptide in the pH 4-10 range, and that there is substantial backbone and side-chain mobility in micelle-bound MLT.     

Dynamics of methyl groups in proteins as studied by proton-detected 13C NMR spectroscopy. Application to the leucine residues of staphylococcal nuclease.

This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having Mr = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-13C2]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the 13C T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau f (5-80 ps) and tau s (less than 2 ns). The fast motion is identified with the rapid rotation of the methyl group about the C gamma-C delta bond axis, while the slow motion is associated with reorientation of the C gamma-C delta bond axis itself. The mean squared order parameters associated with the latter motion, Ss2, lie in the range 0.34-0.92. The values of Ss2 correlate reasonably well with the temperature factors of the leucine methyl carbons obtained from the crystal structures, but some are smaller than anticipated on the basis of the fact that nearly all leucine methyl carbons are buried and have temperature factors no larger than that of the leucine backbone atoms. Five leucine residues in liganded SNase and eight in unliganded SNase have values of Ss2 less than 0.71. These order parameters correspond to large amplitude motions (angular excursions of 27-67 degrees) of the C gamma-C delta bond axis. These results indicate that, in solution, the internal motions of the leucine side chains of SNase are significantly larger than suggested by the X-ray structures or by qualitative analysis of NOESY spectra. Comparison of Ss2 values obtained from liganded and unliganded SNase reveals a strong correlation between delta Ss2 and distance between the leucine methyl carbon and the ligands.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Solution conformation and dynamics of a fungal cell wall polysaccharide isolated from Microsporum gypseum

The conformational and dynamical features of a branched mannan isolated from a fungal cell wall have been analysed by homo and heteronuclear NMR methods, employing different magnetic fields. 1HNMR cross relaxation times have been obtained for this polysaccharide and have been interpreted qualitatively using different motional models. 13C NMR relaxation parameters (T1, T2, NOE) have also been measured and interpreted using different approximations based on the Lipari and Szabo model free approach. The analysis of the data indicate the existence of important flexibility for the different linkages of the polysaccharide. Motions in the range of 4–6 ns contribute to the relaxation of the macromolecule, although faster internal motions in the 500 ps and 100 ps timescales are also present. These time scales indicate that segmental motions as well as internal motions around the glycosidic linkages are the major sources of relaxation for this molecule at 318 K. Molecular dynamics simulations have also been performed. The obtained results also indicate that the polysaccharide possess a substantial amount of conformational freedom.     

Mobility of individual 5-fluorouridine residues in 5-fluorouracil-substituted Escherichia coli valine transfer RNA. A 19F nuclear magnetic resonance relaxation study

19F nuclear magnetic resonance (n.m.r.) relaxation parameters of 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 were measured and used to characterize the internal mobility of individual 5-fluorouridine (FUrd) residues in terms of several models of molecular motion. Measured relaxation parameters include the spin-lattice (T1) relaxation time at 282 MHz, the 19F(1H) NOE at 282 MHz, and the spin-spin (T2) relaxation time, estimated from linewidth data at 338 MHz, 282 MHz and 84 MHz. Dipolar and chemical shift anisotropy contributions to the 19F relaxation parameters were determined from the field-dependence of T2. The results demonstrate a large chemical shift anisotropy contribution to the 19F linewidths at 282 and 338 MHz. Analysis of chemical shift anisotropy relaxation data shows that, relative to overall tumbling of the macromolecule, negligible torsional motion occurs about the glycosidic bond of FUrd residues in 19F-labeled tRNA(Val)1, consistent with the maintenance of base-base hydrogen-bond and/or stacking interactions at all fluorouracil residues in the molecule. The dipolar relaxation data are analyzed by using the "two-state jump" and "diffusion in a cone" formalisms. Motional amplitudes (theta) are interpreted as being due to pseudorotational fluctuations within the ribose ring of the fluorinated nucleoside. These amplitudes range from approximately 30 degrees to 60 degrees, assuming a correlation time (tau i,2) of 1.6 ns. By using available 19F n.m.r. assignment data for the 14 FUrd residues in 5-fluorouracil-substituted tRNA(Val)1, these motional amplitudes can be correlated directly with the environmental domain of the residue. Residues located in tertiary and helical structural domains show markedly less motion (theta approximately equal to 30 to 35 degrees) than residues located in loops (theta approximately equal to 45 to 60 degrees). A correlation between residue mobility and solvent exposure is also demonstrated. The amplitudes of internal motion for specific residues agree quite well with those derived from X-ray diffraction and molecular dynamics data for yeast tRNA(Phe).     

Cholesterol dynamics in membranes.

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.     

Fast internal main-chain dynamics of human ubiquitin.

The fast internal dynamics of human ubiquitin have been studied by the analysis of 15N relaxation of backbone amide nitrogens. The amide 15N resonances have been assigned by use of heteronuclear multiple-quantum spectroscopy. Spin lattice relaxation times at 60.8 and 30.4 MHz and the steady-state nuclear Overhauser effect at 60.8 MHz have been determined for 67 amide 15N sites in the protein using two-dimensional spectroscopy. These data have been analyzed in terms of the model free treatment of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The global motion of the protein is shown to be isotropic and is characterized by a correlation time of 4.1 ns rad-1. The generalized order parameters (S2) of backbone amide N-H vectors in the globular region of the protein range from 0.5 to 0.95. No apparent correlation between secondary structure and generalized order parameters is observed. There is, however, a strong correlation between the magnitude of the generalized order parameters of a given N-H vector and the presence of hydrogen bonding of the amide hydrogen or its peptide bond associated carbonyl. Using a chemical shift tensor breadth of 160 ppm, the N-H vectors of peptide linkages participating in one or more hydrogen bonds to the main chain show an average generalized order parameter of 0.80 (SD 0.06), while those amide NH of peptide linkages free of hydrogen-bonding interactions with the main chain show an average order parameter of 0.69 (SD 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)