Generalized Order Parameters Research Articles

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 (<S(NH)(2)> 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.

(15)N backbone dynamics of ferricytochrome b(562): comparison with the reduced protein and the R98C variant.

The backbone dynamics of ferricytochrome b(562), a four-helix bundle protein from Escherichia coli, have been studied by NMR spectroscopy. The consequences of the introduction of a c-type thioether linkage between the heme and protein and the reduction to the ferrous cytochrome have also been analyzed. (15)N relaxation rates R(1) and R(2) and (1)H-(15)N NOEs were measured at proton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced protein as well as for the oxidized R98C variant. In the latter protein, an "artificial" thioether covalent bond has been introduced between the heme group and the protein frame [Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., de Lumley Woodyear, T., Johnson, C. M., and Barker, P. D. (2000) Biochemistry 39, 1499-1514]. The (15)N relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanosecond time scale were compared for the three species. The three forms are rather rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochrome b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system. Lower order parameters (S(2)) are found for residues belonging to loops 1 and 2. Higher mobility, as indicated by lower order parameters, is found for heme binding helices alpha 1 and alpha 4 in the R98C variant with respect to the wild-type protein. The analysis requires a relatively long rotational correlation time (tau(m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular shape and the high phosphate concentration needed to ensure the occurrence of monomer species. A parallel study of motions in the millisecond to microsecond time scale has also been performed on oxidized wild-type and R98C cytochrome b(562). In a CPMG experiment, decay rates were analyzed in the presence of spin-echo pulse trains of variable spacing. The dynamic behavior on this time scale is similar to that observed on the sub-nanosecond time scale, showing an increased mobility in the residues connected to the heme ligands in the R98C variant. It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity.

NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor

The ligand-binding region of the low-density lipoprotein (LDL) receptor is formed by seven N-terminal, imperfect, cysteine-rich (LB) modules. This segment is followed by an epidermal growth factor precursor homology domain with two N-terminal, tandem, EGF-like modules that are thought to participate in LDL binding and recycling of the endocytosed receptor to the cell surface. EGF-A and the concatemer, EGF-AB, of these modules were expressed in Escherichia coli. Correct protein folding of EGF-A and the concatemer EGF-AB was achieved in the presence or absence of calcium ions, in contrast to the LB modules, which require them for correct folding. Homonuclear and heteronuclear 1H- 15N NMR spectroscopy at 17.6 T was used to determine the three-dimensional structure of the concatemer. Both modules are formed by two pairs of short, anti-parallel β-strands. In the concatemer, these modules have a fixed relative orientation, stabilized by calcium ion-binding and hydrophobic interactions at the interface. 15N longitudinal and transverse relaxation rates, and { 1H}- 15N heteronuclear NOEs were used to derive a model-free description of the backbone dynamics of the molecule. The concatemer appears relatively rigid, particularly near the calcium ion-binding site at the module interface, with an average generalized order parameter of 0.85±0.11. Some mutations causing familial hypercholesterolemia may now be rationalized. Mutations of D41, D43 and E44 in the EGF-B calcium ion-binding region may affect the stability of the linker and thus the orientation of the tandem modules. The diminutive core also provides little structural stabilization, necessitating the presence of disulfide bonds. The structure and dynamics of EGF-AB contrast with the N-terminal LB modules, which require calcium ions both for folding to form the correct disulfide connectivities and for maintenance of the folded structure, and are connected by highly mobile linking peptides.

A comprehensive analysis of multifield 15N relaxation parameters in proteins: determination of 15N chemical shift anisotropies.

This study deals with the exploitation of the three classical 15N relaxation parameters (the longitudinal relaxation rate, R1, the transverse relaxation rate, R2, and the 1H-15N cross-relaxation rate, sigmaNH) measured at several magnetic fields in uniformly 15N-labeled proteins. Spectral densities involved in R1, R2 and sigmaNH are analyzed according to the functional form A + B/(1 + omega(2) taus(2)), where taus is the correlation time associated with slow motions sensed by the NH vector at the level of the residue to which it belongs. The coefficient B provides a realistic view of the backbone dynamics, whereas A is associated with fast local motions. According to the "model free approach", B can be identified with 2tausS(2) where S is the generalized order parameter. The correlation time taus is determined from the field dependency of the relaxation parameters while A and B are determined through linear equations. This simple data processing is needed for obtaining realistic error bars based on a statistical approach. This proved to be the key point for validating an extended analysis aiming at the determination of nitrogen chemical shift anisotropy. The protein C12A-p8(MTCP1) has been chosen as a model for this study. It will be shown that all data (obtained at five magnetic field strengths corresponding to proton resonance of 400, 500, 600, 700, and 800 MHz) are very consistently fitted provided that a specific effective correlation time associated with slow motions is defined for each residue. This is assessed by small deviations between experimental and recalculated values, which, in all cases, remain within experimental uncertainty. This strategy makes needless elaborate approaches based on the combination of several slow motions or their possible anisotropy. Within the core of the protein taus fluctuates in a relatively narrow range (with a mean value of 6.15 ns and a root-mean-square deviation of 0.36 ns) while it is considerably reduced at the protein extremities (down to approximately 3 ns). To a certain extent, these fluctuations are correlated with the protein structure. A is not obtained with sufficient accuracy to be valuably discussed. Conversely, order parameters derived from B exhibit a significant correlation with the protein structure. Finally, the multi-field analysis of the evolution of longitudinal and transverse relaxation rates has been refined by allowing the 15N chemical shift anisotropy (csa) to vary residue by residue. Within uncertainties (derived here on a statistical basis) an almost constant value is obtained. This strongly indicates an absence of correlation between the experimental value of this parameter obtained for a given residue in the protein, the nature of this residue, and the possible involvement of this residue in a structured area of the protein.

Model‐free analysis of a thermophilic Fe7S8 protein compared with a mesophilic Fe4S4 protein

15N T1, T2 and 1H-15N NOE were measured for the thermophilic Fe7S8 protein from Bacillus schlegelii and for the Fe4S4 HiPIP protein from Chromatium vinosum, which is a mesophilic protein. The investigation was performed at 276, 300, and 330 K at 11.7 T for the former, whereas only the 298 K data at 14.1 T for the latter were acquired. The data were analyzed with the model-free protocol after correcting the measured parameters for the effect of paramagnetism, because both proteins are paramagnetic. Both thermophilic and mesophilic proteins are quite rigid, with an average value of the generalized order parameter S2at room temperature of 0.92 and 0.94 for Fe7S8 and Fe4S4 proteins, respectively. The analyzed nitrogens for the Fe7S8 protein showed a significant decrease in S2with increasing temperature, and at the highest temperature >70% of the residues had an internal correlation time. This research shows that subnanosecond rigidity is not related to thermostability and provides an estimate of the effect of increasing temperature on this time scale. Proteins 2000;41:75–85. © 2000 Wiley-Liss, Inc.

Conformational Flexibility of the Disaccharide α-d-Manp-(1→3)-β-d-Glcp-OMe Employing Molecular Dynamics Simulations and trans-Glycosidic 3JC,H from NMR Experiment

Molecular dynamics simulations of the disaccharide α-d-Manp-(1→3)-β-d-Glcp-OMe have been performed in vacuo and in water solution. Heteronuclear scalar coupling constants were measured across the glycosidic linkage. In conjunction with previously obtained experimental data, namely a trans-glycosidic proton−proton distance and the generalized order parameter a description of the molecular system is presented. The disaccharide exists in a major conformational state and has limited flexibility on a shorter time scale, i.e., less than its rotational correlation time (τM). Excursions to other conformational states are required to obtain agreement between simulation and experiment. Thus, on a longer time scale (>τM) these conformational states are populated to some extent. The combined approach of molecular dynamics simulations and NMR experiments is therefore able to generate a consistent molecular picture.

Backbone dynamics of escherichia coli adenylate kinase at the extreme stages of the catalytic cycle studied by (15)N NMR relaxation.

Adenylate kinase from Escherichia coli (AKeco), consisting of a single 23.6 kDa polypeptide chain folded into domains CORE, AMPbd, and LID, catalyzes the reaction AMP + ATP --> 2ADP. Domains LID and AMPbd execute large-scale movements during catalysis. Backbone dynamics of ligand-free and AP(5)A-inhibitor-bound AKeco were studied comparatively with (15)N NMR relaxation methods. Overall diffusion with correlation times of 15.05 (11.42) ns and anisotropy D(parallel)/D(perp) = 1.25 (1.10), and fast internal motions with correlation times up to 100 ps (50 ps), were determined for AKeco (AKecoAP(5)A). Fast internal motions affect 93% of the AKeco sites, with pronounced preference for domains AMPbd and LID, and 47% of the AKecoAP(5)A sites, with limited variability along the chain. The mean squared generalized order parameters, <S(2)>, of secondary structure elements and loops are affected by ligand binding differentially and in a domain-specific manner. Nanosecond motions predominate within AMPbd. Prominent exchange contributions, associated in particular with residue G10 of the nucleotide-binding P-loop motif, are interpreted to reflect hydrogen-bond dynamics at the inhibitor-binding site. The hypothesis of energetic counter balancing of substrate binding based on crystallographic data is strongly supported by the solution NMR results. Correlations between backbone dynamics and domain displacement are established.

Dynamics of the Hck-SH3 domain: comparison of experiment with multiple molecular dynamics simulations.

Molecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X-ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481-493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted from these calculations to those determined through experimental means. Here, we investigate the dynamic behavior of the Src homology 3 (SH3) domain of hematopoietic cell kinase (Hck) via 5N backbone relaxation NMR studies and a set of four independent 4 ns solvated molecular dynamics calculations. We also find that molecular dynamics simulations accurately reproduce fast motion dynamics as estimated from generalized order parameter (S2) analysis for regions of the protein that have experimentally well-defined coordinates (i.e., stable secondary structural elements). However, for regions where the coordinates are not well defined, as indicated by high local root-mean-square deviations among NMR-determined structural family members or high B-factors/low electron density in X-ray crystallography determined structures, the parameters calculated from a short to moderate length (less than 5-10 ns) molecular dynamics trajectory are dependent on the particular coordinates chosen as a starting point for the simulation.

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.

Distinguishing Anisotropy and Flexibility of the Pentasaccharide LNF-1 in Solution by Carbon-13 NMR Relaxation and Hydrodynamic Modeling

Carbon-13 spin−lattice (T1) and spin−spin (T2) relaxation times and 1H−13C NOEs have been measured at four magnetic field strengths (7.0, 9.4, 11.7, and 14.1 T) for a 30 °C D2O solution of lacto-N-fucopentaose-1, a pentasaccharide from human milk. There are significant differences between the residues, with the largest T1, T2, and NOE values for carbons with axial C−H bonds of the 4-substituted glucose unit (E) at the reducing end. The data were fit using model free formalism, first with overall rotation assumed to be isotropic and then with rotational anisotropy taken into account. Somewhat improved fits were obtained with the latter, which yielded an anisotropy D||/D⊥ = 1.35−1.5. The generalized order parameters, S2, for ring carbons of residues A−D were similar in the two models, and approximately equal to 0.7, though significant differences in both order parameters and internal relaxation times were obtained for residue E. As supported by Langevin dynamics simulations, the experimentally derived S2 for the ring carbons of residue E, 0.68 for C1 (α-anomeric configuration) and 0.43 for C2 and C3, are best explained by large fluctuations within the minimum of a single dominant conformation. The translational diffusion coefficient, Dt, obtained from NMR measurements equaled 1.8 × 10-6 cm2 s-1. Hydrodynamic calculations on the energy minimized dominant conformation yielded D||/D⊥ = 1.8 and Dt = 2.0 × 10-6 cm2 s-1, assuming one bound water per hydroxyl group and rigid geometry. The difference between the anisotropy fitted from relaxation data and that calculated by hydrodynamics is proposed to be associated with the substantial flexibility at the reducing end.

Multiple field carbon-13 NMR relaxation of calix[4]arene in solution

Carbon-13 relaxation times and nuclear Overhauser enhancement values were determined for the carbons of calix[4]arene in both CDCl3 and DMSO-d6 solvents at magnetic field strengths of 5.9, 9.4, and 14.1 T at 30 °C. The relaxation data for the CDCl3 solution were well described by an isotropic motional model indicating motions in the extreme narrowing limit with a rotational correlation time of 32–38 ps. The effective shielding anisotropies for the substituted aromatic carbons were found to be in the range 150–170 ppm while the unsubstituted aromatic carbons had values in the range 180–200 ppm. In DMSO-d6 solution, the dipolar relaxation is out of the extreme narrowing limit and it was possible to apply a Lipari–Szabo treatment of the relaxation data. This model demonstrated through the value of the generalized order parameter, S2, a slight increase in the range of motion for the aliphatic CH2 carbon over the unsubstituted aromatic carbons. It was not possible, however, to determine reliable values for the effective shielding anisotropies for the aromatic carbons. An earlier report of carbon-13 relaxation for calix[4]arene in CDCl3 had indicated distinctly different relaxation times for the two unsubstituted aromatic carbons. We found virtually identical relaxation times for both carbons at all three magnetic fields used in this study. Copyright © 1999 John Wiley & Sons, Ltd.

Backbone dynamics of inactive, active, and effector-bound Cdc42Hs from measurements of (15)N relaxation parameters at multiple field strengths.

Cdc42Hs, a member of the Ras superfamily of GTP-binding proteins, initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously determined the structure of Cdc42Hs and found that the regions involved in effector (Switch I) and regulator (Switch II) actions are partially disordered [Feltham, J. L., et al. (1997) Biochemistry 36, 8755-8766]. Recently, we used a 46-amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs, which includes the beta2 strand and a portion of Switch I [Guo, W., et al. (1998) Biochemistry 37, 14030-14037]. Here we describe the backbone dynamics of three constructs of [(15)N]Cdc42Hs (GDP-, GMPPCP-, and GMPPCP- and PBD46-bound) using (15)N-(1)H NMR measurements of T(1), T(1)(rho), and the steady-state NOE at three magnetic field strengths. Residue-specific values of the generalized order parameters (S(s)(2) and S(f)(2)), local correlation time (tau(e)), and exchange rate (R(ex)) were obtained using the Lipari-Szabo model-free formalism. Residues in Switch I were found to exhibit high-amplitude (low-order) motions on a nanosecond time scale, whereas those in Switch II experience low-amplitude motion on the nanosecond time scale and chemical (conformational) exchange on a millisecond time scale. The Insert region of Cdc42Hs-GDP exhibits high-order, nanosecond motions; the time scale of motion in the Insert is reduced in Cdc42Hs-GMPPCP and Cdc42Hs-PBD46. Overall, significant flexibility was observed mainly in the regions of Cdc42Hs that are involved in protein-protein interactions (Switch I, Switch II, and Insert), and flexibility was reduced upon interaction with a protein ligand. These results suggest that protein flexibility is important for high-affinity binding interactions.

Establishing a degree of order: obtaining high-resolution NMR structures from molecular alignment

I would like to thank Eva de Alba for providing structural data as well as Ad Bax and James Baber for their critical comments.

Backbone dynamics and energetics of a Calmodulin domain mutant exchanging between closed and open conformations

Previous studies have suggested that the Ca 2+-saturated E140Q mutant of the C-terminal domain of calmodulin exhibits equilibrium exchange between “open” and “closed” conformations similar to those of the Ca 2+-free and Ca 2+-saturated states of wild-type calmodulin. The backbone dynamics of this mutant were studied using 15N spin relaxation experiments at three different temperatures. Measurements at each temperature of the 15N rate constants for longitudinal and transverse auto-relaxation, longitudinal and transverse cross-correlation relaxation, and the 1H- 15N cross-relaxation afforded unequivocal identification of conformational exchange processes on microsecond to millisecond time-scales, and characterization of fast fluctuations on picosecond to nanosecond time-scales using model-free approaches. The results show that essentially all residues of the protein are involved in conformational exchange. Generalized order parameters of the fast internal motions indicate that the conformational substates are well folded, and exclude the possibility that the exchange involves a significant population of unfolded or disordered species. The temperature dependence of the order parameters offers qualitative estimates of the contribution to the heat capacity from fast fluctuations of the protein backbone, revealing significant variation between the well-ordered secondary structure elements and the more flexible regions. The temperature dependence of the conformational exchange contributions to the transverse auto-relaxation rate constants directly demonstrates that the microscopic exchange rate constants are greater than 2.7×10 3s −1at 291 K. The conformational exchange contributions correlate with the chemical shift differences between the Ca 2+-free and Ca 2+-saturated states of the wild-type protein, thereby substantiating that the conformational substates are similar to the open and closed states of wild-type calmodulin. Taking the wild-type chemical shifts to represent the conformational substates of the mutant and populations estimated previously, the microscopic exchange rate constants could be estimated as 2×10 4to 3×10 4s −1at 291 K for a subset of residues. The temperature depen dence of the exchange allows the characterization of apparent energy barriers of the conformational transition, with results suggesting a complex process that does not correspond to a single global transition between substates.

Analysis of the relationship between enzyme activity and its internal motion using nuclear magnetic resonance: 15N relaxation studies of wild-type and mutant lysozyme

A mutant lysozyme where R14 and H15 are deleted together has higher activity and a similar binding ability to an inhibitor, trimer of N-acetylglucosamine ((NAG)3), compared with wild-type lysozyme. Since this has been attributed to intrinsic protein dynamic properties, we investigated the relationship between the activity and the internal motions of proteins. Backbone dynamics of the free and the complex forms with the (NAG)3 have been studied by measurement of the 15N T1 and T2 relaxation rates and NOE determinations at 600 MHz. Analysis of the data using the model-free formalism showed that the generalized order parameters (S2) were almost the same in wild-type and mutant lysozyme in unbound state, indicating that the mutation had little effect on the global internal motions. On the other hand, in the presence of (NAG)3, although some signals located around the active site were broadened or decreased in intensity because of strong perturbation by (NAG)3, there were several residues that showed increased or decreased backbone S2 in the complexed lysozymes. A comparison of the internal motions of the wild-type and mutant complexes showed a number of distinct dynamic differences between them. In particular, many residues located at or near active-site regions (turn 1, strand 2, turn 2 and long loop), displayed greater backbone dynamics reflecting the order parameter in mutant complex relative to mutant free. Furthermore, the Rex values at the loop C-D region, which was considered to be important for enzymatic activity, significantly increased. From these results, it was suggested that variations in the dynamics of these regions may play an important role in the enzyme activity.

Dynamics of a bis-intercalator DNA complex by 1H-detected natural abundance 13C NMR spectroscopy.

The dynamics of the DNA oligomer d(CGCTAGCG)2 (CTSYM) and its complex with the dye 1,1-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1, 3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO) (CTSYMTOTO) bis-intercalated at the 5'-CT-3' sequence steps have been determined from NMR relaxation parameters. Longitudinal and transverse 13C relaxation rates and heteronuclear NOE relaxation data were acquired and have been analyzed in the context of the Lipari and Szabo model-free formalism. The overall rotational correlation time for the CTSYM is 3.44 ns and the CTSYMTOTO is 3.48 ns. The generalized order parameters (S2) for methine carbons in the CTSYM and CTSYMTOTO are relatively high but nonuniform for the molecules and show sequence context and conformation-dependent variations. Average values of S2 = 0.79 +/- 0.02 for the CTSYM, S2 = 0.80 +/- 0.04 for the CTSYMTOTO aromatic spins, S2 = 0.76 +/- 0.02 for the CTSYM, and S2 = 0.83 +/- 0.05 for the CTSYMTOTO deoxyribose spins were found. The S2 values for the 5'-terminal deoxyribose are lower than for the other residues. The DNA backbone in CTSYMTOTO is distorted and elongated at the site of intercalation, and the C3' atom of the C3 deoxyribose residue has a very low S2 = 0.57 +/- 0.06. The low order for this spin is interpreted in terms of exchange between the C2'-endo and O1'-endo conformations of the C3 deoxyribose. Significant chemical exchange processes were found for most of the aromatic spins in CTSYM that are interpreted in terms of microsecond to millisecond time scale dynamics. The microsecond to millisecond dynamics of the bases in CTSYM are quenched upon TOTO complex formation due to unwinding of the helix and an increase in the surface area of the bases in mutual contact and the large surface area in contact with the intercalated dye. The derived order parameters combined with the solution structure provide motional models for conformational changes induced in the backbone in response to the ligand binding.

Heteronuclear relaxation study of the PH domain of β-spectrin: restriction of loop motions upon binding inositol trisphosphate

The structural dynamics of protein ligand binding sites is one factor determining the specificity towards related ligands. In this context, the spectrin PH domain, which binds to a number of phosphatidylinositol lipid head groups, was investigated with respect to the dynamics of the binding loops. The latter were found to be of intermediate flexibility on a picosecond to nanosecond time-scale in the free protein and become more rigid upon ligand binding. Significant 15N and proton chemical shift changes occur in the binding loops. The internal correlation time, determined from 15N heteronuclear relaxation data using the standard model-free approach, decreases upon ligand binding. For several residues a concomitant rise in the generalized order parameter is observed. This is interpreted as a dampening effect of the ligand on a slow loop motion, while a fast component is not affected. Molecular dynamics simulations were performed to further investigate this situation. In fact, two time-scales of loop motions in the free state are observed in a 9 ns molecular dynamics trajectory. Agreement with generalized order parameters obtained from the experiment improves when a subtrajectory is analyzed that excludes rare dihedral transitions.

Self-association and backbone dynamics of the hck SH2 domain in the free and phosphopeptide-complexed forms.

Decreased dynamic motion in the peptide backbone of proteins may accompany ligand binding and influence the thermodynamic and kinetic stability of the resulting complexes. We have investigated the diffusional behavior and backbone dynamics of the free and phosphopeptide (EPQpYEEIPIYL) complexed Hck SH2 domain using NMR spectroscopy. Both the free domain and its phosphopeptide complex self-associate at higher protein concentrations. Diffusional measurements and surface analysis indicate that charged side-chain groups are probably responsible for self-association. Higher order aggregation, such as trimer and tetramer, also occurs at elevated protein concentrations. Dynamic motion in the peptide backbone of Hck SH2 was determined from 15N relaxation data fit using extended model-free parameters. The rotational correlation time (taum) for uncomplexed Hck SH2 was 6.8 ns while taum for peptide-bound Hck SH2 was 7.6 ns. Generalized order parameters (S2) increased for most residues upon binding of the phosphopeptide, consistent with peptide binding restricting motion of the NH bond vectors on the picosecond time scale. These studies suggest that complexation increases internal order in Hck SH2 and that internal dynamic motions contribute to the activation of Src-family kinases in vivo.

Structural stability and internal motions of Escherichia coli ribonuclease HI: 15N relaxation and hydrogen-deuterium exchange analyses

The relationship between the structural stability and the internal motions of proteins was investigated through measurements of15N relaxation and hydrogen-deuterium exchange rates of ribonuclease HI from Escherichia coli and its thermostable quintuple mutant (Gly23→Ala, His62→Pro, Val74→Leu, Lys95→Gly, and Asp134→His), which has a higher melting temperature by 20.2°C. For most of the residues, the generalized order parameters (S2) obtained from15N relaxation analyses as well as the localized hydrogen-bond-breaking motions (local breathing) observed as fast HD exchange rates were largely unaffected by the mutations, indicating no global mutational effect on the internal motions. Several local mutational effects were observed for residues close to the mutation sites as follows. The S2value significantly increased for Lys96 and Val98, which indicated that motions on the pico- to nanosecond time-scale became restricted within a protruding region including the Lys95→Gly mutation site. In contrast, slight decreases in S2, and drastic increases in the chemical exchange motion on the micro- to millisecond time-scale (Δex), were observed for residues located in the joining region between the protrusion and the major domain of the protein. These changes may be caused by the elimination of the bulky Lys95 side-chain at the center of the protrusion. Δex observed for residues in α-helix I of the wild-type protein was reduced for the mutant, probably because a cavity in the hydrophobic core is filled by the Val74→Leu mutation. The local breathing at position 134 was restricted by the Asp134→His mutation, probably because the reduction of the negative charge repulsion contributes to the stability of the native major conformation relative to the breathing conformations around position 134.

Dynamics in psoralen-damaged DNA by 1H-detected natural abundance 13C NMR spectroscopy.

The dynamics of the DNA oligomer d(GCGTACGC)2 and the 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen-DNA furanside monoadduct (MAf) of this oligomer have been determined from NMR relaxation parameters. Longitudinal and transverse 13C relaxation rates and heteronuclear NOE relaxation data have been measured at natural abundance and have been analyzed in the context of the Lipari and Szabo model-free formalism. The generalized order parameters for methine carbons in the octamer sequence d(GCGTACGC)2 (UM) are relatively and uniformly high for the entire molecule. The generalized order parameters for methine carbons in the MAf are significantly lower for the deoxyribose bearing the damaged thymidine base and for the bases flanking the lesion on the undamaged strand, indicating additional conformational flexibility due to the lesion. The order parameters for the bases on the damaged strand flanking the lesion remain high. Analysis of the relaxation data indicates substantial chemical exchange for the adenosine residues in the UM TpA site, and this chemical exchange is quenched upon MAf formation. These data are discussed in terms of a model for DNA damage recognition by the nucleotide excision repair system.