The anisotropy of fast backbone dynamics of ubiquitin is explored by 15N and 13CO nuclear spin relaxation and molecular dynamics (MD) simulation. T1, T1rho, and NOE data were collected at 400 and 600 MHz, and a 3ns MD simulation of ubiquitin was performed using CHARMM 24 explicitely including the water solvent. Analysis of the MD trajectory suggests that the relaxation-active motion of the 15N and 13CO spins is dominated for 82% of the residues by anisotropic Gaussian axial fluctuations (3D GAF) of the peptide planes about three orthogonal axes. The dominant fluctuation axes are on the average parallel to the Calpha-Calpha axes. The rest of the residues belongs to more flexible regions of the backbone and cannot be described by this type of motion exclusively. A 3D GAF motional model [1] was applied to the relaxation data of those experimentally accessible peptide planes which show a 3D GAF motion according to the MD analysis and do not exhibit conformational exchange contributions in their T1rho values. The fluctuation amplitudes which were extracted for 62% of the residues by least-squares fitting to the experimental data show a significant anisotropy of the internal motion similar to the one found in the MD simulation. This analysis demonstrates that a combined interpretation of the relaxation data of the 15N and 13CO spins belonging to the same peptide bond significantly enhances our insight into polypeptide backbone dynamics from NMR data. [1] Bremi, T.; Brueschweiler, R.; J. Am. Chem. Soc., 1997, 119, 6672-6673
The backbone dynamics of uniformly 15N-labeled reduced and oxidized putidaredoxin (Pdx) have been studied by 2D 15N-relaxation measurements. T1, T2, and 1H-15N NOEs have been measured for the diamagnetic region of the protein. The data were analyzed by using a model free dynamics formalism1 to determine the generalized order parameters (S2), the effective correlation time for internal motions (taue) and 15N exchange broadening contributions (Rex) for each residue, as well as the overall correlation time (taum). Order parameters for the reduced Pdx are generally higher than for the oxidized Pdx, and there is increased mobility on both the ns-ps and ms-us timescales for the oxidized Pdx in comparison with the reduced Pdx. The 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 exchange2. In addition, we observed very high T1/T2 ratios for residues 33 and 34, which indicate a large Rex contribution. Residue 34 especially, is believed3 to be involved in the binding of Pdx with cytochrome P450cam (CYP101). The differences in the backbone dynamics will be discussed in relation to the oxidation states of Pdx and their impact on electron transfer. 1. Arthur M. Mandel, Mikael Akke, and Arthur G. Palmer III, J. Mol. Biol. 246, 144-163 (1995). 2. Teresa A. Lyons, Gayathri Ratnaswamy, and Thomas C. Pochapsky, Protein Sci. 5, 627-639 (1996). 3. T.C. Pochapsky, T.A. Lyons, S. Kazanis, T. Arakaki, and G. Ratnaswamy, Biochimie 78, 723-733 (1996).
Backbone dynamics is an integral part of structure studies of biomolecules by NMR. The backbone dynamics of a 2H and 15N-labelled winged helix protein, Genesis, and its DNA complex was studied through the 15N relaxation experiments of R1, R2 and heteronuclear 1H-15N NOE at a 600 MHz spectrometer. The model_free approach was employed to analysis the experimental data. The results show that for the single domain protein without DNA, multi overall correlation times were needed to explain the experimental results. This indicates that there exist relative motions between regions of the protein. Further, By comparing the backbone dynamics of the Genesis/DNA complex with DNA-free Genesis, the significant differences were observed for internal motions.
The pleckstrin homology (PH) domain of the G-protein coupled receptor kinase 2 ([beta]ARK1) consists of 119 amino acid residues. Its three-dimensional structure was recently solved by NMR spectroscopy [1]. The backbone dynamics of the [beta]ARK1 PH domain were investigated by 15N NMR relaxation (R1 and R2) and steady state heteronuclear 15N {1H} nuclear Overhauser effect measurements at 400, 500 and 600 MHz. Furthermore, the dynamics of the [beta]ARK1 PH domain were simulated using different protocols of molecular dynamics (MD) simulations and two different force fields (AMBER94 and CHARMM22). The experimental and simulated data are compared in order to assess the quality of the MD simulations and to complete the description of the backbone dynamics of the [beta]ARK1 PH domain. The interrelationship of experimental and simulated dynamics permits more accurate assessment of the static and flexible features of protein structures [2]; and the role of flexibility in loops in PH domains in general, and in the "molten helix" and flexible C-terminus of [beta]ARK1 PH domain in particular. The latter contains significant parts of the molecule contributing to its protein-protein interaction with G[beta][gamma] subunits. [1] Fushman, D. et al. (1998) J. Biol. Chem., Vol. 273, in press. [2] Pfeiffer, S. et al. (1997) J. Mol. Biol. 266, 400-423
Cucurbita maxima trypsin inhibitor V (CMTI-V; Mr 7 kDa) of the potato I family provides an excellent model system for investigating structural determinants of internal dynamics and the effects thereof on protein function and stability toward proteolysis. CMTI-V is a canonical inhibitor of serine proteases, including the activated form of blood coagulation factor XII. Its binding loop (residues 39-48), which contains the scissile bond between K44 and D45, is anchored to the protein scaffold by R50 and R52 by means of hydrogen bonds to D45 and T43, respectively. We have delineated the importance of these two anchors by evaluating the structural, dynamic, and functional properties of the mutants, R50A-CMTI-V and R52A-CMTI-V. Compared to the R50A mutant, the R52A variant is more stable to trypsin proteolysis, shows greater enzyme-binding affinity, and gains less binding loop flexibility. Generalized order parameters (S2) of side-chain NHs reveal that removal of the R50 hydrogen bond to the reactive-site loop weakens considerably the R52 hydrogen bond, whereas abolition of the R52 hydrogen bond has almost no effect on the R50 anchor. Comparison of the chemical shift and interresidue NOE data between the two mutants and the wild-type inhibitor indicates that the overall tertiary structure is preserved in the mutant proteins; however, the R50A mutant displays more pronounced changes in the chemical shifts and S2 values of the binding loop residues, in addition to chemical shift perturbations corresponding to some structured regions of the protein scaffold. The present work underscores the differential roles played by the two binding loop anchors in influencing protein structure, function, dynamics, and proteolytic stability. It also suggests that in serine protease inhibitors, binding loop - scaffold interactions influence local flexibility and stability of distant regions.
Cytochrome b5 is a heme protein that acts as an electron transfer partner in a large number of physiological reactions. We believe that the backbone dynamics of cytochrome b5 may play an important role in the thermodynamic optimization of some of these reactions. We have characterized the dynamic behaviour of the A and B forms of ferro- and ferricytochrome b5 through the use of 15N relaxation data. Multiple field strengths were used so as to facilitate the application of minor corrections to the relaxation rates of the oxidized protein due to the paramagnetic iron center. The modelfree analysis was performed taking rotational diffusion anisotropy into consideration. An analysis of the square of the generalized order parameters revealed that the oxidized protein was more dynamic than the reduced. A set of deuterium exchange experiments were performed to corroborate this observation. This experiment showed that there was a dramatic decrease in the strength of the hydrogen bonding networks in the case of the oxidized protein. The entropy change associated with the oxidation of cytochrome b5 was calculated from a van't Hoff like plot where the temperature dependent reduction potential can be converted to a free energy term using the Nernst equation. This result indicated a positive entropy term for the oxidation of cytochrome b5. Our results suggest that changes in backbone dynamics could play a significant role in determining the thermodynamic driving force for the reaction which involves the reduction of methemoglobin by cytochrome b5 in the erythrocytes. Preliminary results show that in the case of myoglobin, a negative entropy term is associated with oxidation and once again this is compatible with the physiological situation where myoglobin binds to oxygen and still resists oxidation at the iron center.
The calcium-saturated E140Q mutant of the C-terminal domain of calmodulin exhibits global conformational exchange between at least two different states (Evens et al. (1997) Biochemistry, 36, 3448). These two states appear similar to the closed and open conformations of the apo and calcium-saturated states of wild-type calmodulin, as indicated by chemical shifts and NOEs. Thus, the observed exchange appears to mimic the calcium-induced structural transition in wild-type calmodulin. The exchange is fast to intermediate on the chemical shift time scale (kex~ 104 s-1), and the population of the open state is estimated to approximately 65%. Here, we characterize further the timescales and energetics of this conformational exchange process. We measured the backbone 15N T1,T2 and steady-state NOE values for the E140Q mutant at 18, 28 and 35oC. Using the model-free approach we calculated order parameters, effective internal correlation times and Rex terms describing motions on the microsecond-millisecond time scale. Apparent activation energies of 30-100 kJ/mol were derived from the temperature dependence of the Rex terms, following methods presented by Mandel et al. (1996, Biochemistry, 35, 16009). Residues with energies of similar magnitude are found to be clustered in the three-dimensional structures of calmodulin.
13C - 17O distances between bound retinal and water in the active site of rhodopsin will be measured using rotational echo adiabatic-passage double resonance (REAPDOR) NMR. Two all-trans retinals containing selective labels were prepared: one with labels at C(15), C(19) and C(20), and one with labels at C(15), and C(19). Additionally, these retinals have 17O labels incorporated in the aldehyde functional group. Results of 13C - 17O distances measured in the free retinals will be used as calibration standards for the measurements on reconstituted rhodopsin, and compared to X-ray distances. 13C - 17O distances between 17O labeled bound water in the rhodopsin binding pocket and the labeled 11-cis retinal in rhodopsin will yield information regarding the orientation of the retinal in the binding site of rhodopsin. In the same manner, results will be obtained for the all-trans retinal in the photoreaction intermediates of rhodopsin to establish how the retinal orientation changes upon absorption of light.
Recently, we reported the solution structure of LSIII, a long neurotoxin from the venom of Laticauda semifasciata(1). Here, we report the results of heteronuclear 13C relaxation experiments at natural abundance and of a 1.6 nsec molecular dynamics trajectory. We derive dynamical parameters on the basis of T1, T2, and NOE measurements and compare the results to those obtained from the trajectory and inferred from the structure. Possible implications of these results for receptor binding and evidence for rigid-body motion of Loop II are discussed. 1) Connolly, P.C., Stern, A.S., Hoch, J.C., Biochemistry, 35, 418, (1996).