Vladislav Yu. Orekhov

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

SummaryThe 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 α-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 α-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 α-helixes of (1–71)bacterioopsin. The internal dynamics of the α-helixes on the millisecond and nanosecond time scales should be taken into account in the development of a model of the functioning bacteriorhodopsin.

SAMPLING OF PROTEIN DYNAMICS IN NANOSECOND TIME SCALE BY 15N NMR RELAXATION AND SELF-DIFFUSION MEASUREMENTS

This paper presents a procedure for detection of intermediate nanosecond internal dynamics in globular proteins. The procedure uses 1H-15N relaxation measurements at several spectrometer frequencies and hydrodynamic calculations based on experimental self-diffusion coefficients. New heteronuclear experiments, using pulse field gradients, are introduced for the measurement of translation diffusion coefficients of 15N labeled proteins. An advanced interpretation of recently published (Luginbühl et al., Biochemistry, 36, 7305-7312 (1997)) backbone amide 15N relaxation data, measured at two spectrometers (400 and 750 MHz for 1H) for N-terminal DNA-binding domain (1-63) of 434 repressor, is presented. Non-applicability of commonly used fast (picosecond) dynamics model (FD) was justified by (i) poor fit of relaxation data by the FD model-free spectral density function both for isotropic and anisotropic models of the overall molecular tumbling; (ii) specific dependence of the overall rotation correlation times calculated from T1/T2 ratio on the spectrometer frequency; (iii) mismatch of the ratio of longitudinal 15N relaxation times T1, measured at different spectrometer frequencies, in comparison with that anticipated for the FD model; (iv) significantly underestimated overall rotation correlation time provided by the FD model (5.50+/-0.15 and 5.80+/-0.15 ns for 750 and 400 MHz spectrometer frequency respectively) in comparison with correlation time obtained from hydrodynamics. On the other hand, all relaxation and hydrodynamics data are in good correspondence with the model of intermediate (nanoseconds) dynamics. Overall rotation correlation time of 7.5+/-0.7 ns was calculated from experimental translation self-diffusion rate using hydrodynamics formalism (Garcia de la Torre, J. and Bloomfield, V.A. Quart. Rev. Biophys., 14, 81-139 (1981)). The statistical analysis of 15N relaxation data along with the hydrodynamic consideration clearly revealed that most of the residues in 434(1-63) repressor are involved in the nanosecond internal dynamics characterized by the the mean order parameters of 0.59+/-0.06 and the correlation times of ca. 5 ns.

Pressure effect on the dynamics of an isolated α-helix studied by 15N-1H NMR relaxation

Dynamics and structure of (1–36)bacteriorhodopsin solubilized in chloroform/methanol mixture (1:1) were investigated by 1H-15N NMR spectroscopy under a hydrostatic pressure of 2000xa0bar. It was shown that the peptide retains its spatial structure at high pressure. 15N transverse and longitudinal relaxation times, 15N{1H} nuclear Overhauser effects, chemical shifts and the translation diffusion rate of the peptide at 2000 bar were compared with the respective data at ambient pressure [Orekhov etxa0al. (1999) J. Biomol. NMR, 14, 345–356]. The model free analysis of the relaxation data for the helical 9–31 fragment revealed that the high pressure decreases the overall rotation and translation diffusion, as well as apparent order parameters of fast picosecond internal motions (S2f) but has no effect on internal nanosecond motions (S2s and τs) of the peptide. The decrease of translation and overall rotation diffusion was attributed to the increase in solvent viscosity and the decrease of apparent order parameters S2f to a compression of hydrogen bonds. It is suggested that this compression causes an elongation of H-N bonds and a decrease of absolute values of chemical shift anisotropy (CSA). In particular, the observed decrease of S2f at 2000 bar can be explained by 0.001xa0nm increase of N-H bond lengths and 10xa0ppm decrease of 15N CSA values.

1H-15N NMR dynamic study of an isolated α-helical peptide (1–36)- bacteriorhodopsin reveals the equilibrium helix-coil transitions

The backbone dynamics of the bacteriorhodopsin fragment (1–36)BR solubilized in a 1:1 chloroform/methanol mixture were investigated by heteronuclear 1H-15N NMR spectroscopy. The heteronuclear 15N longitudinal and transverse relaxation rates and 15N{1H} steady-state NOEs were measured at three magnetic fields (11.7, 14.1, and 17.6xa0T). Careful statistical analysis resulted in the selection of the extended model-free form of the spectral density function [Clore etxa0al. (1990) J. Am. Chem. Soc., 112, 4989–4991] for all the backbone amides of (1–36)BR. The peptide exhibits motions on the micro-, nano-, and picosecond time scales. The dynamics of the α-helical part of the peptide (residues 9–31) are characterised by nanosecond and picosecond motions with mean order parameters Ss2 = 0.60 and Sf2 = 0.84, respectively. The nanosecond motions were attributed to the peptides helix-coil transitions in equilibrium. Residues 3–7 and 30–35 also exhibit motions on the pico- and nanosecond time scales, but with lower order parameters. Residue 10 at the beginning of the α-helix and residues 30–35 at the C-terminus are involved in conformational exchange processes on the microsecond time scale. The implications of the obtained results for the studies of helix-coil transitions and the dynamics of membrane proteins are discussed.

1H-15N backbone resonance assignments of bacteriorhodopsin

Des-(232-248)-bacteriorhodopsin was solubilized in a membrane mimicking environment of methanol-chloroform (1:1) containing 0.1 M 2HCO2N2H4 and 1H-15N backbone resonance assignment was obtained using 2D HMQC, 3D NOESY-HMQC, 3D TOCSY-HMQC and 3D HMQC-NOESY-HMQC NMR experiments. 87 cross-peaks out of 117 present in the HMQC spectrum were assigned to particular residues in 1-73 and 195-231 parts of the protein. For these residues also signals of C alpha H and C beta H protons were assigned.

Manifestation of intramolecular motions on pico- and nanosecond time scales in 1H−15N NMR relaxation: Analysis of dynamic models of one- and two-helical subunits of bacterioopsin

SummaryThe influence of the internal dynamics of two polypeptides comprising transmembrane α-helix A or two α-helices A and B of bacterioopsin on experimentally accessible 15N NMR relaxation rates was investigated by molecular dynamics (MD) simulations, combined with more simple mechanic considerations. ‘Model-free’ order parameters and correlation times of internal motions [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559] were calculated for these models. It was found that both peptides exhibit two types of internal motions of the amide bonds, on the pico- and nanosecond time scales, affecting 15N NMR relaxation. The fast fluctuations are local and correspond to the librational motions of the individual N−H vectors in an effective potential of atoms of the surrounding matrix. In contrast, the motions on the nanosecond time scale imply concerted collective vibrations of a large number of atoms and could be represented as bending oscillation of α-helices, strongly overdamped by the ambient solvent. A few other molecular mechanisms of slow internal motion were found, such as local distortions of the α-helices (e.g., α-aneurysm), delocalized distortions of the α-helical backbone, as well as oscillations of the tilt angle between the axes of the α-helices A and B. The results are compared with 15N NMR relaxation data measured for the (1–36)bacterioopsin and (1–71)bacterioopsin polypeptides in chloroform-methanol (1:1) and in SDS micelles [Orekhov, V.Yu., Pervushin, K.V. and Arseniev, A.S. (1994) Eur. J. Biochem., 219, 887–896].

An estimate of spin diffusion in a spin subset: Application to iterative distance calculation from 3D 15N NOESY-HMQC

SummaryA method for quantification of distances between amide hydrogens using only the 3D NOESY-HMQC experiment recorded on a 15N-labelled protein is presented. This method is based on an approximate expression of the NOE intensities between amide hydrogens obtained from continuum modelling of the non-amide spins; this expression is used in a distance calculation algorithm. The algorithm has been named CROWD, standing for Continuum approximation of Relaxati On path Ways between Dilute spins. This approximation as well as the CROWD algorithm are tested on a simulated case; the CROWD algorithm is then applied to experimental data, measured on a fragment of bacteriorhodopsin.