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Modeling the effects of structure and dynamics of the nitroxide side chain on the ESR spectra of spin-labeled proteins
No full textIn the companion paper (J. Phys. Chem. B 2006, 110, jp0629487), a study of the conformational dynamics of methanethiosulfonate spin probes linked at a surface-exposed alpha-helix has been presented. Here, on the basis of this analysis, X-band ESR spectra of these spin labels are simulated within the framework of the Stochastic Liouville equation (SLE) methodology. Slow reorientations of the whole protein are superimposed on fast chain motions, which have been identified with conformational jumps and fluctuations in the minima of the chain torsional potential. Fast chain motions are introduced in the SLE for the protein reorientations through partially averaged magnetic tensors and relaxation times calculated according to the motional narrowing theory. The 72R1 and 72R2 mutants of T4 lysozyme, which bear the spin label at a solvent-exposed helix site, have been taken as test systems. For the side chain of the R2 spin label, only a few noninterconverting conformers are possible, whose mobility is limited to torsional fluctuations, yielding almost identical spectra, typical of slightly mobile nitroxides. In the case of R1, more complex spectra result from the simultaneous presence of constrained and mobile chain conformers, with relative weights that can depend on the local environment. The model provides an explanation for the experimentally observed dependence of the spectral line shapes on temperature, solvent, and pattern of substituents in the pyrroline ring. The relatively simple methodology presented here allows the introduction of realistic features of the spin probe dynamics into the simulation of ESR spectra of spin-labeled proteins; moreover, it provides suggestions for a proper account of such dynamics in more sophisticated approaches
Chain dynamics and the simulation of electron spin resonance spectra from oriented phospholipid membranes
No full textA model previously developed for describing the dynamics of flexible alkyl chains that is based on Flory's rotational isomeric state approximation is adapted and applied to the analysis of electron spin resonance (ESR) spectra obtained from a phospholipid spin label in a macroscopically aligned phospholipid membrane, In this model, rotation around each C-C bond of the labeled alkyl chain is characterized by three inequivalent minima, with one end of the chain fixed to mimic the phospholipid headgroup, and with the dynamic effects of the nitroxide label explicitly included. This model is integrated with that for the overall rotation of the phospholipid in the mean orientational potential of the aligned membrane, and it is incorporated into the stochastic Liouville equation which describes the ESR line shape in the presence of these dynamic processes. The analysis is simplified by introducing the fact that the relatively rapid internal modes of motion can be treated by motional narrowing theory and a time scale separation can be made with respect to the much slower overall motions of the phospholipid. A series of ESR spectra from the spin label 16-PC in the lipid dimyristoylphosphatidylcholine were obtained over a range of temperatures (35-65 degrees C) in the L-alpha phase for various orientations of the normal to the bilayer plane relative to the magnetic field. Very good agreement with experiment is obtained from this model by using least squares fitting procedures for the overall motional dynamics. One finds an order parameter of [D-00(2)] that is constant throughout the phase and the perpendicular component for rotational diffusion, R-perpendicular to, that ranges from about 1-3 x 10(7)s(-1) (which corresponds to the ESR slow motional regime). Fits to the ESR spectra were also obtained from a simple but standard model wherein a single overall rotational diffusion tensor is used to describe the combined effects of internal and overall dynamics. These fits were almost as good, but they lead to a much larger R-perpendicular to approximate to 3-6 x 10(8)s(-1) and a smaller [D-00(2)] = 0.1, since these parameters now include the composite effects of both types of processes. New ESR experiments are proposed to provide more critical tests of these models
Studies of spin relaxation and molecular dynamics in liquid crystals by two-dimensional Fourier transform electron spin resonance .2. Perdeuterated-tempone in butoxy benzylidene octylaniline and dynamic cage effects
No full textTwo-dimensional Fourier transform (2D-FT)-electron spin resonance (ESR) studies on the small globular spin probe perdeuterated tempone (PDT) in the liquid crystal solvent 4O,8 (butoxy benzylidene octylaniline) are reported. These experiments, over the temperature range of 95 degrees C to 24 degrees C, cover the isotropic (I), nematic (N), smectic A (S-A), smectic B (S-B), and crystal (C) phases. The 2D-ELDOR (two-dimensional electron-electron double resonance) spectra confirm the anomalously rapid reorientation of PDT, especially in the lower temperature phases. The model of a slowly relaxing local structure (SRLS) leads to generally very good non-linear least squares (NLLS) global fits to the sets of 2D-ELDOR spectra obtained at each temperature. These fits are significantly better than those achieved by the standard model of Brownian reorientation in a macroscopic orienting potential. The SRLS model is able to account for anomalies first observed in an earlier 2D-ELDOR study on PDT in a different liquid crystal in its smectic phases. Although it is instructional to extract the various spectral densities from the COSY (correlation spectroscopy) and 2D-ELDOR spectra, the use of NLLS global fitting to a full set of 2D-ELDOR spectra is shown to be more reliable and convenient for obtaining optimum model parameters, especially in view of possible (incipient) slow motional effects from the SRLS or dynamic cage. The cage potential is found to remain fairly constant at about k(B)T over the various phases (with only a small drop in the S-B phase), but its asymmetry increases with decreasing temperature T. This value is significantly larger than the weak macroscopic orienting potential which increases from 0.1 to 0.3k(B)T with decreasing T. The cage relaxation rate, given by R(c) is about 3x10(7) s(-1) the I phase, but increases to about 10(8) s(-1) in the S-A, S-B, and C phases. The rotational diffusion tensor for PDT shows only a small T-independent asymmetry, and its mean rotational diffusion coefficient is of order 10(10) s(-1), with however, a small increase in the S-B phase. These results are consistent with a model previously proposed for PDT in benzylidene liquid crystal solvents, that as T is reduced the PDT molecules are partially expelled from the hard core (dipolar) region of the liquid crystalline molecules toward the more flexible aliphatic chain region as a result of increased core packing from smectic layer formation, and it thus experiences a more fluid (for a given temperature) local cage structure
Methyl Dynamics of a Ca(2+)-Calmodulin-Peptide Complex from NMR/SRLS
No full textWe developed the slowly relaxing local structure (SRLS) approach for analyzing NMR spin relaxation in proteins. SRLS accounts for dynamical coupling between the tumbling of the protein and the local motion of the probe and for general tensorial properties. It is the generalization of the traditional model-free (MF) method, which does not account for mode-coupling and treats only simple tensorial properties. SRLS is applied herein to 2H relaxation of 13CDH2 groups in the complex of Ca2+−calmodulin with the peptide smMLCKp. Literature data comprising 2H T1 and T2 acquired at 14.1 and 17.6 T, and 288, 295, 308, and 320 K, are used. We find that mode-coupling is a small effect for methyl dynamics. On the other hand, general tensorial properties are important. In particular, it is important to allow for the asymmetry of the local spatial restrictions, which can be represented in SRLS by a rhombic local ordering tensor with components S02 and S22. The principal axes frame of this tensor is obviously different from the axial frames of the magnetic tensors. Here, we find that −0.2 ≤ S02 ≤ 0.5 and −0.4 ≤ S22 ≤ 0. MF features a single “generalized” order parameter, S, confined to the 0−0.316 range; the local geometry is inherently simple. The parameter S is inaccurate, having absorbed unaccounted for effects, notably S22 ≠ 0. We find that the methionine methyls (the other methyl types) reorient with rates of 8.6 × 109 to 21.4 × 109 (0.67 × 109 to 6.5 × 109) 1/s. The corresponding activation energies are 10 (10−27) kJ/mol. By contrast, MF yields inaccurate effective local motional correlation times, τe, with nonphysical temperature dependence. Thus, the problematic S- and τe-based MF picture of methyl dynamics has been replaced with an insightful physical picture based on a local ordering tensor related to structural features, and a local diffusion tensor that yields accurate activation energies
Dynamics of the nitroxide side chain in spin-labeled proteins
No full textThe dynamics of the tether linking methanethiosulfonate (MTSSL) spin probes to alpha-helices has been investigated with the purpose of rationalizing its effects on ESR line shapes. Torsional profiles for the chain bonds have been calculated ab initio, and steric interactions with the alpha-helix and the neighboring residues have been introduced at the excluded-volume level. As a consequence of the restrictions deriving from chain geometry and local constraints, a limited number of allowed conformers has been identified that undergo torsional oscillations and conformational jumps. Torsional fluctuations are described as damped oscillations, while transition rates between conformers are calculated according to the Langer multidimensional extension of the Kramers theory. The time scale and amplitude of the different motions are compared; the major role played by rotations of the outermost bonds of the side chain emerges, along with the effects of substituents in the pyrroline ring on the conformer distribution and dynamics. The extent and symmetry of magnetic tensor averaging produced by the side chain motions are estimated, the implications for the ESR spectra of spin-labeled proteins are discussed, and suggestions for the introduction of realistic features of the spin probe dynamics into the line shape simulation are presented
Conformational Motion of the ABC Transporter MsbA Induced by ATP Hydrolysis
Get PDFWe measured the amplitude of conformational motion in the ATP-binding cassette (ABC) transporter MsbA upon lipopolysaccharide (LPS) binding and following ATP turnover by pulse double electron-electron resonance and fluorescence homotransfer. The distance constraints from both methods reveal large-scale movement of opposite signs in the periplasmic and cytoplasmic part of the transporter upon ATP hydrolysis. LPS induces distinct structural changes that are inhibited by trapping of the transporter in an ATP post-hydrolysis intermediate. The formation of this intermediate involves a 33-angstrom distance change between the two ABCs, which is consistent with a dimerization-dissociation cycle during transport that leads to their substantial separation in the absence of nucleotides. Our results suggest that ATP-powered transport entails LPS sequestering into the open cytoplasmic chamber prior to its translocation by alternating access of the chamber, made possible by 10 -20-angstrom conformational changes
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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