Influence of the disulfide bond configuration on the dynamics of the spin label attached to cytochrome <i>c</i>

Murzyn, Krzysztof; Róg, Tomasz; Blicharski, Wojciech; Dutka, Małgorzata; Pyka, Janusz; Szytuła, Sebastian; Froncisz, Wojciech

doi:10.1002/prot.20838

Cited by 25 publications

(35 citation statements)

References 56 publications

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“…Thus, the results of our analysis support the so-called 'χ 4 , χ 5 ' model, which has been suggested by a number of experimental studies 46,48,51 and by MD simulations. 24,25 In our approach, we have focussed on a simple model, that can be worked out in detail, and it offers the advantage of an easier interpretation of the determining factors of the conformational dynamics. Our description of the conformational dynamics of the side-chain, although approximate, contains substantial realistic features, and it has led to some rather general results on the geometry and kinetics of such motions.…”

Section: Discussionmentioning

confidence: 99%

“…The magnetic tensors partially averaged by chain motions can be expressed in terms of these order parameters: (24) where μ = g or μ = A, for the g and the A hyperfine tensor, respectively.…”

Section: Orientational Distribution Of the Spin Labelmentioning

confidence: 99%

“…19 An account of the dynamics of the spin probe beyond the simplifying assumptions used here could be obtained in the form of trajectories by MD or Brownian Dynamics simulations. [20][21][22][23][24][25] A detailed picture of the local environment probed by the spin label and of the various processes modulating its orientations can be attained by such means, but at a computational cost which can be very great, due to the huge number of degrees of freedom. Conformational transitions in the side-chain occur on the nanosecond time-scale; therefore, even long trajectories can sample just a few conformations.…”

Section: Introductionmentioning

confidence: 99%

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Dynamics of the Nitroxide Side Chain in Spin-Labeled Proteins

2006

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The dynamics of the tether linking methanethiosulfonate (MTSSL) spin probes to α-helices has been investigated, with the purpose of rationalizing its effects on ESR lineshapes. Torsional profiles for the chain bonds have been calculated ab initio, and steric interactions with the α-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, which 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 lineshape simulation are presented.

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Section: Discussionmentioning

confidence: 99%

Section: Orientational Distribution Of the Spin Labelmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Dynamics of the Nitroxide Side Chain in Spin-Labeled Proteins

2006

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“…The point charges for MTSL (previously reported 65 and recalculated by us using Gaussian 66 ) were not included in the final series of calculations. Indeed, it was found that including the charges leads to some instances where the MTSL ring becomes tightly packed against the protein chain (essentially, an artefact from the structure calculations in vacuo).…”

Section: Model 4: Diffusion In Numerically Defined Realistic Potentialmentioning

confidence: 99%

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

et al. 2009

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Site-directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as 1 H N is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean-force potential (Smoluchowski equation); (iv) explicit-atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the 1 H N residue number and static magnetic field strength; the results are appreciably different from the Gillespie-Shortle model. At the same time, the Gillespie-Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between 1 H N spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin-labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme-using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates-lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random-coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random-coil protein in good solvent/poor solvent.Additional Supporting Information may be found in the online version of this article.Abbreviations: drkN SH3, N-terminal SH3 domain of the Drosophila adapter protein drk; EDTA, ethylenediaminetetraacetic acid; ESR, electron spin resonance; FRET, fluorescence resonance energy transfer; MTSL, methanethiosulfonate spin label; NOE, nuclear Overhauser effect; NAG, N-acetyl-glycine.

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“…The matrix elements , which will be designated as order parameters, reflect the orientational distribution of the magnetic frame in the frame of the amino acid residue. They are related to the magnetic tensors partially averaged by chain motions: (16) Even for the evaluation of the spectral densities, eq 13, it is convenient to make explicit the dependence upon the variables which are modulated by chain motions; the following form is obtained: (17) where j q,k,q′,k′ is the reduced zero-frequency spectral density: (18) with .…”

Section: B Fast Side-chain Motions: Average Magnetic Tensors and Spementioning

confidence: 99%

Modeling the Effects of Structure and Dynamics of the Nitroxide Side Chain on the ESR Spectra of Spin-Labeled Proteins

2006

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In a companion paper (Tombolato, F.; Ferrarini, A.; Freed, J.H., ... ) a study of the conformational dynamics of methanethiosulfonate spin probes linked at a surface exposed α-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 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 non-interconverting 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 which can depend on the local environment. The model provides an explanation for the experimentally observed dependence of the spectral lineshapes 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.

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Influence of the disulfide bond configuration on the dynamics of the spin label attached to cytochrome c

Cited by 25 publications

References 56 publications

Dynamics of the Nitroxide Side Chain in Spin-Labeled Proteins

Dynamics of the Nitroxide Side Chain in Spin-Labeled Proteins

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Modeling the Effects of Structure and Dynamics of the Nitroxide Side Chain on the ESR Spectra of Spin-Labeled Proteins

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