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

Abstract

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.

Hydrodynamic modeling of diffusion tensor properties of flexible molecules

“…Discussion about this aspect beyond the purposes of the present article, but a large literature is available concerning the use of the SRLS model in the interpretation of magnetic resonance data, both electronic and nuclear. [21][22][23][24][32][33][34] Diffusion coefficients can also be obtained from the analysis of electron spin resonance (ESR) spectra. We present here two examples, taken from recent studies where we integrated different ab initio techniques to set up an effective protocol for interpreting continuous wave (CW) ESR spectra of single and double paramagnetic probes in liquids.…”

“…34 Analysis of the diffusion tensor for each conformation shows that the global rotational diffusion tensor is negligibly affected by the internal rotations (deviations under 5%) because of the very small size of the three rotating fragments with respect to the overall extension of the molecule. Moreover, the internal part of the diffusion tensor reveals that the three motions are practically decoupled, with the off-diagonal terms 2-3 orders of magnitude smaller than the diagonal values: this can be attributed to both the small dimensions of the rotating fragments and to their large distances.…”

Chapter 2. Computational approaches for simulating motional EPR spectra

“…Although this approach provides all the information about tilt angles and ordering directly from MD calculations, the method still relied on varying the principle diffusion tensor rates as adjustable parameters to fit EPR spectra. Freed and co-workers 85,86 have extended the application of SLE approach to spin labelled proteins by taking into consideration discreet rotameric states for both MTSL R1 and R2 (1-oxyl-2,2,4,5,5-pentamethyl-3-pyrroline-3-(methyl)-methanethiosulfonate) spin labels. In their work the refined SLE model of slow reorientation of the whole protein is 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 were introduced in the SLE for the protein reorientations through partially averaged magnetic tensors and relaxation times (T 2 ) calculated according to the motional narrowing theory. 85,86 The effects of side chain dynamics on the spectral line shape have been analyzed as a function of temperature and solvent for the 72R1 and 72R2 mutants of T4L phage lysozyme taken as test cases, and compared with experimental data. Steinhoff and Hubbell in their seminal paper 49 presented a method for the simulation of EPR spectra using a single particle model in the Langevin-LvN approach with the ordering potential reconstructed from the actual MD simulations results on spin-labelled proteins.…”

Cu(ii)–porphyrin molecular dynamics as seen in a novel EPR/Stochastic Liouville equation study

“…8,9 The partial averaging has been treated for EPR problems by considering a nitroxide spin label in biomolecules. 51,52 A general form of Liouvillian corresponding to fast processes in SLEL takes the form L the orientation dependence in Redfield terms derived in ref. 51 and 52.…”