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.

We report a study on charged, filamentous virus called M13, whose suspensions in water exhibit a chiral nematic (cholesteric) phase. In spite of the right-handed helicity of the virus, a left-handed phase helicity is found, with a cholesteric pitch which increases with temperature and ionic strength. Several sources of chirality can be devised in the system, ranging from the subnanometer to the micrometer length scale. Here an explanation is proposed for the microscopic origin of the cholesteric organization, which arises from the helical arrangement of coat proteins on the virus surface. The phase organization is explained as the result of the competition between contributions of opposite handedness, deriving from best packing of viral particles and electrostatic interparticle repulsions. This hypothesis is supported by calculations based on a coarse-grained representation of the virus.

B-DNA solutions of suitable concentration form left-handed chiral nematic phases (cholesterics).Such phases have also been observed in solutions of other stiff or semiflexible chiral polymers; magnitude and handedness of the cholesteric pitch are uniquely related to the molecular features. In this work we present a theoretical method and a numerical procedure which, starting from the structure of polyelectrolytes, lead to the prediction of the cholesteric pitch. Molecular expressions for the free energy of the system are obtained on the basis of steric and electrostatic interactions between polymers; the former are described in terms of excluded volume, while a mean field approximation is used for the latter. Calculations have been performed for 130 bp fragments of B-DNA. The theoretical predictions provide an explanation for the experimental behavior, by showing the counteracting role played by shape and charge chirality of the molecule.2

The region 35-43 of human alpha-Synuclein bound to small unilamellar lipid vesicles and to sodium dodecyl sulfate micelles has been investigated by site-directed spin labeling and electron paramagnetic resonance spectroscopy. The distance distributions obtained from spectral fitting have been analyzed on the basis of the allowed rotamers of the spin-label side-chain. Very similar results have been obtained in the two environments: an unbroken helical structure of the investigated region can be ruled out. The distance distributions are rather compatible with the presence of conformational disorder, in agreement with previous findings for micelle-bound alpha-Synuclein. The propensity for helix breaking is confirmed by molecular dynamics simulations.

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.