Mapping local protein electrostatics by EPR of pH-sensitive thiol-specific nitroxide.

Biochemistry
Maxim A VoinovAlex I Smirnov

Abstract

A first thiol-specific pH-sensitive nitroxide spin-label of the imidazolidine series, methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester (IMTSL), has been synthesized and characterized. X-Band (9 GHz) and W-band (94 GHz) EPR spectral parameters of the new spin-label in its free form and covalently attached to an amino acid cysteine and a tripeptide glutathione were studied as a function of pH and solvent polarity. The pKa value of the protonatable tertiary amino group of the spin-label was found to be unaffected by other ionizable groups present in side chains of unstructured small peptides. The W-band EPR spectra were shown to allow for pKa determination from precise g-factor measurements. Is has been demonstrated that the high accuracy of pKa determination for pH-sensitive nitroxides could be achieved regardless of the frequency of measurements or the regime of spin exchange: fast at X-band and slow at W-band. IMTSL was found to react specifically with a model protein, iso-1-cytochrome c from the yeast Saccharomyces cerevisiae, giving EPR spectra very similar to those of the most commonly employed cysteine-specific label MTSL. CD data indicated no perturbations to the overall protein struct...Continue Reading

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Citations

Sep 4, 2009·Photosynthesis Research·Johann P Klare, Heinz-Jürgen Steinhoff
Feb 7, 2012·Proceedings of the National Academy of Sciences of the United States of America·Aaron T FafarmanSteven G Boxer
Dec 12, 2020·Journal of the American Chemical Society·Boris DzikovskiJack H Freed
Jan 10, 2012·Journal of Chemical Theory and Computation·Zilvinas RinkeviciusHans Ågren

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