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10.1016/bs.mie.2015.08.007

http://scihub22266oqcxt.onion/10.1016/bs.mie.2015.08.007
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C5008871!5008871!26477252
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suck abstract from ncbi


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pmid26477252      Methods+Enzymol 2015 ; 564 (ä): 191-217
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  • Ionizable Nitroxides for Studying Local Electrostatic Properties of Lipid Bilayers and Protein Systems by EPR #MMPMID26477252
  • Voinov MA; Smirnov AI
  • Methods Enzymol 2015[]; 564 (ä): 191-217 PMID26477252show ga
  • Electrostatic interactions are known to play one of the major roles in the myriad of biochemical and biophysical processes. In this Chapter we describe biophysical methods to probe local electrostatic potentials of proteins and lipid bilayer systems that is based on an observation of reversible protonation of nitroxides by EPR. Two types of the electrostatic probes are discussed. The first one includes methanethiosulfonate derivatives of protonatable nitroxides that could be used for highly specific covalent modification of the cysteine?s sulfhydryl groups. Such spin labels are very similar in magnetic parameters and chemical properties to conventional MTSL making them suitable for studying local electrostatic properties of protein-lipid interfaces. The second type of EPR probes is designed as spin-labeled phospholipids having a protonatable nitroxide tethered to the polar head group. The probes of both types report on their ionization state through changes in magnetic parameters and a degree of rotational averaging, thus, allowing one to determine the electrostatic contribution to the interfacial pKa of the nitroxide, and, therefore, determining the local electrostatic potential. Due to their small molecular volume these probes cause a minimal perturbation to the protein or lipid system while covalent attachment secure the position of the reporter nitroxides. Experimental procedures to characterize and calibrate these probes by EPR and also the methods to analyze the EPR spectra by least-squares simulations are also outlined. The ionizable nitroxide labels and the nitroxide-labeled phospholipids described so far cover an exceptionally wide pH range from ca. 2.5 to 7.0 pH units making them suitable to study a broad range of biophysical phenomena especially at the negatively charged lipid bilayer surfaces. The rationale for selecting proper electrostatically neutral interface for calibrating such probes and example of studying surface potential of lipid bilayer is also described.
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