Mapping Local Protein Electrostatics by EPR of pH-Sensitive Thiol-Specific Nitroxide

Voinov, Maxim A.; Ruuge, Andres E.; Резников, В. А.; Григорьев, И. А.; Smirnov, Alex I.

doi:10.1021/bi800272f

Cited by 22 publications

(35 citation statements)

References 49 publications

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“…These studies have inferred electrostatic potentials based on free energy measurements [e.g., pK a shifts of ionizable residues (9-11), reactivity differences among cysteines (12,13), equilibrium binding constants of charged ligands (14), and redox potential shifts (15)] or spectroscopic variations in probes incorporated into or bound to proteins [e.g., fluorescent dyes (16), 19 F and 13 C chemical shifts (17)(18)(19)(20)(21), and EPR-measured g-factor perturbations of nitroxides (22)]. These studies have spurred the ongoing development of electrostatic calculation methodology (6,7) and contributed greatly to our general recognition that the solvation environment within proteins is very different from water.…”

mentioning

confidence: 99%

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Fafarman¹,

Sigala²,

Schwans³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

216

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Understanding the electrostatic forces and features within highly heterogeneous, anisotropic, and chemically complex enzyme active sites and their connection to biological catalysis remains a longstanding challenge, in part due to the paucity of incisive experimental probes of electrostatic properties within proteins. To quantitatively assess the landscape of electrostatic fields at discrete locations and orientations within an enzyme active site, we have incorporated site-specific thiocyanate vibrational probes into multiple positions within bacterial ketosteroid isomerase. A battery of X-ray crystallographic, vibrational Stark spectroscopy, and NMR studies revealed electrostatic field heterogeneity of 8 MV∕cm between active site probe locations and widely differing sensitivities of discrete probes to common electrostatic perturbations from mutation, ligand binding, and pH changes. Electrostatic calculations based on active site ionization states assigned by literature precedent and computational pK a prediction were unable to quantitatively account for the observed vibrational band shifts. However, electrostatic models of the D40N mutant gave qualitative agreement with the observed vibrational effects when an unusual ionization of an active site tyrosine with a pK a near 7 was included. UV-absorbance and 13 C NMR experiments confirmed the presence of a tyrosinate in the active site, in agreement with electrostatic models. This work provides the most direct measure of the heterogeneous and anisotropic nature of the electrostatic environment within an enzyme active site, and these measurements provide incisive benchmarks for further developing accurate computational models and a foundation for future tests of electrostatics in enzymatic catalysis. E xtensive structural studies of enzymes have revealed that biological catalysis occurs within sequestered active site crevices that solvate reacting substrates with an anisotropic and chemically complex constellation of charged, polar, and hydrophobic groups. But beyond visualization of the chemical composition and architecture of active sites permitted by the rich library of available protein structures, our understanding of the electrostatic nature and properties of this highly heterogeneous environment and its role in molecular recognition and catalysis is largely based on simulations. Computations using three-dimensional structures routinely provide electrostatic potentials, as in Fig. 1A. These potentials have been used to identify and characterize protein-protein and protein-ligand binding sites, and interaction energies derived from these calculations have suggested key contributions from the electrostatic environment to enzymatic rate enhancement and specificity (1-5). However, even the most sophisticated computational methods have multiple approximations and limitations (6-8) and, most importantly, have not been rigorously evaluated through cycles of nontrivial computational predictions and experimental tests. Incisive and quantitative experimental measures o...

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mentioning

confidence: 99%

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Fafarman¹,

Sigala²,

Schwans³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

216

View full text Add to dashboard Cite

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“…For example, it was reported that above neutral pH IMTSL would rapidly form biradicals which became evident from characteristic five-line EPR spectra (not shown). (40) Formation of the biradicals has also been observed for MTSL under similar conditions. It was speculated that at basic pH, some of the MTS groups are hydrolyzed to thiols, which would then react with the remaining methanethiosulfonates forming disulfide biradicals.…”

Section: Epr Characterization Of Ph-sensitive Thiol-specific Nitromentioning

confidence: 54%

“…It was speculated that at basic pH, some of the MTS groups are hydrolyzed to thiols, which would then react with the remaining methanethiosulfonates forming disulfide biradicals. (40) Thus, for the optimal labeling it is desirable avoiding pH above the neutral values.…”

Section: Epr Characterization Of Ph-sensitive Thiol-specific Nitromentioning

confidence: 99%

“…While MTSL has been proven to be a very useful probe of molecular structure and dynamics, it cannot be used for local pH determination because it does not have any groups that are ionized within the useful biochemical range. In order to overcome this deficiency, we have introduced cysteine-specific labels (39, 40) and lipid-mimicking EPR probes (41, 42) that are based on derivatives of imidazoline and imidazolidine nitroxides. These nitroxides contain basic functionalities in the heterocycles making there EPR spectra sensitive to pH changes near the probe p K a .…”

Section: Introductionmentioning

confidence: 99%

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Ionizable Nitroxides for Studying Local Electrostatic Properties of Lipid Bilayers and Protein Systems by EPR

Voinov

Smirnov

2015

Methods in Enzymology

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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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“…c Chemical structure of the MTS-4-oxyl spin label. d Reaction of a maleimide spin label N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide with the sulfhydryl group of a cysteine side chain 2004) or of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae (Voinov et al 2008).…”

Section: Methods Of Spin Labelingmentioning

confidence: 99%

Spin labeling EPR

2009

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Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy has emerged as an efficient tool to elucidate the structure and conformational dynamics of biomolecules under native-like conditions. This article summarizes the basics as well as recent progress of site-directed spin labeling. Continuous wave EPR spectra analyses and pulse EPR techniques are reviewed with special emphasis on applications to the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis and the photosynthetic reaction center from Rhodobacter sphaeroides R26.

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Mapping Local Protein Electrostatics by EPR of pH-Sensitive Thiol-Specific Nitroxide

Cited by 22 publications

References 49 publications

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Ionizable Nitroxides for Studying Local Electrostatic Properties of Lipid Bilayers and Protein Systems by EPR

Spin labeling EPR

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