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

Abstract

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.

Electrostatic Environment of Proteorhodopsin Affects the pKa of Its Buried Primary Proton Acceptor

“…The protonation state of embedded charged residues in TMPs reconstituted in such an environ-ment could potentially be affected by the charge of lipid headgroups, the orientation of water at the lipid bilayer surface (46), the population of penetrated water inside lipid bilayers, or ion condensation around the surface of lipid bilayers including protons (25). In fact, it has been suggested that differences in the local proton concentration on a charged lipid bilayer surface, which give rise to a spatially varying surface proton concentration compared to the bulk pH, affect the apparent pKa of embedded charged residues in TMPs (47)(48)(49)(50). Motivated by biophysical understanding objectives, we ask in this study whether the deviation of surface proton concentration is the only major factor that affects the protonation behavior of embedded charged residues or whether the change of external factors, such as lipid compositions, buffer and salt type, and/or salt concentration, that affect the local proton concentration can also affect the microenvironment around the embedded charge through structural rearrangement to change its intrinsic pKa.…”

EPR Oximetry with Nitroxides: Effects of Molecular Structure, pH, and Electrolyte Concentration

“…6, which shows a progressive decrease in A iso upon lowering pH below ≈5.5. This decrease is associated with the protonation of the tertiary amino group of the nitroxide, and, therefore, is well fitted to a modified Henderson-Hasselbalch equation (e.g., [51]); the result of the fit is shown as a solid line. The pK a of nitroxide 4 (pK a = 3.47 ± 0.02) is close to pK a = 3.33 ± 0.03 determined for IMTSL-ME (i.e., the same nitroxide but without the isotopic substitution) at slightly lower temperature (19.0 °C) [39].…”

“…Effects of molecular oxygen on protonated and non-protonated forms of ionizable nitroxides were further investigated for solutions of isotopically substituted nitroxide 4 (IMTSL-ME-d 16 -15 N). First, an EPR titration experiment with an aqueous solution of 4 was conducted at 21.0 °C following the experimental protocols and spectral fitting methods described earlier (e.g., [51]). In brief, pH of the nitroxide aqueous solution was adjusted from ca.…”

Dielectric and Electrostatic Properties of the Silica Nanoparticle–Water Interface by EPR of pH-Sensitive Spin Probes

“…An EPR spectrum of a nitroxide spin probe changes when it acquires a charge because of changes in both the nitroxide magnetic and rotational diffusion parameters. 48 In an EPR titration experiments, we are primarily interested in a fraction of the nonprotonated species, f, as a function of pH rather than in determining complete set of the nitroxide rotational motion and magnetic parameters from the experimental spectra. Then, assuming that a slow chemical exchange model is satisfied for the probe protonation, an experimental EPR spectrum at an intermediate pH, I(B), is approximated by a superposition of the spectra from the neutral and the ionized species, F R (B) and F RH+ (B), respectively:…”