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  • Book
    Aaron Thomas Fafarman.
    Electrostatic fields in the interior of proteins, the consequence of the charged, polar and polarizable matter they are comprised of, have been hypothesized to vary on the order of tens of megavolts per centimeter and thus to be of tremendous consequence to biological processes. It is intuitively apparent that the rate of electron transfer in photosynthesis, the rate constant for catalysis by an enzyme, the flux through an ion channel, or the affinity between a drug molecule and its target, each involving a translocation of charged or polar species, would depend strongly on the energetic contribution from the electrostatic fields exerted by the surroundings. Despite a proliferation of calculations aimed at rationalizing the energetics of these processes, there remains a paucity of direct measurements of the electrostatic fields on which these calculations depend. By Stark spectroscopy, the directional and linear sensitivity of certain vibrational transitions to externally applied electric fields has been demonstrated, and a calibration obtained, in the form of the linear Stark tuning rate. The hypothesis has been previously submitted that for such probes, incorporated into proteins, spectroscopically observed band shifts could be quantitatively translated into changes in the electrostatic fields experienced by the probe. Carbon-fluorine and carbon-deuterium oscillators are examined as probes of electrostatic field and the means to circumvent the limitations of spectral congestion for the former and low oscillator strength for the latter are demonstrated. As an alternative solution to both problems, a straightforward and general method for the incorporation of thiocyanate electric field probes at any location in a protein by post-translational cysteine modification is presented. Incorporating nitrile probes into many locations in the proteins ribonuclease S and ketosteroid isomerase, the Stark model for vibrational band shifts is evaluated more critically than has been done previously for these probes. In ribonuclease, vibrational Stark spectra are used to calibrate multiple types of nitrile-modified proteins. The results provide evidence that the simple response to external electric fields of small, nitrile-containing molecules immobilized in frozen organic glasses can be generalized to nitriles in the interior of a protein, a requisite condition for the simple interpretation of band shifts in terms of changes in the internal electrostatic field. With this point established, the accuracy of the electrostatic force model incorporated in a molecular dynamics force field is evaluated by comparing observed spectral shifts to those calculated using simulated electrostatic fields in conjunction with the Stark model. Qualitative agreement is observed. However, the simplicity of the Stark model is complicated by the possibility of direct hydrogen-bond formation to the nitrile. This limitation is overcome using a method to both detect cases where this occurs, and to quantitatively account for this effect: a comparison of nitrile chemical shifts by NMR and frequencies by IR, each calibrated in turn by a solvatochromic model. With this additional observable, we are able to confidently ascribe spectral shifts due to mutation, pH titration and ligand binding to changes in the electrostatic fields experienced by the probes. Efforts towards employing nitrile probes to measure electric fields in the complex environment of the photosynthetic reaction center are presented.