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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 pKa 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 pKa near 7 was included. UV-absorbance and 13C 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.

Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol–Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.

Prion diseases are characterized by an accumulation of PrPSc, a misfolded isoform of the normal cellular prion protein, PrPC. We previously reported the bioactivity of acridine-based compounds against PrPSc replication in scrapie-infected neuroblastoma cells and now report the improved potency of bis-acridine compounds. Bis-acridines are characterized by a dimeric motif, comprising two acridine heterocycles tethered by a linker. A library of bis-(6-chloro-2-methoxy-acridin-9-yl) and bis-(7-chloro-2-methoxy-benzo[b][1,5]-naphthyridin-10-yl) analogs was synthesized to explore the effect of structurally diverse linkers on PrPSc replication in scrapie-infected neuroblastoma cells. Structure-activity analysis revealed that linker length and structure are important determinants for inhibition of prion replication in cultured scrapied cells. Three bis-acridine analogs, (6-chloro-2-methoxy-acridin-9-yl)-(3-{4-[3-(6-chloro-2-methoxy-acridin-9- ylamino)-propyl]-piperazin-1-yl}-propyl)-amine, N,N ′-bis-(6-chloro-2-methoxy-acridin-9-yl)-1,8-diamino-3,6- dioxaoctane, and (1-{[4-(6-chloro-2-methoxy-acridin-9-ylamino)-butyl]-[3- (6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-carbamoyl}-ethyl)-carbamic acid tert-butyl ester, showed half-maximal inhibition of PrPSc formation at 40, 25, and 30 nM, respectively, and were not cytotoxic to uninfected neuroblastoma cells at concentrations of 500 nM. Our data suggest that bis-acridine analogs may provide a potent alternative to the acridine-based compound quinacrine, which is currently under clinical evaluation for the treatment of prion disease.

Perovskite solar cells are noted for their high performance and ease of synthesis, but are still plagued by concerns over their stability. Researchers are now demonstrating why higher performance and increased stability go hand-in-hand — and how to continue improving both.

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.