Solvent-mediated forces in protein dielectrophoresis.

DEP is an established method to manipulate micrometer-sized particles, but standard continuum theories predict only negligible effects for nanometer-sized proteins despite contrary experimental evidence. A theoretical description of protein DEP needs to consider details on the molecular scale. Previous work toward this goal addressed the role of orientational polarization of static protein dipole moments for dielectrophoretic effects, which successfully predicts the general magnitude of dielectrophoretic forces on proteins but does not readily explain negative DEP forces observed for proteins in some experiments. However, contributions to the protein chemical potential due to protein-water interactions have not yet been considered in this context. Here, we utilize atomistic molecular dynamics simulations to evaluate polarization-induced changes in the protein solvation free energy, which result in a solvent-mediated contribution to dielectrophoretic forces. We quantify solvent-mediated dielectrophoretic forces for two proteins and a small peptide in water, which follow expectations for protein-water dipole-dipole interactions. The magnitude of solvent-mediated dielectrophoretic forces exceeds predictions of nonmolecular continuum theories, but plays a minor role for the total dielectrophoretic force for the simulated proteins due to dominant contributions from the orientational polarization of their static protein dipoles. However, we extrapolate that solvent-mediated contributions to negative protein DEP forces will become increasingly relevant for multidomain proteins, complexes and aggregates with large protein-water interfaces, as well as for high electric field frequencies, which provides a potential mechanism for corresponding experimental observations of negative protein DEP.

Interfacial structural crossover and hydration thermodynamics of charged C60 in water.

Classical molecular dynamics simulations of the hydration thermodynamics, structure, and dynamics of water in hydration shells of charged buckminsterfullerenes are presented in this study. Charging of fullerenes leads to a structural transition in the hydration shell, accompanied by creation of a significant population of dangling O-H bonds pointing toward the solute. In contrast to the well accepted structure-function paradigm, this interfacial structural transition causes nearly no effect on either the dynamics of hydration water or on the solvation thermodynamics. Linear response to the solute charge is maintained despite significant structural changes in the hydration shell, and solvation thermodynamic potentials are nearly insensitive to the altering structure. Only solvation heat capacities, which are higher thermodynamic derivatives of the solvation free energy, indicate some sensitivity to the local hydration structure. We have separated the solvation thermodynamic potentials into direct solute-solvent interactions and restructuring of the hydration shell and analyzed the relative contributions of electrostatic and nonpolar interactions to the solvation thermodynamics.

Marcus Bell-Shaped Electron Transfer Kinetics Observed in an Arrhenius Plot.

The Marcus theory of electron transfer predicts a bell-shaped dependence of the reaction rate on the reaction free energy. The top of the "inverted parabola" corresponds to zero activation barrier when the electron-transfer reorganization energy and the reaction free energy add up to zero. Although this point has traditionally been reached by altering the chemical structures of donors and acceptors, the theory suggests that it can also be reached by varying other parameters of the system including temperature. We find here dramatic evidence of this phenomenon from experiments on a fullerene-porphyrin dyad. Following photoinduced electron transfer, the rate of charge recombination shows a bell-shaped dependence on the inverse temperature, first increasing with cooling and then decreasing at still lower temperatures. This non-Arrhenius rate law is a result of a strong, approximately hyperbolic temperature variation of the reorganization energy and the reaction free energy. Our results provide potentially the cleanest confirmation of the Marcus energy gap law so far since no modification of the chemical structure is involved.

Digital analysis of the prostate tumor microenvironment with high-order chromogenic multiplexing.

As our understanding of the tumor microenvironment grows, the pathology field is increasingly utilizing multianalyte diagnostic assays to understand important characteristics of tumor growth. In clinical settings, brightfield chromogenic assays represent the gold-standard and have developed significant trust as the first-line diagnostic method. However, conventional brightfield tests have been limited to low-order assays that are visually interrogated. We have developed a hybrid method of brightfield chromogenic multiplexing that overcomes these limitations and enables high-order multiplex assays. However, how compatible high-order brightfield multiplexed images are with advanced analytical algorithms has not been extensively evaluated. In the present study, we address this gap by developing a novel 6-marker prostate cancer assay that targets diverse aspects of the tumor microenvironment such as prostate-specific biomarkers (PSMA and p504s), immune biomarkers (CD8 and PD-L1), a prognostic biomarker (Ki-67), as well as an adjunctive diagnostic biomarker (basal cell cocktail) and apply the assay to 143 differentially graded adenocarcinoma prostate tissues. The tissues were then imaged on our spectroscopic multiplexing imaging platform and mined for proteomic and spatial features that were correlated with cancer presence and disease grade. Extracted features were used to train a UMAP model that differentiated healthy from cancerous tissue with an accuracy of 89% and identified clusters of cells based on cancer grade. For spatial analysis, cell-to-cell distances were calculated for all biomarkers and differences between healthy and adenocarcinoma tissues were studied. We report that p504s positive cells were at least 2× closer to cells expressing PD-L1, CD8, Ki-67, and basal cell in adenocarcinoma tissues relative to the healthy control tissues. These findings offer a powerful insight to understand the fingerprint of the prostate tumor microenvironment and indicate that high-order chromogenic multiplexing is compatible with digital analysis. Thus, the presented chromogenic multiplexing system combines the clinical applicability of brightfield assays with the emerging diagnostic power of high-order multiplexing in a digital pathology friendly format that is well-suited for translational studies to better understand mechanisms of tumor development and growth.

Effective Dielectric Constant of Water at the Interface with Charged C60 Fullerenes.

The dipolar susceptibility of interfacial water and the corresponding interface dielectric constant were calculated from numerical molecular dynamics simulations for neutral and charged states of buckminsterfullerene C60. Dielectric constants in the range 10-22, depending on temperature and solute charge, were found. These values are consistent with recent reports for biological and nanometer-scale interfaces. The hydration water undergoes a structural crossover as a function of the surface charge of the charged fullerene. Its main signatures include the release of dangling O-H bonds pointing toward the solute and the change in the preferential orientations of hydration water from those characterizing hydrophobic to charged substrates. The interface dielectric constant marks the structural transition with a spike showing a Curie-type phenomenology. The computational formalism adopted here provides direct access to interface susceptibility from configurations produced by computer simulations. The required property is the cross-correlation between the radial projection of the dipole moment of the solvation shell with the electrostatic potential of the solvent inside the solute.

Wetting of the Protein Active Site Leads to Non-Marcusian Reaction Kinetics.

Enzymes exist in continuously fluctuating water bath dramatically affecting their function. Water not only forms the solvation shell but also penetrates into the protein interior. Changing the wetting pattern of the protein's active site in response to altering redox state initiates a highly nonlinear structural change and non-Gaussian electrostatic fluctuations at the active site. The free-energy surfaces of electron transfer are highly nonparabolic (non-Marcusian), as shown by atomistic molecular dynamics simulations of hydrated ferredoxin protein and by an analytical model in agreement with simulations. The reorganization energy of electron transfer passes through a spike marking equal probabilities of the wet and dry states of the active site. The activation thermodynamics affected by wetting leads to a non-Arrhenius, passing through a maximum, plot for the reaction rate vs the inverse temperature.

Impact of Temperature and Non-Gaussian Statistics on Electron Transfer in Donor-Bridge-Acceptor Molecules.

A combination of experimental data and theoretical analysis provides evidence of a bell-shaped kinetics of electron transfer in the Arrhenius coordinates ln k vs 1/T. This kinetic law is a temperature analogue of the familiar Marcus bell-shaped dependence based on ln k vs the reaction free energy. These results were obtained for reactions of intramolecular charge shift between the donor and acceptor separated by a rigid spacer studied experimentally by Miller and co-workers. The non-Arrhenius kinetic law is a direct consequence of the solvent reorganization energy and reaction driving force changing approximately as hyperbolic functions with temperature. The reorganization energy decreases and the driving force increases when temperature is increased. The point of equality between them marks the maximum of the activationless reaction rate. Reaching the consistency between the kinetic and thermodynamic experimental data requires the non-Gaussian statistics of the donor-acceptor energy gap described by the Q-model of electron transfer. The theoretical formalism combines the vibrational envelope of quantum vibronic transitions with the Q-model describing the classical component of the Franck-Condon factor and a microscopic solvation model of the solvent reorganization energy and the reaction free energy.

Theory and Electrochemistry of Cytochrome c.

Extensive simulations of cytochrome c in solution are performed to address the apparent contradiction between large reorganization energies of protein electron transfer typically reported by atomistic simulations and much smaller values produced by protein electrochemistry. The two sets of data are reconciled by deriving the activation barrier for electrochemical reaction in terms of an effective reorganization energy composed of half the Stokes shift (characterizing the medium polarization in response to electron transfer) and the variance reorganization energy (characterizing the breadth of electrostatic fluctuations). This effective reorganization energy is much smaller than each of the two components contributing to it and is fully consistent with electrochemical measurements. Calculations in the range of temperatures between 280 and 360 K combine long, classical molecular dynamics simulations with quantum calculations of the protein active site. The results agree with the Arrhenius plots for the reaction rates and with cyclic voltammetry of cytochrome c immobilized on self-assembled monolayers. Small effective reorganization energy, and the resulting small activation barrier, is a general phenomenology of protein electron transfer allowing fast electron transport within biological energy chains.