This study concerned the effects of short-term exposures to continuous (10 kv/meter) and pulsed 20 volts at 640 cps/100 msecs) electrostatic fields on the EEG recorded from external electrodes and hypothalamic activity recorded from implanted electrodes in rats. Each experiment lasted at least 90 minutes. The total energies of the waveforms recorded were integrated and printed out for plotting and analysis. Besides the brain activity, the ECG, respiration, and temperature of the animals were also monitored before, during,and after exposure to the electrostatic fields.

Noncovalent factors, such as shape complementarity and electrostatic driving forces, almost exclusively cause the affinity and specificity for which two or more biological macromolecules organize into a functioning complex. The human oncoprotein p21Ras (Ras) and a structurally identical but functionally distant analog, Rap1A (Rap), exhibit high selectivity and specificity when binding to downstream effector proteins that cannot be explained through structural analysis alone. Both Ras and Rap bind to Ral guanine nucleotide dissociation stimulator (RalGDS) with affinities that differ tenfold instigating diverse cellular functions; it is hypothesized that this specificity of RalGDS to discriminate between GTPases is largely electrostatic in nature. To investigate this hypothesis, electrostatic fields at the binding interface between mutants of RalGDS bound to Rap or Ras are measured using vibrational Stark effect (VSE) spectroscopy, in which spectral shifts of a probe oscillator’s energy is related directly to that probe’s local electrostatic environment and measured by Fourier transform infrared spectroscopy (FTIR). After calibration, the probe is inserted into a known position in RalGDS where it becomes a highly local, sensitive, and directional reporter of fluctuations of the protein’s electrostatic field caused by structural or chemical perturbations of the protein. The thiocyanate (SCN) vibrational spectroscopic probe was systematically incorporated throughout the binding interface of RalGDS. Changes in the absorption energy of the thiocyanate probe upon binding were directly related to the change of the strength of the local electrostatic field in the immediate vicinity of the probe, thereby creating a comprehensive library of the binding interactions between Ras-RalGDS and Rap-RalGDS. The measured SCN absorption energy on the monomeric protein was compared with solvent-accessible surface area (SASA) calculations with the results highlighting the complex structural and electrostatic nature of protein-water interface. Additional SASA studies of the nine RalGDS mutants that bind to Ras or Rap verified that experimentally measured thiocyanate absorption energies are negatively correlated with exposure to water at the protein-water interface. By changing the solvent composition, we confirmed that the cyanocysteine residues that are more exposed to solvent experienced a large difference in absorption energy. These studies reinforce the hypothesis that differences in the electrostatic environment at the binding interfaces of Ras and Rap are responsible for discriminating binding partners. / text

This examines the interplay of structure and local electrostatic fields in protein-protein and protein-solvent interactions. The partial charges of the protein amino acids and the polarization of the surrounding solvent create a complex system of electrostatic fields at protein-protein and protein-solvent interfaces. An approach incorporating vibrational Stark effect (VSE) spectroscopy, dissociation constant measurements, and molecular dynamics (MD) simulations was used to investigate the electrostatic interactions in these interfaces. Proteins p21Ras (Ras) and Rap1A (Rap) have nearly identical amino acid sequences and structures along the effector-binding region but bind with different affinities to Ral guanine nucleotide dissociation stimulator (RalGDS). A charge reversion mutation at position 31 alters the binding affinity of Ras and Rap with RalGDS from 0.1 [mu]M and 1 [mu]M, to 1 [mu]M and 0.5 [mu]M, respectively. A spectral probe was placed at various locations along the binding interface on the surface of RalGDS as it was docked with Ras and Rap single (position 30 or 31) and double mutants (both positions). By comparing the probes' absorption energies with the respective wild-type (WT) analogs, VSE spectroscopy was able to measure molecular-level electrostatic events across the protein-protein interface. MD simulations provided a basis for deconvoluting the structural and electrostatic changes observed by the probes. The mutation at position 31 was found to be responsible for both structural and electrostatic changes compared to the WT analogs. Furthermore, previous identification of positions N27 and N29 on RalGDS as "hot spots" that help discriminate between structurally similar GTPases was supported. The RalGDS probe-containing variants and three model compounds were placed in aqueous solvents with varying dielectric constants to measure changes in absorption energy. We investigated the ability of the Onsager solvent model to describe the solvent induced changes in absorption energy, while MD simulations were employed to determine the location and solvation of the probes at the protein-solvent interface. The solvent accessible-surface area, a measure of hydration, was determined to correlate well with the change in magnitude of the probe's absorption energy and the displaced solvent by the probe. / text