The measurement and prediction of electric fields in the active site of human aldose reductase

The electric potentials and fields produced by the organization of charged and polar groups in folded proteins influence nearly every aspect of protein function. While a large number of computational techniques have been developed to estimate potentials and fields in proteins relatively few experiments measure quantities directly related to these calculations. Vibrational spectroscopy is used to measure changes in field due to mutation in an enzyme's active site. The sensitivity of various bond types to electric fields is calibrated by observing the change in the infrared absorption spectra of immobilized samples upon application of an external electric field. The nitrile vibration is identified as the most general probe due to its ideal frequency, relatively large intensity and sensitivity to electric fields. Several methods of incorporating nitriles into proteins are explored and infrared spectra of nitrile containing proteins are presented.; To investigate electric fields in the active site human aldose reductase the nitrile containing inhibitor IDD743 is used. The sensitivity IDD743's nitrile stretching frequency to electric fields is determined and absorption spectra are obtained for the inhibitor bound to wild-type and several mutants. From the calibrated sensitivity and the observed frequency shifts the change in field along IDD743's nitrile for these mutations is determined. The experimentally determined fields are compared to those predicted by continuum electrostatics calculations. It is found that the calculated fields are extremely sensitive to the side chain conformation selected for mutant structures. Molecular mechanics simulations are used to relax side chain conformations from initial models and continuum electrostatic calculations are performed along these trajectories. Distributions of field for wild-type and mutants are calculated for trajectories at long time. It is argued that these distributions provide a much better description of the experimentally determined changes in field than electrostatics calculations performed on single static structures.