Abstract:
Electrostatic interactions in proteins play a crucial role in determining the structure–function relation in biomolecules. In recent years, fluorescent probes have been extensively employed to interrogate the polarity in biological cavities through dielectric constants or semiempirical polarity scales. A choice of multiple spectroscopic methods, not limited by fluorophores, along with a molecular level description of electrostatics involving solute–solvent interactions, would allow more flexibility to pick and choose the experimental technique to determine the local electrostatics within protein interiors. In this work we report that ultraviolet/visible-absorption, infrared-absorption, or 13C NMR can be used to calibrate the local electric field in both hydrogen bonded and non-hydrogen bonded protein environments. The local electric field at the binding site of a serum protein has been determined using the absorption wavelength as well as the carbonyl stretching frequency of its natural steroid substrate, testosterone. Excellent agreement is observed in the results obtained from two independent spectroscopic techniques.
