This preview has intentionally blurred sections. Sign up to view the full version.View Full Document
Unformatted text preview: Electrostatic Fields Near the Active Site of Human Aldose Reductase: 1. New Inhibitors and Vibrational Stark Effect Measurements ² Lauren J. Webb and Steven G. Boxer* Department of Chemistry, Stanford Uni V ersity, Stanford, California 94305-5080 Recei V ed August 22, 2007; Re V ised Manuscript Recei V ed No V ember 19, 2007 ABSTRACT : Vibrational Stark effect spectroscopy was used to measure electrostatic fields in the hydrophobic region of the active site of human aldose reductase ( h ALR2). A new h ALR2 inhibitor was designed and synthesized that contains a nitrile probe with a Stark tuning rate of 0.77 cm- 1 /(MV/cm). Mutations to amino acid residues in the vicinity of the nitrile functional group were selected based on electrostatics calculations, possible complications from hydrogen bonds near the nitrile, and comparison with the active site of human aldehyde reductase, whose structure is very similar. Changes in the absorption energy of the nitrile probe when bound to those mutated proteins were then used to quantify perturbations to the protein’s electrostatic field. Electrostatic field changes as large as- 10 MV/cm were observed. Measured electrostatic fields were compared to predictions based on continuum electrostatics calculations, revealing that substantial modifications to the calculation strategy are necessary. The effects of hydrogen bonding of amino acid side chains to the nitrile probe are considered, and applications of vibrational Stark effect spectroscopy to investigations of ligand binding and biological function are discussed. The highly organized three-dimensional structure of a protein can support large variations in internal electrostatic fields that influence every aspect of the protein’s function ( 1- 3 ) including folding ( 4 ), chemical reactivity and kinetics ( 5- 7 ), and protein- protein interactions ( 8 ). Measurements of the strength and direction of these fields are therefore of great importance, and represent a long-standing biophysical challenge ( 9- 16 ). One strategy that has been developed in recent years is vibrational Stark effect (VSE 1 ) spectroscopy, which describes the influence of an electric field on a molecular vibrational frequency and can be observed through infrared spectroscopy ( 17, 18 ). VSE spectroscopy is a two- step experiment. In the first step, a vibrational probe is selected and the effect of a known external applied electric field on the infrared absorption frequency is used to calibrate sensitivity of the oscillator’s vibrational frequency to an electric field. The magnitude of this effect is called the Stark tuning rate, and is taken as an intrinsic property of the oscillator. In the second step, the calibrated vibrational probe is inserted into a protein of interest and used as a highly local, sensitive, and directional reporter of changes of the protein’s electrostatic field as perturbations are made to the protein, for example by amino acid mutagenesis. We use the term “vibrational Stark effect” to describe both the effect...
View Full Document
- Fall '09
- Electrostatics, Enzyme inhibitor, ELECTROSTATIC FIELD