Human aldose reductase (hALR2) reduces glucose to sorbitol under hyperglycemic conditions, and is thought to be a cause of long-term health effects of diabetes including cataracts and nephropathy. One key to finding strong inhibitors of hALR2 that do not also bind to a structurally similar protein, human aldehyde reductase (hALR1), is to measure the electrostatic field of the inhibitor binding site. Measuring these fields in such a complicated system will be done using vibrational Stark effect (VSE) spectroscopy of hALR2 bound with inhibitors which contain a cyano functional group, which has a vibrational absorption frequency in a region of the spectrum that is free of protein absorption. First, a series of inhibitor molecules will be synthesized in which the location of the cyano group is moved around the hydrophobic pocket of the protein active site. Second, a crystal structure of the protein-inhibitor complex will be obtained. Finally, amino acid residues near the active site will be mutated to alter the local electrostatic field surrounding the probe. VSE spectroscopy will then be conducted on protein mutants with inhibitor bound to the active site. Results will be compared to predictions from current computational models of electrostatic fields in hALR2. ? ?
| Webb, Lauren J; Boxer, Steven G (2008) Electrostatic fields near the active site of human aldose reductase: 1. New inhibitors and vibrational stark effect measurements. Biochemistry 47:1588-98 |
