Studies of electrostatics and hydrogen bonds in human aldose and aldehyde reductase using a nitrile-containing inhibitor
Abstract/Contents
- Abstract
- Human aldose reductase (hALR2) and human aldehyde reductase (hALR1) are both aldo-keto reductases with highly conserved tertiary structures. hALR2 is involved in the development of long-term, secondary diabetes complications. In light of the biomedical interest in developing selective hALR2 inhibitors against hALR1, nitrile vibrational probes were introduced into hALR2 and hALR1 through the binding of nitrile-containing inhibitors. These probes can provide information on microenvironments (e.g. electrostatics, hydrogen bonds) in the specificity pockets of similar proteins and help us understand inhibitor selectivity mechanisms. A new nitrile-containing inhibitor was designed and synthesized. The x-ray structure of its complex with wild type (WT) hALR2, along with cofactor NADP+, was determined. The nitrile is found to be in close proximity to T113, consistent with a hydrogen bond interaction. Two vibrational absorption peaks were observed at room temperature in the nitrile region when the inhibitor binds to WT hALR2, which were empirically assigned to hydrogen bonded and non-hydrogen bonded populations. IR studies on hALR2 mutants, classical molecular dynamics (MD) simulations, temperature dependent IR measurements, and 13C NMR-IR correlation experiments provide consistent, supportive evidence for this assignment from different perspectives. Hydrogen bonding interactions usually help to improve inhibitor potency; meanwhile, it also complicates the simplest analysis of vibrational frequency shifts as being due solely to electrostatic interactions through the vibrational Stark effect (VSE). VSE spectroscopy is an experimental technique which allows direct measurements of protein electrostatic fields. In order to examine the role of electrostatics as a possible selectivity determinant, VSE spectroscopy was used to measure electric fields in the active sites of hALR2 and hALR1 with nitrile vibrational probes. X-ray structures of two mutants, hALR2:T113A and hALR1:Y115A, in complex with the inhibitor and NADP+, were solved. Mutations to amino acid residues near the nitrile probe were made to convert the specificity pocket of one protein to the other, and vibrational frequency shifts were used to quantify the electrostatic differences in these two structurally similar proteins. MD simulations were performed to calculate protein electric fields, which were compared with measured fields. The applications of VSE spectroscopy in the investigation of important biological processes such as inhibitor selectivity are discussed.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2013 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Xu, Lin |
|---|---|
| Associated with | Stanford University, Department of Chemistry. |
| Primary advisor | Boxer, Steven G. (Steven George), 1947- |
| Thesis advisor | Boxer, Steven G. (Steven George), 1947- |
| Thesis advisor | Cegelski, Lynette |
| Thesis advisor | Pecora, Robert, 1938- |
| Advisor | Cegelski, Lynette |
| Advisor | Pecora, Robert, 1938- |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Lin Xu. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2013. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2013 by Lin Xu
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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