# Conformations and Dynamics of Cytochrome P450s via 2D Infrared Spectroscopy

> **NIH NIH R01** · TRUSTEES OF INDIANA UNIVERSITY · 2021 · $222,156

## Abstract

PROJECT SUMMARY
Cytochrome P450s (P450s) are oxidases involved in a wide variety of synthetic and metabolic biochemical
reactions in organisms throughout the kingdoms of life. Importantly, P450s are the central enzymes of drug
metabolism. Despite similar structures and catalytic mechanisms, P450 homologs vary greatly in their specificity
for and catalytic selectivity on different substrates, and these differences can have critical therapeutic
consequences. A major obstacle in predicting P450 reactivity on any given substrate is our incomplete
understanding of the involvement of protein dynamics - the population of multiple states and their interconversion
- in the mechanisms of regioselectivity. Specifically, the population of multiple bound states could orient multiple
parts of the substrate with respect to the reactive intermediate compound I and lead to multiple products. In
addition, flexibility of the active site within one bound state could permit multiple positions of the substrate to
approach the reactive compound I intermediate during its lifetime. Experimentally testing these possibilities is
however challenging due to the complex, heterogeneous nature of the proteins and the contribution of motion
on very fast timescales to protein flexibility. Infrared (IR) spectroscopy can resolve protein conformations and
dynamics that interconvert on even the fastest timescales and, furthermore, 2D techniques can quantify
conformational heterogeneity, as well as the frequency fluctuation amplitudes and timescales with which they
are sampled. When combined with the spatial precision provided by site-selective labeling with IR probes, the
approach should enable unprecedented description of the energy landscapes of P450s. This application is
directed at three P450s which vary in flexibility: P450cam, 3A4, and 2C9 and their complexes with substrates
that are hydroxylated with differing levels of regioselectivity. Measurement and comparison of the dynamics of
the substrate complexes will provide information for evaluating how dynamics are involved in their different
activity. The new information about P450s will advance our understanding of the biophysical mechanisms that
underlie enzyme function, as well as improve our ability to predict P450 activity on a given molecule, and thus
develop better drugs with improved pharmacokinetics.

## Key facts

- **NIH application ID:** 9873047
- **Project number:** 5R01GM114500-04
- **Recipient organization:** TRUSTEES OF INDIANA UNIVERSITY
- **Principal Investigator:** Megan Corrine Thielges
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $222,156
- **Award type:** 5
- **Project period:** 2017-05-01 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9873047

## Citation

> US National Institutes of Health, RePORTER application 9873047, Conformations and Dynamics of Cytochrome P450s via 2D Infrared Spectroscopy (5R01GM114500-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9873047. Licensed CC0.

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