The proposed research will examine how dynamics influence molecular recognition and catalysis in cytochrome (cyt) P450s. While this family of enzymes catalyzes the hydroxylation of a wide variety of substrates, different cyt P450 homologs show varying degrees of substrate specificity. Understanding this specificity is critical for human health as cyt P450s are involved in many biochemical processes, such as the metabolism of xenobiotics and the biosynthesis of hormones and fatty acids. Importantly, cyt P450s also metabolize the majority of clinically used drugs, so their activity contributes directly to bioavailability and toxicity. While dynamics have been implicated in the substrate specificity of cyt P450s, previous experimental studies have lacked the time resolution to measure the fastest motions that make critical contribution to substrate recognition. However, the newly developed techniques of two dimensional infrared vibrational echo spectroscopy have the potential to directly measure the fast dynamics of cyt P450s and how they might contribute to substrate specificity. The proposed experiments will test the hypothesis that cyt P450s are highly dynamic and that the binding of different substrates is associated with varying changes in dynamics. First, the dynamics at the active site of the relatively substrate-specific cyt P450cam will be characterized using a heme-bound CO probe in the unbound protein and in the protein bound to its substrate, camphor, and several substrate analogs. These experiments will be extended to specific sites throughout the tertiary structure of cyt P450cam by the use of site-specifically incorporated azidophenylalanine probes. Finally, to explore the dynamics of the medically important human cyt P450s, homebound CO will be used to measure the active site dynamics of cyt P450 3A4, one of the most promiscuous cyt P450s that metabolizes a large variety of drug molecules. With both cyt P450s, correlations between the observed dynamics and the presence and nature of bound substrates will support the hypothesis that protein dynamics are important for controlling activity. This would have important ramifications for our understanding of drug metabolism. Thus, the proposed research will further our understanding of dynamics of cyt P450s and how they might contribute to their critical biological functions.

Public Health Relevance

Two dimensional infrared vibrational echo spectroscopy will be used to study how protein motions affect the substrate repertoires of cytochrome P450s. These enzymes are required to process metabolites and toxins, and they play an essential role in drug bioavailability and toxicity. Thus, understanding their activity would be of direct utility in the design of therapeutics with increased efficacy and reduced toxicity.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F04B-B (20))
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Flicker, Paula F
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Stanford University
Schools of Arts and Sciences
United States
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Thielges, Megan C; Fayer, Michael D (2012) Protein dynamics studied with ultrafast two-dimensional infrared vibrational echo spectroscopy. Acc Chem Res 45:1866-74
Thielges, Megan C; Fayer, Michael D (2011) Time-dependent fifth-order bands in nominally third-order 2D IR vibrational echo spectra. J Phys Chem A 115:9714-23
Thielges, Megan C; Chung, Jean K; Axup, Jun Y et al. (2011) Influence of histidine tag attachment on picosecond protein dynamics. Biochemistry 50:5799-805
Thielges, Megan C; Chung, Jean K; Fayer, Michael D (2011) Protein dynamics in cytochrome P450 molecular recognition and substrate specificity using 2D IR vibrational echo spectroscopy. J Am Chem Soc 133:3995-4004
Thielges, Megan C; Axup, Jun Y; Wong, Daryl et al. (2011) Two-dimensional IR spectroscopy of protein dynamics using two vibrational labels: a site-specific genetically encoded unnatural amino acid and an active site ligand. J Phys Chem B 115:11294-304