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Rapid protein dynamics and catalysis: modulation by laboratory evolution, designed mutation, and protein control of electric field environment

Award Number: R01GM127594
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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The goal of the research program described in this application is to obtain a deeper understanding of how rapid protein dynamics, so called promoting vibrations on a sub picosecond timescale, are employed and incorporated into both natural and artificially designed enzymes. Over the years we have identified such motions in a variety of enzymatically catalyzed reactions, and also found them missing in at least one. Our goal for the long term is to decipher how nature has used this approach as an engineering principle and how it has been incorporated as a part of the function. For example, many studies have now found that in hydride transfer enzymes such as alcohol dehydrogenase, rapid motion of the donor to the acceptor facilitates the chemical step by lowering the effective adiabatic barrier. We also study two seemingly dichotomous views of enzyme function – the electrostatic view, and the dynamic view. It is entirely possible that the electric field milieu both contributes strongly to catalysis, and in certain cases is modulated by the same types of motions that for example control donor acceptor distance. Because our methods allow us to harvest ensembles of reactive trajectories, exactly such mechanisms can be studied. Finally, there exist cases in which naturally occurring enzymes exhibit simple and direct chemistry, while single mutations cause significant complexities in kinetic analysis. It is simply not clear how this can come about. In order to pursue this research we propose to implement the following three specific aims: Aim 1: We will study one of the more successful attempts in synthetic enzymatic chemistry – the artificial creation of retro-aldolases. We will ascertain whether theoretical design coupled to laboratory evolution of these artificial protein catalysts caused change in the coupling of protein dynamics to reaction. Aim 2: We will study how electric field varies in the active site of an enzyme as the reaction proceeds from reactants to products through a transition state. Catechol-O-methyltransferase (COMT) is an enzyme in which both protein dynamics and electrostatic preorganization have been stated emphatically by other groups to produce the catalytic effect. In particular we will identify if there is a promoting vibration as part of the reaction coordinate in this enzyme, and how it may help to control the field environment. Aim 3: We will analyze reactions catalyzed by the poorly understood “ene-reductase” family. We will address the importance of protein dynamics and the ability of a single point mutation to create multiple reaction “configurations” with highly divergent kinetic behavior. After decades of study, the deceptively simple question of how enzymes work is still a hotbed of debate. Via such studies as here proposed we contribute to both basic knowledge and eventual practical control application.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2023 ( Subtotal = $0 )
2023	2022	UNIVERSITY OF ARIZONA	845 N PARK AVE RM 538	TUCSON	AZ	85721	PIMA	USA	Biomedical Research and Research Training	000	4	4/4/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2022 ( Subtotal = $297,047 )
2022	2022	UNIVERSITY OF ARIZONA	888 N EUCLID AVE RM 510	TUCSON	AZ	85719	PIMA	USA	Biomedical Research and Research Training	001	4	6/27/2022	NON-COMPETING CONTINUATION	$29,705
2022	2022	UNIVERSITY OF ARIZONA	888 N EUCLID AVE RM 510	TUCSON	AZ	85719	PIMA	USA	Biomedical Research and Research Training	000	4	11/17/2021	NON-COMPETING CONTINUATION	$267,342
														Subtotal = $297,047

Issue Date FY: 2021 ( Subtotal = $297,500 )
2021	2021	UNIVERSITY OF ARIZONA	888 N EUCLID AVE RM 510	TUCSON	AZ	85719	PIMA	USA	Biomedical Research and Research Training	001	3	3/1/2021	NON-COMPETING CONTINUATION	$29,750
2021	2021	UNIVERSITY OF ARIZONA	888 N EUCLID AVE RM 510	TUCSON	AZ	85719	PIMA	USA	Biomedical Research and Research Training	000	3	11/16/2020	NON-COMPETING CONTINUATION	$267,750
														Subtotal = $297,500

Issue Date FY: 2020 ( Subtotal = $297,920 )
2020	2020	UNIVERSITY OF ARIZONA	888 N EUCLID AVE RM 510	TUCSON	AZ	85719	PIMA	USA	Biomedical Research and Research Training	001	2	3/10/2020	NON-COMPETING CONTINUATION	$29,792
2020	2020	UNIVERSITY OF ARIZONA	888 N EUCLID AVE RM 510	TUCSON	AZ	85719	PIMA	USA	Biomedical Research and Research Training	000	2	11/19/2019	NON-COMPETING CONTINUATION	$268,128
														Subtotal = $297,920

Issue Date FY: 2019 ( Subtotal = $496,308 )
2019	2019	UNIVERSITY OF ARIZONA	888 N EUCLID AVE RM 510	TUCSON	AZ	85719	PIMA	USA	Biomedical Research and Research Training	001	1	9/9/2019	SUPPLEMENT FOR EXPANSION	$198,000
2019	2019	UNIVERSITY OF ARIZONA	888 N EUCLID AVE RM 510	TUCSON	AZ	85719	PIMA	USA	Biomedical Research and Research Training	000	1	12/17/2018	NEW	$298,308
														Subtotal = $496,308

Grand Total All Awards = $1,388,775

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Rapid protein dynamics and catalysis: modulation by laboratory evolution, designed mutation, and protein control of electric field environment

Award Number: R01GM127594

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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