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Ultrafast dynamics in enzyme catalysis

Award Number: R35GM119818
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/07/2016
PERIOD OF PERFORMANCE END DATE: 05/31/2023

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Ultrafast dynamics in enzyme catalysis - PROJECT SUMMARY Enzyme dynamics span a broad range of timescales, from milliseconds and microseconds down to picoseconds and femtoseconds. Catalysis of biological reactions is linked to these motions, but the role of ultrafast dynamics in the femtosecond and picosecond range remains controversial. Because diverse fields in the biological sciences, such as the study of metabolism and drug design, depend on a detailed understanding of enzyme function, resolving this controversy could have important implications for our understanding of many human diseases. We approach this problem using a combination of traditional protein chemistry techniques and 2D IR spectroscopy to correlate dynamics measurements with biologically-relevant function. In this proposal, we describe our current and future efforts to characterize differences in the ultrafast motions of enzymes in sets of mutants with different degrees of activity. The long-term goal of this project is to discover how protein sequence variations, including disease-causing mutations, can influence the catalytic function of enzymes via motions on a similar timescale as bond formation and breaking in an enzyme’s active site. Our direct objectives in this research are to develop the experimental tools needed to observe communication between an enzyme’s active site and scaffold, both via mutagenesis and spectroscopy. We hypothesize that trends in the activities of mutants will also be reflected in their ultrafast dynamics due to modulation of barrier crossing during reactions. We will use protein synthesis and labeling techniques to prepare a set of model enzymes with active site vibrational labels, and will use 2D IR spectroscopy to characterize local electric field dynamics. We will utilize vibrationally-labeled substrate analogs as well as protein-based labels in concert with variations in pump-rpbe waiting time to characterize these dynamics. Using random mutagenesis and activity screens, we will identify mutants of these enzymes with altered catalytic rates. Dynamics measurements of active site labels will be correlated to enzyme activities and other properties such as fold stability. Our research program also includes experiments designed to measure non-equilibrium dynamics that may influence catalysis. We will also use our labeled systems to investigate the origins of non-exchangeable heavy isotope effects on catalytic efficiency. Later, we will examine the efficiency of energy transfer within the active site, and between the active site and scaffold, using variations on 2D IR spectroscopy. We will use dual- frequency 2D IR spectroscopy to probe pairs of labels for evidence of vibrational energy transfer pathways. Finally, we will use transient 2D IR spectroscopy to examine pathways for energy relaxation through the protein scaffold after electronic photoexcitation of active site moieties, which we use as a proxy for relaxation after a reaction step. In all cases, we will perform these experiments against mutant backgrounds in which catalysis is altered.


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: 2024 ( Subtotal = -$7,730 )
2024	2020	BOARD OF TRUSTEES OF SOUTHERN ILLINOIS UNIVERSITY	900 S NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	5	12/6/2023	NON-COMPETING CONTINUATION	-$7,730
														Subtotal = -$7,730

Issue Date FY: 2022 ( Subtotal = $0 )
2022	2020	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	5	6/2/2022	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2020 ( Subtotal = $328,145 )
2020	2020	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	5	7/30/2020	NON-COMPETING CONTINUATION	$328,145
														Subtotal = $328,145

Issue Date FY: 2019 ( Subtotal = $335,161 )
2019	2019	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	4	6/7/2019	NON-COMPETING CONTINUATION	$335,161
														Subtotal = $335,161

Issue Date FY: 2018 ( Subtotal = $337,125 )
2018	2018	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	3	5/21/2018	NON-COMPETING CONTINUATION	$337,125
														Subtotal = $337,125

Issue Date FY: 2017 ( Subtotal = $313,579 )
2017	2017	SOUTHERN ILLINOIS UNIVERSITY	900 SOUTH NORMAL AVE	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	2	5/22/2017	NON-COMPETING CONTINUATION	$313,579
														Subtotal = $313,579

Issue Date FY: 2016 ( Subtotal = $289,496 )
2016	2016	SOUTHERN ILLINOIS UNIVERSITY-CARBONDALE	RESEARCH & PROJECTS FISCAL M	CARBONDALE	IL	62901	JACKSON	USA	Biomedical Research and Research Training	000	1	9/6/2016	NEW	$289,496
														Subtotal = $289,496

Grand Total All Awards = $1,595,776

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Ultrafast dynamics in enzyme catalysis

Award Number: R35GM119818

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 09/07/2016

PERIOD OF PERFORMANCE END DATE: 05/31/2023

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