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Structure function investigations of radical transfer and disulfide exchange in a class Ia ribonucleotide reductase

Award Number: F32GM145072
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 01/01/2022
PERIOD OF PERFORMANCE END DATE: 12/31/2024

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Structure function investigations of radical transfer and disulfide exchange in a class Ia ribonucleotide reductase - PROJECT SUMMARY/ABSTRACT Class Ia ribonucleotide reductases (RNRs) are anti-cancer and antibiotic drug targets due to their role in the biosynthesis of deoxyribonucleotides. They convert ribonucleotides to deoxyribonucleotides using radical- based chemistry with reducing equivalents coming from a pair of cysteine residues in the active site that form a disulfide upon enzyme turnover. Recent structural work elucidating the active state structure of the class Ia RNR of Escherichia coli has revealed for the first time, the intact 32-Å-long pathway for radical transfer between subunits that is responsible for the generation of a catalytically essential radical species in the enzyme active site. In this proposed research, I will investigate the role of water channels in the proton transfer that occurs concomitant with electron transfer. I will also investigate the role of a tryptophan residue (W48) in a radical transfer pathway that otherwise involves tyrosine residues. Furthermore, I will probe the structural rearrangements that occur in order to facilitate re-reduction of the active site disulfide. This re-reduction pathway is known to be dependent on the C-terminal tail of the α subunit of RNR, which shuttles reducing equivalents from thioredoxin to the active site, but no structural snapshots of this process have ever been captured. Here, I propose to use site-specific mutagenesis to investigate the involvement of water channels in proton transfer by assessing the catalytic competency of RNR variants in which the water networks have been disrupted. I intend to assess the role of W48 in the PCET pathway through the site-specific incorporation of unnatural W analogues which will give us a spectroscopic handle with which to directly monitor radical transfer to and from W48. Finally, I propose to capture structural snapshots of the re-reduction steps of RNR, including a structure of RNR with thioredoxin using cryogenic electron microscopy. These studies will enable us to answer several of the major outstanding questions of this prototypical RNR. This work will also enable me, a physical inorganic and materials chemist by training, to gain expertise in biochemical and structural biology methods which will inform new research directions that will benefit from a synthesis of these two skill sets. As one of the world’s foremost research institutions, MIT is an ideal and highly collaborative environment in which to pursue the studies described in this proposal. Furthermore, as an internationally recognized leader in the field of structural biology, Prof. Drennan’s scientific mentorship will enable me to excel in my undertaking of this research.


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 = $76,756 )
2024	2024	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	3	11/20/2023	NON-COMPETING CONTINUATION	$71,792
2024	2024	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	001	3	5/14/2024	NON-COMPETING CONTINUATION	$4,964
														Subtotal = $76,756

Issue Date FY: 2023 ( Subtotal = $69,500 )
2023	2023	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	001	2	2/9/2023	NON-COMPETING CONTINUATION	$1,918
2023	2023	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	2	12/1/2022	NON-COMPETING CONTINUATION	$67,582
														Subtotal = $69,500

Issue Date FY: 2022 ( Subtotal = $67,174 )
2022	2022	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	1	12/16/2021	NEW	$65,994
2022	2022	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	001	1	12/16/2021	NEW	$0
2022	2022	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	002	1	5/30/2022	NEW	$1,180
														Subtotal = $67,174

Grand Total All Awards = $213,430

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Structure function investigations of radical transfer and disulfide exchange in a class Ia ribonucleotide reductase

Award Number: F32GM145072

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 01/01/2022

PERIOD OF PERFORMANCE END DATE: 12/31/2024

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