SINGLE-MOLECULE LEVEL STUDY OF THE INTERACTION BETWEEN ATR AND VITAMIN B12 - CONFORMATIONAL ALLOSTERY FOR MISCELLANEOUS FUNCTION

Simple item page
dc.contributor.advisorChen, Tai-Yen
dc.contributor.committeeMemberBaldelli, Steven
dc.contributor.committeeMemberYang, Ding-Shyue
dc.contributor.committeeMemberZastrow, Melissa L.
dc.contributor.committeeMemberWang, Yuhong
dc.creatorPan, Manhua
dc.creator.orcid0000-0002-1367-0279
dc.date.accessioned2023-05-31T21:28:00Z
dc.date.createdDecember 2022
dc.date.issued2022-11-09
dc.date.updated2023-05-31T21:28:01Z
dc.description.abstractVitamin B12 is an essential cofactor for methylmalonyl-CoA mutase (MCM) to catalyze the conversions of methylmalonyl-CoA to succinyl-CoA in the mitochondria. The Cob(I)alamin adenosyltransferase (ATR) initiates this metabolic process by synthesizing and delivering 5-deoxyadenosylcobalamin (AdoCbl) to MCM. Failure of AdoCbl delivery to MCM will eventually form the oxidative inactive species HOCbl. The homo-trimeric ATR maximally binds two AdoCbl in its subunit interface with negative cooperativity for the MCM delivery function. A recent crystallographic study further reveals that ATR may regulate its reactivity and transportability by modulating its mobile loops. Despite the valuable thermodynamic and protein structural information, how ATR binding kinetics correlate with the protein allosteric regulation and the origin of the negative cooperativity remains unknown. Further, with the presence of HOCbl, it is also unclear how ATR could preferentially interact with AdoCbl over HOCbl to maximize the transferring efficiency. This research aims to address these questions using single-molecule approaches. Quantitatively measuring the binding/unbinding process is needed to address these questions. By developing a FRET-based single-molecule relative fluorescence (SRF) approach with the single-molecule interaction simulation (SMIS), we probe the interactions between ATR with two B12 derivatives, AdoCbl and HOCbl. The SRF trajectories capture the interactions and report the microscopic interacting dwell-time distributions, which quantify the binding and unbinding kinetic parameters and reveal a two-step binding mechanism. SMIS simulates and gives the expected kinetic characteristics based on the assigned kinetic model, and it can also be used to dissect the interaction kinetics from single-molecule FRET trajectories. Our data indicate that ATR undergoes a conformational change before binding substrates. Such conformational change slows down the second substrate binding. This negative cooperativity favors the AdoCbl release or delivery, which benefits the AdoCbl synthesis by emptying a reaction site. The comparative analysis of interaction kinetics between AdoCbl and HOCbl also informs that the apo-ATR preferentially binds AdoCbl through a conformational sampling mechanism. These findings illustrated important molecular dynamic insight into how the dynamic ATR-AdoCbl interaction mediates AdoCbl transferring. The developed SRF assay also offers a possibility to investigate protein-substrate interactions with relevant time resolutions, and SMIS enables the precise quantification of the interaction kinetic rate constants without using the traditional single-molecule analytical solution.
dc.description.departmentChemistry, Department of
dc.format.digitalOriginborn digital
dc.format.mimetypeapplication/pdf
dc.identifier.citationPortions of this document appear in: Pan, Manhua, Yuteng Zhang, Guangjie Yan, and Tai-Yen Chen. "Dissection of interaction kinetics through single-molecule interaction simulation." Analytical chemistry 92, no. 17 (2020): 11582-11589.
dc.identifier.urihttps://hdl.handle.net/10657/14386
dc.language.isoeng
dc.rightsThe author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. UH Libraries has secured permission to reproduce any and all previously published materials contained in the work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).
dc.subjectB12
dc.subjectATR
dc.subjectSMIS
dc.subjectSingle-molecule fluorescent quenching
dc.titleSINGLE-MOLECULE LEVEL STUDY OF THE INTERACTION BETWEEN ATR AND VITAMIN B12 - CONFORMATIONAL ALLOSTERY FOR MISCELLANEOUS FUNCTION
dc.type.dcmiText
dc.type.genreThesis
dcterms.accessRightsThe full text of this item is not available at this time because the student has placed this item under an embargo for a period of time. The Libraries are not authorized to provide a copy of this work during the embargo period.
local.embargo.lift2024-12-01
local.embargo.terms2024-12-01
thesis.degree.collegeCollege of Natural Sciences and Mathematics
thesis.degree.departmentChemistry, Department of
thesis.degree.disciplineChemistry
thesis.degree.grantorUniversity of Houston
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy

Files

License bundle

Now showing 1 - 2 of 2
No Thumbnail Available
Name:
PROQUEST_LICENSE.txt
Size:
4.43 KB
Format:
Plain Text
Description:
Download
No Thumbnail Available
Name:
LICENSE.txt
Size:
1.81 KB
Format:
Plain Text
Description:
Download

Collections

Published ETD Collection