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Kettering, Regina Dawn (2012) THE PROTEIN RELATIONSHIPS IN RESPIRATION REGULATION ELUCIDATED BY X-RAY CRYSTALLOGRAPHY. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Many pathways feed into the cellular respiration system. Hemoglobin supplies the oxygen necessary for aerobic respiration. Glycerol metabolism contributes electrons to the electron transport chain. Rap1B regulates superoxide production and platelet activation. I have determined 14 mutant structures that demonstrate how changes affect the regulation and function of the above systems: structural models of Enterococcus casseliflavus glycerol kinase mutants with glycerol or ethylene glycol, Pseudomonas aeruginosa glycerol kinase with glycerol or ethylene glycol, octameric human hemoglobin mutants, and two Rap1B mutants. The structures demonstrate that small changes in one region of the model can significantly affect the protein activity and adopted conformation of other portions of the model.
In the case of En. casseliflavus GK, the presence of loop interactions creates a domino effect that closes the active cleft and orients the substrates properly for glycerol phosphorylation. The His232Arg mutants with glycerol or ethylene glycol contain contacts between the activation loop and Domain I that stabilize a conformation that causes closure of the glycerol binding cleft. The P. aeruginosa GK adopts similar conformations to the E. coli structure in the glycerol-bound form. The activation cleft closes when bound to glycerol rather than ethylene glycol as in the En. casseliflavus structures, but requires ATP to stabilize the area close to the active site entrance.
The octameric structure of the hemoglobin mutants are affected by the presence of ligand in the molecule. The αAsn78Cys mutant does not show the same type of variability in the disulfide bridges, and therefore it does not have as much variability in the tetramers as the βGly83Cys mutants. The structures and crystallographic results suggest that the ligand stabilizes the βGly83Cys mutant relative tetramer motion.
The Rap1B models demonstrate changes in the switch II region determined by the phosphorylation of Ser179 rather than the presence of GTP. The negatively-charged Ser179Asp residue changes the interaction between the C-terminal loop and the core of the protein, presenting a binding area that is inaccessible in the Ser179Ala mutant.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Kettering, Regina
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee CoChairYeh, Joanne I.jiyeh@pitt.eduJIYEH
Committee CoChairHendrix, Roger W.rhx@pitt.eduRHX
Committee MemberGrabowski, Paula J.pag4@pitt.eduPAG4
Committee MemberSchwacha, Anthonyschwacha@pitt.eduSCHWACHA
Committee MemberVandemark, Andrewandyv@pitt.eduANDYV
Date: 27 September 2012
Date Type: Publication
Defense Date: 2 August 2012
Approval Date: 27 September 2012
Submission Date: 17 August 2012
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 194
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: glycerol kinase, octameric hemoglobin, Rap1, G-protein, x-ray crystallography
Date Deposited: 28 Sep 2012 00:43
Last Modified: 27 Sep 2017 05:15


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