Structural Studies of Thioredoxin S-nitrosation and Detection of Protein S-nitrosothiols by Phosphine Derivatization

Persistent Link:
http://hdl.handle.net/10150/312667
Title:
Structural Studies of Thioredoxin S-nitrosation and Detection of Protein S-nitrosothiols by Phosphine Derivatization
Author:
The, Juliana
Issue Date:
2013
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Embargo:
Release 14-Jan-2015
Abstract:
S-nitrosylation (or S-nitrosation) has emerged as an important pathway of non-classical nitric oxide signaling. This post-translational modification involves the transfer of a nitroso group onto a cysteine residue and has been shown to regulate protein function. However, very little is known about the mechanism and structure-dependent factors of the modification. Understanding of S-nitrosothiol chemistry has lagged behind that for the classical nitric oxide signaling pathway due to challenges and limitations of current detection methods of S-nitrosothiols. The S-N bond is typically labile and indirect detection by traditional biotin switch method has low sensitivity and is prone to false positives. In this work, I have explored phosphine derivatization as a new direct approach to labeling protein S-nitrosothiols. Syntheses of aza-ylide derivatives of small organic S-nitrosothiols were successful and the termolecularity of the reaction was overcome by using a bisphosphine. Similarly, S-nitrosated cysteines of thioredoxin were successfully derivatized with the phosphine TCEP and identified by tandem mass spectrometry of the digested protein. Surprisingly, derivatization of S-nitrosoglutathione was found to be unsuccessful and ¹⁸O-labeling of the reaction indicated hydrolysis of the aza-ylide product. We hypothesize that solvent effects are the source of this discrepancy. In addition, x-ray crystallography studies were undertaken to investigate structural rearrangement of a thioredoxin helix to expose residue Cys 62 to S-nitrosation. A new structure of thioredoxin Q63A/C69S/C73S mutant was found to exhibit a highly dynamic N-terminal loop surrounding the pocket of Cys 62 which could have an effect on S-nitrosation of this residue.
Type:
text; Electronic Dissertation
Keywords:
phosphine derivatization; S-nitrosation; S-nitrosylation; thioredoxin; Chemistry; aza ylide
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry and Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Montfort, William R.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen
dc.titleStructural Studies of Thioredoxin S-nitrosation and Detection of Protein S-nitrosothiols by Phosphine Derivatizationen_US
dc.creatorThe, Julianaen_US
dc.contributor.authorThe, Julianaen_US
dc.date.issued2013-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.releaseRelease 14-Jan-2015en_US
dc.description.abstractS-nitrosylation (or S-nitrosation) has emerged as an important pathway of non-classical nitric oxide signaling. This post-translational modification involves the transfer of a nitroso group onto a cysteine residue and has been shown to regulate protein function. However, very little is known about the mechanism and structure-dependent factors of the modification. Understanding of S-nitrosothiol chemistry has lagged behind that for the classical nitric oxide signaling pathway due to challenges and limitations of current detection methods of S-nitrosothiols. The S-N bond is typically labile and indirect detection by traditional biotin switch method has low sensitivity and is prone to false positives. In this work, I have explored phosphine derivatization as a new direct approach to labeling protein S-nitrosothiols. Syntheses of aza-ylide derivatives of small organic S-nitrosothiols were successful and the termolecularity of the reaction was overcome by using a bisphosphine. Similarly, S-nitrosated cysteines of thioredoxin were successfully derivatized with the phosphine TCEP and identified by tandem mass spectrometry of the digested protein. Surprisingly, derivatization of S-nitrosoglutathione was found to be unsuccessful and ¹⁸O-labeling of the reaction indicated hydrolysis of the aza-ylide product. We hypothesize that solvent effects are the source of this discrepancy. In addition, x-ray crystallography studies were undertaken to investigate structural rearrangement of a thioredoxin helix to expose residue Cys 62 to S-nitrosation. A new structure of thioredoxin Q63A/C69S/C73S mutant was found to exhibit a highly dynamic N-terminal loop surrounding the pocket of Cys 62 which could have an effect on S-nitrosation of this residue.en_US
dc.typetexten
dc.typeElectronic Dissertationen
dc.subjectphosphine derivatizationen_US
dc.subjectS-nitrosationen_US
dc.subjectS-nitrosylationen_US
dc.subjectthioredoxinen_US
dc.subjectChemistryen_US
dc.subjectaza ylideen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistry and Biochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorMontfort, William R.en_US
dc.contributor.committeememberMontfort, William R.en_US
dc.contributor.committeememberGlass, Richard S.en_US
dc.contributor.committeememberCordes, Matthew H. J.en_US
dc.contributor.committeememberMiesfeld, Roger L.en_US
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