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Improving the Usage of Unnatural Amino Acids in Proteins : = Thioamides and Other Biophysical Probes.

Record Type:	Language materials, manuscript : Monograph/item
Title/Author:	Improving the Usage of Unnatural Amino Acids in Proteins :/
Reminder of title:	Thioamides and Other Biophysical Probes.
Author:	Walters, Christopher R.
Description:	1 online resource (323 pages)
Notes:	Source: Dissertation Abstracts International, Volume: 79-07(E), Section: B.
Contained By:	Dissertation Abstracts International79-07B(E).
Subject:	Chemistry. -
Online resource:	click for full text (PQDT)
ISBN:	9780355618273

Improving the Usage of Unnatural Amino Acids in Proteins : = Thioamides and Other Biophysical Probes.
Walters, Christopher R.

Improving the Usage of Unnatural Amino Acids in Proteins :Thioamides and Other Biophysical Probes. - 1 online resource (323 pages)

Source: Dissertation Abstracts International, Volume: 79-07(E), Section: B.

Thesis (Ph.D.)--University of Pennsylvania, 2017.

Includes bibliographical references

Methods for genetically and synthetically manipulating protein composition enable a greater flexibility in the study of protein dynamics and function. However, current techniques for incorporating biophysical probes in the form of unnatural amino acids (Uaas) can suffer from poor yield, limited selectivity for the desired probe, and an insufficient understanding of the impact that the probe has on protein structure and function. Each of the studies discussed herein addresses one or more of these shortcomings in an effort to improve the usage of Uaas in biochemistry. We have shown that using inteins as traceless, cleavable purification tags enables the separation of full length Uaa containing proteins from their corresponding truncation products. This method has been used to incorporate Uaas in previously unattainable positions in a variety of proteins using a myriad of Uaas, inteins, and purification tags. In other applications, we have used E. coli aminoacyl transferase (AaT) to selectively modify the N-termini of proteins with Uaas to be used in native chemical ligation or "click" chemistry reactions. Finally, we have previously used backbone thioamide modifications to enable biophysical studies of proteins by taking advantage of their properties as fluorescence quenchers. However, the impact of thioamides on the stability of proteins rich in secondary and tertiary structure has yet to be understood in detail. In this work, we have conducted the most comprehensive studies to date on the effect of thioamides on the structure and thermostability of the full-length proteins, using calmodulin and the B1 domain of protein G. We have found that the thioamide can have destabilizing, neutral, or even stabilizing effects depending on the position of substitution within alpha-helical and beta-sheet folds. Moreover, the advances we have made in thioamide peptide synthesis and protein ligation will enable us to install thioamides with increased efficiency, permitting the first syntheses of proteins with multiple thioamides. In general, by working at the interface of several protein modification technologies, we have developed rigorous methodologies for the incorporation of side chain and backbone modifications while scrutinizing the effects that these modifications may have on protein structure and stability.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2018

Mode of access: World Wide Web

ISBN: 9780355618273Subjects--Topical Terms:

593913
Chemistry.
Index Terms--Genre/Form:

554714
Electronic books.

Improving the Usage of Unnatural Amino Acids in Proteins : = Thioamides and Other Biophysical Probes.
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520 $a Methods for genetically and synthetically manipulating protein composition enable a greater flexibility in the study of protein dynamics and function. However, current techniques for incorporating biophysical probes in the form of unnatural amino acids (Uaas) can suffer from poor yield, limited selectivity for the desired probe, and an insufficient understanding of the impact that the probe has on protein structure and function. Each of the studies discussed herein addresses one or more of these shortcomings in an effort to improve the usage of Uaas in biochemistry. We have shown that using inteins as traceless, cleavable purification tags enables the separation of full length Uaa containing proteins from their corresponding truncation products. This method has been used to incorporate Uaas in previously unattainable positions in a variety of proteins using a myriad of Uaas, inteins, and purification tags. In other applications, we have used E. coli aminoacyl transferase (AaT) to selectively modify the N-termini of proteins with Uaas to be used in native chemical ligation or "click" chemistry reactions. Finally, we have previously used backbone thioamide modifications to enable biophysical studies of proteins by taking advantage of their properties as fluorescence quenchers. However, the impact of thioamides on the stability of proteins rich in secondary and tertiary structure has yet to be understood in detail. In this work, we have conducted the most comprehensive studies to date on the effect of thioamides on the structure and thermostability of the full-length proteins, using calmodulin and the B1 domain of protein G. We have found that the thioamide can have destabilizing, neutral, or even stabilizing effects depending on the position of substitution within alpha-helical and beta-sheet folds. Moreover, the advances we have made in thioamide peptide synthesis and protein ligation will enable us to install thioamides with increased efficiency, permitting the first syntheses of proteins with multiple thioamides. In general, by working at the interface of several protein modification technologies, we have developed rigorous methodologies for the incorporation of side chain and backbone modifications while scrutinizing the effects that these modifications may have on protein structure and stability.
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