Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs

Bibliographic Details
Main Author:	Glidden, Michael D., II
Language:	English
Published:	Case Western Reserve University School of Graduate Studies / OhioLINK 2018
Subjects:	Biophysics Biochemistry single-chain insulin analog hormone diabetes mellitus protein engineering protein stability protein structure and folding protein dynamics nuclear magnetic resonance single-chain insulin pharmacology
Online Access:	http://rave.ohiolink.edu/etdc/view?acc_num=case1522270994798884

id	ndltd-OhioLink-oai-etd.ohiolink.edu-case1522270994798884
record_format	oai_dc
spelling	ndltd-OhioLink-oai-etd.ohiolink.edu-case15222709947988842021-08-03T07:05:31Z Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs Glidden, Michael D., II Biophysics Biochemistry single-chain insulin analog hormone diabetes mellitus protein engineering protein stability protein structure and folding protein dynamics nuclear magnetic resonance single-chain insulin pharmacology Single-chain insulin (SCI) analogs have long provided a valuable model for structure-function studies of insulin. Recently, a biologically active SCI containing a six-residue connecting (C) domain (inserted between the B- and A chains of two-chain insulin) demonstrated marked resistance to thermal degradation. Such a design has potential to circumvent the cold-storage requirements of current insulin therapeutic formulations which hinder their distribution, storage, and use in developing regions. An understanding of how this SCI scaffold can be engineered to confer desired biophysical and pharmacological properties would enable rational design, development, and clinical implementation of SCIs. This Thesis leverages principles of protein folding, structure, dynamics, stability and function to modulate SCI properties. Following an introduction of insulin analog design with focus on how SCIs could address a global need for ultra-stable formulations (Chapter 1), a synthetic single-chain [des-B29,des-B30]-insulin platform was utilized to model an oxidative intermediate in the folding of wild-type insulin. This system enabled comparative studies of an unstable TyrB16->Pro variant, which exhibited a severe folding defect (Chapter 2). Chapters 3-4 contain studies of two novel biologically active SCIs: SCI-a and SCI-b. These analogs exhibited remarkable resistance to thermal inactivation; SCI-a had native-like duration of hormone action in diabetic rats. Collaborative X-ray crystallographic studies of SCI-a revealed a novel zinc-free T6 hexamer (Chapter 3) whereas NMR and molecular dynamics studies of SCI-b provided insight into its mechanisms of enhanced stability and receptor binding (Chapter 4). Studies of variant SCIs containing amino-acid substitutions at position A14 (Chapter 5) suggested that the reverse hydrophobic effect, a general biophysical principle uncommonly observed, was operative. This principle could be leveraged to enhance SCI resistance to chemical degradation. An unexpected prolongation of hormone action observed upon intravenous injection of SCI-b in diabetic rats (Chapters 3 and 4) motivated mutagenesis of A- and C domain sites (Chapter 6). Our results identified a set of novel fast-acting SCIs. A Thesis summary (Chapter 7) is followed by a discussion of how the findings presented herein may motivate future biophysical studies. Implications for the development of SCI therapeutic formulations and their potential impact on global diabetes care are discussed. 2018-05-31 English text Case Western Reserve University School of Graduate Studies / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=case1522270994798884 http://rave.ohiolink.edu/etdc/view?acc_num=case1522270994798884 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
collection	NDLTD
language	English
sources	NDLTD
topic	Biophysics Biochemistry single-chain insulin analog hormone diabetes mellitus protein engineering protein stability protein structure and folding protein dynamics nuclear magnetic resonance single-chain insulin pharmacology
spellingShingle	Biophysics Biochemistry single-chain insulin analog hormone diabetes mellitus protein engineering protein stability protein structure and folding protein dynamics nuclear magnetic resonance single-chain insulin pharmacology Glidden, Michael D., II Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs
author	Glidden, Michael D., II
author_facet	Glidden, Michael D., II
author_sort	Glidden, Michael D., II
title	Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs
title_short	Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs
title_full	Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs
title_fullStr	Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs
title_full_unstemmed	Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs
title_sort	single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs
publisher	Case Western Reserve University School of Graduate Studies / OhioLINK
publishDate	2018
url	http://rave.ohiolink.edu/etdc/view?acc_num=case1522270994798884
work_keys_str_mv	AT gliddenmichaeldii singlechaininsulinanalogsasultrastabletherapeuticsandasmodelsofproteinmisfoldingstabilitystructuredynamicsandfunctionofnovelanalogs
_version_	1719453423465660416

Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs

Similar Items