The AAA+ chaperone Hsp104 mediates the reactivation of aggregated proteins in Saccharomyces cerevisiae and is crucial for cell survival after exposure to stress. Protein disaggregation depends on cooperation between Hsp104 and a cognate Hsp70 chaperone system. Hsp104 forms a hexameric ring with a narrow axial channel penetrating the centre of the complex. In Chapter 2, I show that conserved loops in each AAA+ module that line this channel are required for disaggregation and that the position of these loops is likely determined by the nucleotide bound state of Hsp104. This evidence supports a common protein remodeling mechanism among Hsp100 members in which proteins are unfolded and threaded along the axial channel. In Chapter 3, I use a peptide-based substrate mimetic to reveal other novel features of Hsp104’s disaggregation mechanism. An Hsp104-binding peptide selected from solid phase arrays recapitulated several properties of an authentic Hsp104 substrate. Inactivation of the pore loops in either AAA+ module prevented stable peptide or protein binding. However, when the loop in the first AAA+ was inactivated, stimulation of ATPase turnover in the second AAA+ module of this mutant was abolished. Drawing on these data, I propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA+ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA+ module. In Chapter 4, I explore the recognition elements within a model Hsp104-binding peptide that are required for rapid binding to Hsp104. Removal of bulky hydrophobic residues and lysines abrogated the ability of this peptide to function as a peptide-based substrate mimetic for Hsp104. Furthermore, rapid binding of a model unfolded protein to Hsp104 required an intact N-terminal domain and ATP binding at the first AAA+ module. Taken together, I have defined numerous structural features within Hsp104 and its model substrates that are crucial for substrate binding and processing by Hsp104. This work provides a theoretical framework that will encourage research in other protein remodeling AAA+ ATPases.

Misfolding of membrane proteins as a result of mutations that disrupt their functions in substrate transport across the membrane or signal transduction is the cause of many significant human diseases. Yet, we still have a limited understanding of the direct consequences of these mutations on folding and function - a necessary step toward the rational design of corrective therapeutics. This thesis addresses the gap in understanding the residue-specific implications for folding through a series of experiments that utilize the cystic fibrosis transmembrane conductance regulator (CFTR) as a model in various contexts. We first examined the thermodynamic implications of mutations in the soluble nucleotide binding domain 1 (NBD1) of CFTR. We found that mutations can have a significant effect on thermodynamic stability that is masked in non-physiological conditions. Our studies were then focussed on a membrane-embedded hairpin CFTR fragment comprised of transmembrane segments 3 (TM3) and 4 (TM4) to evaluate the direct effects of mutations on folding in a systematic manner. It was found that the translocon-mediated membrane insertion of helices closely parallels a basic hydrophobic-aqueous partitioning event. This study was then extended to determine residue-specific effects on helix-helix association. We found that this process is not solely dependent on hydropathy, but there is a context dependence of these results with regard to residue position within the helix. Overall, these findings constitute a key step in relating mutation-derived effects on membrane protein folding to the underlying basis of human disease such as cystic fibrosis.

Single-molecule fluorescence spectroscopy techniques such as Fluorescence Correlation Spectroscopy (FCS) and single-molecule Förster Resonance Energy Transfer (smFRET) not only possess an unprecedented high sensitivity but also have high temporal and spatial resolution. Therefore, they have an immense potential both in investigation of fundamental biological principles and in clinical applications. FCS analyses are based on both theoretical approximations of the beam geometry and assumptions of the underlying molecular processes. To address the accuracy of analysis, firstly the experimental conditions that should be fulfilled in order to obtain reliable physical parameters are discussed and the input parameters are carefully controlled accordingly to demonstrate the performance of FCS measurements on our home-built confocal multiparameter photon-counting microscope in several in vitro and in-vivo applications. Secondly, we performed a comprehensive FCS analysis of rhodamine family of dyes to evaluate the validity of assigning the correlation relaxation times to the time constant of conformational dynamics of biomolecules. While it is the common approach in literature our data suggests that conformational dynamics mainly appear in the correlation curve via modulation of the dark states of the fluorophores. The size and shape of the folded, unfolded and chemically-denatured states of the N-terminal Src-homology-3 of downstream of receptor kinases (DrkN SH3) were investigated by FCS and smFRET burst experiments. Based on the data, we conclude that a considerable sub-population of the denatured protein is in a closed loop state which is most likely formed by cooperative hydrogen bonds, salt bridges and nonpolar contacts. As a clinical application, we developed and characterized an ultrasensitive capillary electrophoresis method on our multiparameter confocal microscope. This allowed us to perform Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with about 500 times better sensivity than a commercial instrument. Quite remarkably, we were able to analyze samples of cell lysate down to the contents of a single cell.

Peptides are considered as drugs of the future because of their advantageous features of high specificity and low toxicity. However, the complete therapeutic potential of peptides has not yet been realized because of the in vivo instability displayed by most potential peptides. In this thesis, two naturally derived cyclic peptides, cyclotides and sunflower trypsin inhibitor 1 (SFTI-1), were utilized to impart stability to linear bioactive epitopes and enhance their therapeutic potential in a biological environment. Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclised backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Sunflower typsin inhibitor 1 (SFTI-1) is a stable cyclic peptide containing a single disulfide bond. Taking advantage of these stable cyclic peptide frameworks, novel drug leads to inhibit/stimulate angiogenesis were developed by using the approach of ‘epitope grafting’ in which linear epitopes were grafted onto the cyclic peptide frameworks. Angiogenesis is a physiological condition that is unregulated in the progression of many diseases, including cancers and cardiovascular diseases. Thus the drug leads designed in the current project have potential therapeutic applications to combat cancers and cardiovascular diseases. To fully exploit cyclotides as drug scaffolds, it is imperative to understand their folding. Two main subfamilies, referred to as the Möbius and bracelet cyclotides have been identified and interestingly, they require dramatically different in vitro folding conditions to achieve formation of the conserved cyclic cystine knot motif. To determine the underlying structural elements that influence cyclotide folding, the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1 was examined in this thesis. The pathways of folding of the two cyclotide subfamilies were found to be different and primarily dictated by specific residues harboured within inter-cysteine loops 2 and 6. Two changes in these loops, an amino acid substitution in loop 2 and an amino acid addition in loop 6 enabled the folding of cycloviolacin O1 under conditions where folding does not occur in vitro for the native peptide. Thus, the study identified key residues that are not in close proximity in the primary sequence or three-dimensional structure which assist folding in cyclotides. A key intermediate species in the folding pathway was isolated and characterised, and found to contain a native-like hairpin structure that appears to be a nucleation locus early in the folding process. The intermediate does not have native disulfide connectivities, but disulfide shuffling processes ultimately lead to a rearrangement to the native form. Overall these mechanistic findings on the folding of cyclotides are potentially valuable for protein engineering applications that utilize cystine-rich peptides as scaffolds in the design of new drug leads. The current study has also enabled the extention of the grafting studies to the bracelet cyclotide subfamily, which was intractable to grafting prior to this work. Cyclotides are gene encoded macrocyclic proteins and another way to exploit their potential as drug scaffolds, would be to develop combinatorial cyclotide libraries. The most efficient way to generate engineered cyclotides would be via recombinant expression, which currently remains unsuccessful, partly due to lack of understanding of the mechanism of cyclotide backbone cyclization. Understanding how the cyclotide precursor folds may provide clues to how cyliclization occurs. A conserved region known as the N-terminal repeat (NTR) region in the cyclotide precursor has been speculated to play an important role in precursor folding. In this thesis, the function of the NTR in the folding of the cyclotide precursor in vitro was examined via the design of a series of constructs for the precursor protein for the prototypic kalata B1 cyclotide, with incremental additions of the NTR region. Analysis of the constructs by NMR spectroscopy for evidence of secondary structure revealed that the NTR does not assist folding of the cyclotide precursor in vitro. Using diffusion NMR, the unstructured nature of the constructs was localized to the NTR region. In a complementary study, structural analysis of the full length cyclotide precursor was carried out by expressing the precursor gene for kalata B1 in a bacterial expression system. The full-length precursor was found to be unstructured in solution despite approximately half of the precursor comprising the mature domain and NTR, both of which are structured in isolation. The unstructured nature of the cyclotide precursor suggested that a different environment, or indeed interaction of the NTR with a particular enzyme involved in processing, is necessary for it to adopt a well-defined conformation and allow processing to produce the mature circular protein. The information that NTR alone may not assist folding of the cyclotide precursors has provided new impetus to examine the role of other potential folding auxiliaries such as protein disulfide isomerase in cyclotide folding and has indirectly advanced the production of cyclotides via transgenic means. In summary, this thesis has provided a fundamental insight into the folding of cyclotides, both when expressed as part of a precursor protein and in isolation via solid phase chemical synthesis, and has exploited the potential of cyclic peptide scaffolds in drug design applications.

Addendum pasted onto back end-paper. / Bibliography: leaves 146-179. / Bibliography: leaves 146-179. / ix, 179, [66] leaves, [2] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis investigates the effect of mutations and an N-terminal extension on the oxidative folding pathway of IGF-I, analyses the structure of the stable mis-folded molecule in terms of its biological interactions, examines the kinetics of the late stages of oxidative folding and finally attempts to dissect the folding pathway of a mutant of IGF-I. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1996?

Peptides are considered as drugs of the future because of their advantageous features of high specificity and low toxicity. However, the complete therapeutic potential of peptides has not yet been realized because of the in vivo instability displayed by most potential peptides. In this thesis, two naturally derived cyclic peptides, cyclotides and sunflower trypsin inhibitor 1 (SFTI-1), were utilized to impart stability to linear bioactive epitopes and enhance their therapeutic potential in a biological environment. Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclised backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Sunflower typsin inhibitor 1 (SFTI-1) is a stable cyclic peptide containing a single disulfide bond. Taking advantage of these stable cyclic peptide frameworks, novel drug leads to inhibit/stimulate angiogenesis were developed by using the approach of ‘epitope grafting’ in which linear epitopes were grafted onto the cyclic peptide frameworks. Angiogenesis is a physiological condition that is unregulated in the progression of many diseases, including cancers and cardiovascular diseases. Thus the drug leads designed in the current project have potential therapeutic applications to combat cancers and cardiovascular diseases. To fully exploit cyclotides as drug scaffolds, it is imperative to understand their folding. Two main subfamilies, referred to as the Möbius and bracelet cyclotides have been identified and interestingly, they require dramatically different in vitro folding conditions to achieve formation of the conserved cyclic cystine knot motif. To determine the underlying structural elements that influence cyclotide folding, the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1 was examined in this thesis. The pathways of folding of the two cyclotide subfamilies were found to be different and primarily dictated by specific residues harboured within inter-cysteine loops 2 and 6. Two changes in these loops, an amino acid substitution in loop 2 and an amino acid addition in loop 6 enabled the folding of cycloviolacin O1 under conditions where folding does not occur in vitro for the native peptide. Thus, the study identified key residues that are not in close proximity in the primary sequence or three-dimensional structure which assist folding in cyclotides. A key intermediate species in the folding pathway was isolated and characterised, and found to contain a native-like hairpin structure that appears to be a nucleation locus early in the folding process. The intermediate does not have native disulfide connectivities, but disulfide shuffling processes ultimately lead to a rearrangement to the native form. Overall these mechanistic findings on the folding of cyclotides are potentially valuable for protein engineering applications that utilize cystine-rich peptides as scaffolds in the design of new drug leads. The current study has also enabled the extention of the grafting studies to the bracelet cyclotide subfamily, which was intractable to grafting prior to this work. Cyclotides are gene encoded macrocyclic proteins and another way to exploit their potential as drug scaffolds, would be to develop combinatorial cyclotide libraries. The most efficient way to generate engineered cyclotides would be via recombinant expression, which currently remains unsuccessful, partly due to lack of understanding of the mechanism of cyclotide backbone cyclization. Understanding how the cyclotide precursor folds may provide clues to how cyliclization occurs. A conserved region known as the N-terminal repeat (NTR) region in the cyclotide precursor has been speculated to play an important role in precursor folding. In this thesis, the function of the NTR in the folding of the cyclotide precursor in vitro was examined via the design of a series of constructs for the precursor protein for the prototypic kalata B1 cyclotide, with incremental additions of the NTR region. Analysis of the constructs by NMR spectroscopy for evidence of secondary structure revealed that the NTR does not assist folding of the cyclotide precursor in vitro. Using diffusion NMR, the unstructured nature of the constructs was localized to the NTR region. In a complementary study, structural analysis of the full length cyclotide precursor was carried out by expressing the precursor gene for kalata B1 in a bacterial expression system. The full-length precursor was found to be unstructured in solution despite approximately half of the precursor comprising the mature domain and NTR, both of which are structured in isolation. The unstructured nature of the cyclotide precursor suggested that a different environment, or indeed interaction of the NTR with a particular enzyme involved in processing, is necessary for it to adopt a well-defined conformation and allow processing to produce the mature circular protein. The information that NTR alone may not assist folding of the cyclotide precursors has provided new impetus to examine the role of other potential folding auxiliaries such as protein disulfide isomerase in cyclotide folding and has indirectly advanced the production of cyclotides via transgenic means. In summary, this thesis has provided a fundamental insight into the folding of cyclotides, both when expressed as part of a precursor protein and in isolation via solid phase chemical synthesis, and has exploited the potential of cyclic peptide scaffolds in drug design applications.

Peptides are considered as drugs of the future because of their advantageous features of high specificity and low toxicity. However, the complete therapeutic potential of peptides has not yet been realized because of the in vivo instability displayed by most potential peptides. In this thesis, two naturally derived cyclic peptides, cyclotides and sunflower trypsin inhibitor 1 (SFTI-1), were utilized to impart stability to linear bioactive epitopes and enhance their therapeutic potential in a biological environment. Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclised backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Sunflower typsin inhibitor 1 (SFTI-1) is a stable cyclic peptide containing a single disulfide bond. Taking advantage of these stable cyclic peptide frameworks, novel drug leads to inhibit/stimulate angiogenesis were developed by using the approach of ‘epitope grafting’ in which linear epitopes were grafted onto the cyclic peptide frameworks. Angiogenesis is a physiological condition that is unregulated in the progression of many diseases, including cancers and cardiovascular diseases. Thus the drug leads designed in the current project have potential therapeutic applications to combat cancers and cardiovascular diseases. To fully exploit cyclotides as drug scaffolds, it is imperative to understand their folding. Two main subfamilies, referred to as the Möbius and bracelet cyclotides have been identified and interestingly, they require dramatically different in vitro folding conditions to achieve formation of the conserved cyclic cystine knot motif. To determine the underlying structural elements that influence cyclotide folding, the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1 was examined in this thesis. The pathways of folding of the two cyclotide subfamilies were found to be different and primarily dictated by specific residues harboured within inter-cysteine loops 2 and 6. Two changes in these loops, an amino acid substitution in loop 2 and an amino acid addition in loop 6 enabled the folding of cycloviolacin O1 under conditions where folding does not occur in vitro for the native peptide. Thus, the study identified key residues that are not in close proximity in the primary sequence or three-dimensional structure which assist folding in cyclotides. A key intermediate species in the folding pathway was isolated and characterised, and found to contain a native-like hairpin structure that appears to be a nucleation locus early in the folding process. The intermediate does not have native disulfide connectivities, but disulfide shuffling processes ultimately lead to a rearrangement to the native form. Overall these mechanistic findings on the folding of cyclotides are potentially valuable for protein engineering applications that utilize cystine-rich peptides as scaffolds in the design of new drug leads. The current study has also enabled the extention of the grafting studies to the bracelet cyclotide subfamily, which was intractable to grafting prior to this work. Cyclotides are gene encoded macrocyclic proteins and another way to exploit their potential as drug scaffolds, would be to develop combinatorial cyclotide libraries. The most efficient way to generate engineered cyclotides would be via recombinant expression, which currently remains unsuccessful, partly due to lack of understanding of the mechanism of cyclotide backbone cyclization. Understanding how the cyclotide precursor folds may provide clues to how cyliclization occurs. A conserved region known as the N-terminal repeat (NTR) region in the cyclotide precursor has been speculated to play an important role in precursor folding. In this thesis, the function of the NTR in the folding of the cyclotide precursor in vitro was examined via the design of a series of constructs for the precursor protein for the prototypic kalata B1 cyclotide, with incremental additions of the NTR region. Analysis of the constructs by NMR spectroscopy for evidence of secondary structure revealed that the NTR does not assist folding of the cyclotide precursor in vitro. Using diffusion NMR, the unstructured nature of the constructs was localized to the NTR region. In a complementary study, structural analysis of the full length cyclotide precursor was carried out by expressing the precursor gene for kalata B1 in a bacterial expression system. The full-length precursor was found to be unstructured in solution despite approximately half of the precursor comprising the mature domain and NTR, both of which are structured in isolation. The unstructured nature of the cyclotide precursor suggested that a different environment, or indeed interaction of the NTR with a particular enzyme involved in processing, is necessary for it to adopt a well-defined conformation and allow processing to produce the mature circular protein. The information that NTR alone may not assist folding of the cyclotide precursors has provided new impetus to examine the role of other potential folding auxiliaries such as protein disulfide isomerase in cyclotide folding and has indirectly advanced the production of cyclotides via transgenic means. In summary, this thesis has provided a fundamental insight into the folding of cyclotides, both when expressed as part of a precursor protein and in isolation via solid phase chemical synthesis, and has exploited the potential of cyclic peptide scaffolds in drug design applications.

Peptides are considered as drugs of the future because of their advantageous features of high specificity and low toxicity. However, the complete therapeutic potential of peptides has not yet been realized because of the in vivo instability displayed by most potential peptides. In this thesis, two naturally derived cyclic peptides, cyclotides and sunflower trypsin inhibitor 1 (SFTI-1), were utilized to impart stability to linear bioactive epitopes and enhance their therapeutic potential in a biological environment. Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclised backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Sunflower typsin inhibitor 1 (SFTI-1) is a stable cyclic peptide containing a single disulfide bond. Taking advantage of these stable cyclic peptide frameworks, novel drug leads to inhibit/stimulate angiogenesis were developed by using the approach of ‘epitope grafting’ in which linear epitopes were grafted onto the cyclic peptide frameworks. Angiogenesis is a physiological condition that is unregulated in the progression of many diseases, including cancers and cardiovascular diseases. Thus the drug leads designed in the current project have potential therapeutic applications to combat cancers and cardiovascular diseases. To fully exploit cyclotides as drug scaffolds, it is imperative to understand their folding. Two main subfamilies, referred to as the Möbius and bracelet cyclotides have been identified and interestingly, they require dramatically different in vitro folding conditions to achieve formation of the conserved cyclic cystine knot motif. To determine the underlying structural elements that influence cyclotide folding, the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1 was examined in this thesis. The pathways of folding of the two cyclotide subfamilies were found to be different and primarily dictated by specific residues harboured within inter-cysteine loops 2 and 6. Two changes in these loops, an amino acid substitution in loop 2 and an amino acid addition in loop 6 enabled the folding of cycloviolacin O1 under conditions where folding does not occur in vitro for the native peptide. Thus, the study identified key residues that are not in close proximity in the primary sequence or three-dimensional structure which assist folding in cyclotides. A key intermediate species in the folding pathway was isolated and characterised, and found to contain a native-like hairpin structure that appears to be a nucleation locus early in the folding process. The intermediate does not have native disulfide connectivities, but disulfide shuffling processes ultimately lead to a rearrangement to the native form. Overall these mechanistic findings on the folding of cyclotides are potentially valuable for protein engineering applications that utilize cystine-rich peptides as scaffolds in the design of new drug leads. The current study has also enabled the extention of the grafting studies to the bracelet cyclotide subfamily, which was intractable to grafting prior to this work. Cyclotides are gene encoded macrocyclic proteins and another way to exploit their potential as drug scaffolds, would be to develop combinatorial cyclotide libraries. The most efficient way to generate engineered cyclotides would be via recombinant expression, which currently remains unsuccessful, partly due to lack of understanding of the mechanism of cyclotide backbone cyclization. Understanding how the cyclotide precursor folds may provide clues to how cyliclization occurs. A conserved region known as the N-terminal repeat (NTR) region in the cyclotide precursor has been speculated to play an important role in precursor folding. In this thesis, the function of the NTR in the folding of the cyclotide precursor in vitro was examined via the design of a series of constructs for the precursor protein for the prototypic kalata B1 cyclotide, with incremental additions of the NTR region. Analysis of the constructs by NMR spectroscopy for evidence of secondary structure revealed that the NTR does not assist folding of the cyclotide precursor in vitro. Using diffusion NMR, the unstructured nature of the constructs was localized to the NTR region. In a complementary study, structural analysis of the full length cyclotide precursor was carried out by expressing the precursor gene for kalata B1 in a bacterial expression system. The full-length precursor was found to be unstructured in solution despite approximately half of the precursor comprising the mature domain and NTR, both of which are structured in isolation. The unstructured nature of the cyclotide precursor suggested that a different environment, or indeed interaction of the NTR with a particular enzyme involved in processing, is necessary for it to adopt a well-defined conformation and allow processing to produce the mature circular protein. The information that NTR alone may not assist folding of the cyclotide precursors has provided new impetus to examine the role of other potential folding auxiliaries such as protein disulfide isomerase in cyclotide folding and has indirectly advanced the production of cyclotides via transgenic means. In summary, this thesis has provided a fundamental insight into the folding of cyclotides, both when expressed as part of a precursor protein and in isolation via solid phase chemical synthesis, and has exploited the potential of cyclic peptide scaffolds in drug design applications.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.