Submitted in partial fulfilment for the Degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2010. / This study involved a series of molecular dynamics (MD) simulations applied to case studies of small and medium-size polypeptides to assess the thermodynamics of their folding characteristics. Peptide folding is a complex and vital phenomenon taking place in all living systems. Bioactive conformational structures of folded peptides need to be well characterized before using them in computer-aided drug design. The computational procedure was validated on the 10-residue long chignolin-like synthetic mini-protein (CLN025). For this peptide, replica exchange molecular dynamics (REMD) calculations were carried out in explicit and implicit solvents using the generalized Born (GB)/surface area (SA) approximation with different sets of force field parameters. Following this validation procedure, case studies of the folding conformations of peptides of different lengths including the 5-residue met-enkephalin, the 27-residue pituitary adenylate-activating polypeptide 27(PACAP27) and the 28-residue vasoactive intestinal peptide (VIP) were undertaken. The latter two peptides are multifunctional hormones that mediate diverse biological functions, such as the cell cycle, cardiac muscle relaxation, immune response, septic shock, bone metabolism, and endocrine function. Results obtained indicate that when explicit water, methanol and DMSO solvents were used, it appeared that methanol (MeOH) and dimethylsulphoxide (DMSO) afforded met-enkephalin the ability to form more intra-hydrogen bonds than water, producing type I and type III β-turn structures; thus enhancing the helical conformation of the peptide. MD trajectories of longer polypeptides (VIP and PACAP27) were also populated with type I and type III β-turns, which occurred consecutively; with α- and 310-helices occurring from the middle of each peptide towards the C-terminal. Characterization of implicit solvent results, reveal that these simulations have been able to reproduce the same type of conformers obtained by experimental NMR studies published in literature, which structurally resemble the native conformation of the bioactive peptides. These conformational structures will be applied as lead agents in computer-aided drug design. One of the major achievements of this study is the ability to optimize and validate the force field parameter sets to describe the thermodynamic properties of peptide systems in an unbiased manner, a non-trivial task for even the smallest of peptides. These findings re-affirm the notion that computational methods have matured enough to model dynamic biological phenomena such as peptide folding, a feat previously thought to be impossible.

In spite of the presence of much research in the field of foldable structures whose applications have covered most of the requirements of academic and practical aspects of life, there is still a wide domain in which to undertake further studies. One of the required studies is to invest in foldable structures for the process of temporary accommodation. This study endeavours to find solutions for folding pyramidal shapes constructed from stiff panels that can be used as an upper part of temporary accommodation units, e.g., roofs. Several attempts have been made to find a mechanism that realises the folding of a three-dimensional pyramid. These attempts led to suggest a design that represents an initial solution for folding the pyramid. It was taken into consideration in this design that the structure should deploy strain free when the thickness of its panels is not considered. Trigonometry was used to find mathematical equations that can be used to identify the lengths and angles of the proposed design plates. These equations theoretically proved the validity of the proposed mechanism. The proposed design was applied to construct an actual model formed with thick panels. Considering the panel thickness of the model plates led to amendments and improvements to the proposed design. Two actual models were experimentally tested to make sure that they achieve the design concepts in the processes of full folding and deployment. The models were also tested in the laboratory to make sure of the integrity of the panel hinges and resistance of the elements to external loading. The model was constructed in a simulation program in order to verify the foldability of the design, folding efficiency and absence of strain or collisions during the process of folding and deployment at all stages.

The folding station is one step of the packing process taking place in the metal tube filling machines. The purpose of this project is to develop and design a folding station for metal tubes. With this aim, a product design process has been created and as a result of the application of a deductive method, ten concepts have been discovered and the most suitable one has been developed later on.

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

SH3 domains are excellent models for probing folding and protein interactions. This thesis describes NMR studies of several SH3 domains, including the N-terminal SH3 domain of the Drosophila adaptor protein Drk (drkN SH3 domain), the SH3 domain of the proto-oncogene tyrosine-kinase Fyn, and the SH3 domains of the human adaptor protein CIN85, involved in Cbl-mediated downregulation of epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs). The drkN SH3 domain is an ideal system for studying disordered states. The unique quality of this isolated domain is that it exists in an approximately 50/50 equilibrium between its folded and unfolded states under non-denaturating buffer conditions. Interestingly, the single T22G mutation dramatically stabilizes the domain. Here the NMR structures of the drkN SH3 domain and its T22G mutant are determined and compared in order to illuminate the causes of the marginal stability of the domain. Solvent exposure of the folded and the unfolded drkN SH3 domains are probed and compared with a novel NMR technique using molecular oxygen dissolved in solution as a paramagnetic probe. The changes in partial molar volume along the folding trajectories of the drkN SH3 and Fyn SH3 domains are also studied and analyzed here in terms of changes in protein hydration and packing accompanying folding. Finally, the interactions between the SH3 domains of CIN85 and ubiquitin are discussed. All three are shown to bind ubiquitin. The structure of the SH3-C domain in complex with ubiquitin is presented and the effect of disruption of ubiquitin binding on ubiquitination of CIN85 and EGFR in vivo is discussed. SH3 domains are easily amendable to a wide range of NMR approaches and provide a good system for development and testing of novel methods. Through the use of these approaches significant insights into details of SH3 domain structure, stability, mechanisms of folding and cellular function have been gained.

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.

In this thesis, we study foldings of orthogonal polygons into orthogonal polyhedra. The particular problem examined here is whether a paper cutout of an orthogonal polygon with fold lines indicated folds up into a simple orthogonal polyhedron. The folds are orthogonal and the direction of the fold (upward or downward) is also given. We present a polynomial time algorithm to solve this problem. Next we consider the same problem with the exception that the direction of the folds are not given. We prove that this problem is NP-complete. Once it has been determined that a polygon does fold into a polyhedron, we consider some restrictions on the actual folding process, modelling the case when the polyhedron is constructed from a stiff material such as sheet metal. We show an example of a polygon that cannot be folded into a polyhedron if folds can only be executed one at a time. Removing this restriction, we show another polygon that cannot be folded into a polyhedron using rigid material.

Gas-phase analytical techniques provide very valuable tools for tackling the structural complexity of macromolecular structures such as those encountered in biological systems. Conformational dynamics of polypeptides and polypeptide assemblies underlie most biological functionalities, yet great difficulties arise when investigating such phenomena with the well-established techniques of X-ray crystallography and NMR. In areas such as these ion mobility interfaced with mass spectrometry (IMMS) and molecular modelling can make a significant contribution. During an IMMS experiment analyte ions drift in a chamber filled with an inert gas; measurement of the transport properties of analyte ions under the influence of a weak electric field can lead to determination of the orientationally-averaged collision cross-section of all resolved ionic species. A comparison with cross-sections estimated for model molecular geometries can lead to structural assignments. Thus IMMS can be used effectively to separate gas-phase ions based on their conformation. The drift tube employed in the experiments described herein is thermally regulated, which also enables the determination of collision cross-sections over a range of temperatures, and can provide a view of temperature-dependent conformational dynamics over the experimental (low microsecond) timescale. Studies described herein employ IMMS and a gamut of other MS-based techniques, solution spectroscopy and – importantly – molecular mechanics simulations to assess a) conformational stability of isolated peptide ions, with a focus on small model peptides and proteins, especially the Trp cage miniprotein; and b) structural characteristics of oligomeric aggregates of an amyloidogenic peptide. The results obtained serve to clarify the factors which dominate the intrinsic stability of non-covalent structure in isolated peptides and peptide assemblies. Strong electrostatic interactions are found to play a pivotal role in determining the conformations of isolated proteins. Secondary structures held together by hydrogen bonding, such as helices, are stable in the absence of solvent, however gas-phase protein structures display loss of their hydrophobic cores. The absence of a polar solvent, “self-solvation” is by far the most potent force influencing the gas-phase configuration of these systems. Geometries that are more compact than the folded state observed in solution are routinely detected, indicating the existence of intrinsically stable compact non-native states in globular proteins, illuminating the nature of proteins’ ‘unfolded’ states.

Sculptural Textiles is a material investigation exploring sculptural possibilities for machine woven textiles. Two important factors are yarn combinations and textile construction, and how these two together can result in fabrics that can be manipulated by hand into shapes and thus adaptable to various settings. The essence of this project is in the meeting between contrasts such as shiny-dull, elastic-stiff, transparent-opaque, natural and synthetic and how these meetings can create sculptural qualities. The project is aiming to create a range of sculptural textiles as well as being an exploration in material. Construction and density are tools to bring forth the beauty and function of the materials. The result is a range of textiles showing that small changes in material and construction can lead to different sculptural characteristics.

The Malmberget apatite iron ore deposit is one of the most important iron producers within Europe located within an area of world-renowned mines and mining companies. It is becoming increasingly accepted that in order to increase our resources it is essential to gain a better understanding of the formation and evolution of our known mineral deposits. This thesis is part of an ongoing multi-scale 4-dimensional geological modeling project as part of a collaboration between Vinnova, LKAB, Boliden & LTU. The aim of the which is to piece together the series of geological events that are responsible for the entire Gällivare mining district as seen today. This project looks at three smaller old open pits on the outer limbs of the synform fold structure that forms the Malmberget deposit. This thesis aims to gain a better understanding of the structures that have defined this current shape, and relating them to the regional-scale structural evolution. The results show two distinct deformation events, D1and D2, with each event leaving their own signature on the region. D1 deformation resulted in the formation of high strain zones and a gneissic cleavage within the volcanic rocks. D2 deformation subsequently folded the S1 gneissic cleavage and high strain zones but without developing its own fabric. / Multi-scale 4-dimensional geological modeling of the Gällivare area