This thesis describes several molecular dynamics studies of polymers, proteins, and active colloids. These diverse systems fall under the purview of soft matter physics and in Part I, I explain what is soft matter and describe some of its essential features. In Part II, I introduce some basic polymer physics and show how confined polymers can be described using blob theory. I also discuss how phase separation of polymer mixtures can occur. These concepts are applied to systems of mixed polymer brushes on spheroids, objects that have surfaces with non-uniform curvature. I show how the interplay of phase separation and surface curvature give rise to striped patterns, and how an extension of blob theory can give analytical expressions for the free energy. Finally, I show how phase separation of miscible polymers can occur, driven solely by surface curvature. In Part III, I present an overview of self-assembly and describe how active, or self-propelled colloids can be used to assemble new materials. I show how two large colloids immersed in a bath of smaller active colloids exhibit an effective short-ranged repulsion and long-ranged attraction, which stands in contrast to the standard short-ranged depletion attraction. I also explore how self-propulsion changes clustering by focusing on a system with short-ranged attractive and long-ranged repulsive particles, which under equilibrium, exhibit finite-sized clusters. I show that for certain parameters, spheres can form a fluid of living crystals, and dumbbells can form a crystal of rotors. In Part IV, I give a brief introduction to protein folding and describe how molecular chaperones combat misfolding in the human body. Then, taking inspiration from the chaperones, I show that a polymer-grafted “soft” nanopore can be used to unfold misfolded proteins and destroy undesired aggregates. I also show preliminary results for a hydrophobic “smart” nanopore that can selectively capture and unfold misfolded proteins.

Oxidative folding, the process by which folding and disulfide oxidation occur in concert, is a critical step in the production of many extracellular proteins and is therefore centrally linked to a vast multitude of important physiological functions. The primary focus of this dissertation is the remarkable disulfide oxidoreductase DsbA, the sole catalyst of oxidative folding in Escherichia coli. DsbA was the first oxidative folding catalyst to be discovered, and remains the strongest known oxidant among the thioredoxin superfamily of disulfide oxidoreductases due to unique biochemical and biophysical properties. Through the activity of its substrate repertoire, which includes adhesion structures and toxins, DsbA is an essential component of many pathogenic processes and therefore is an active target for the development of novel antibiotics. Though DsbA has been analyzed through a host of biochemical, genetic, and cellular experiments over the quarter-century since its identification, the elucidation of certain mechanistic details of its catalytic process have proven elusive to conventional techniques. This primarily results from the experimental difficulties in independently monitoring the progress of folding and oxidation during oxidative folding that arise with conventional, ensemble-averaged approaches. In this work, single molecule force spectroscopy methods are applied to investigate the process of oxidative folding as catalyzed by DsbA. Through observing single substrate molecules as they undergo DsbA-catalyzed oxidative folding, a precise kinetic analysis of the enzyme is constructed. DsbA is demonstrated to be a highly effective catalyst of oxidative folding, outperforming its eukaryotic counterpart by substantial margins in every metric considered. This efficacy complements the strong preference for simpler disulfide connectivity patterns in the Escherichia coli proteome, which in conjunction likely represent a strategy for navigating the physiological demands that are imposed by the inherent speed of prokaryotic life, in which a generation can be as short as twenty minutes.

Mechanical forces regulate biological processes in unique and unexpected ways, but many biochemical methods are unable to reproduce the vectorial stretching experienced by proteins in cells. Force spectroscopy techniques remedy these shortcomings by utilizing microscopic force probes to stretch and relax single protein, DNA, and RNA molecules. The central focus of this thesis is the development and implementation of a custom-built protein magnetic tweezers for unfolding and refolding Ig domains from titin, a critical filament of the sarcomere, and the longest continuous peptide in the human body. Suspended from the Z-disc to the tip of the thick filament, titin sustains the brunt of intracellular forces during muscle elongation. Since the discovery of titin, it has been widely debated whether Ig domain unfolding contributes to muscle mechanics. A combination of single quantum dot tracking in myofibrils extracted from rabbit muscle and single molecule magnetic tweezers experiments on recombinant titin fragments confirms, for the first time, the presence of titin Ig domain unfolding and refolding at physiological sarcomere lengths and stretching forces. The magnetic tweezers experiments show the surprising ability of titin Ig domains to generate piconewton level forces during folding, and we advance the hypothesis that titin folding is an important source of energy during muscle contraction. Muscle elongation recruits Ig domains to the unfolded state, whereby folding is initiated through reduction of force on titin upon actomyosin crossbridges formation. Magnetic tweezers measurements demonstrate that titin Ig folding generates peak work, velocity, and power output of 64 zeptojoules, 1.9 microns per second, and 6,000 zeptowatts, matching or exceeding the equivalent single molecule measurements from single molecule myosin II powerstrokes. The forces generated by protein folding are therefore likely to be an integral part of the contractile process of animal muscle.

Polyglutamine (polyQ) diseases are progressive fatal neurodegenerative movement disorders. Although many cellular processes are perturbed in polyQ disease, recent studies highlight the importance of protein misfolding as a central event in polyQ toxicity. Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a particularly interesting polyQ disease because of the special qualities of the disease protein ataxin-3, which normally participates in cellular protein quality control. Here I use multiple mouse models of disease to explore toxic protein species and the role of protein homeostasis in SCA3 pathogenesis. In Chapter 1, I review the key features of polyQ disease, and outline the background and rationale behind our strategy for identifying toxic protein species in SCA3. In Chapter 2, I examine the role of the protein quality control ubiquitin ligase, CHIP (C-terminus of Hsp70 interacting protein), in regulating the toxicity of expanded ataxin-3 in vivo. Genetic reduction or removal of CHIP increases formation of detergent-resistant ataxin-3 microaggregates specifically in the brain. Concomitant with this, reduction or removal of CHIP exacerbates the phenotype of SCA3 mice, revealing a correlation between high levels of microaggregates and phenotypic severity. Additional cell-based studies confirm that CHIP may not directly mediate ataxin-3 degradation, suggesting that CHIP reduces expanded ataxin-3 toxicity in the brain primarily by enhancing ataxin-3 solubility. In Chapter 3, I use various biochemical techniques to reveal the presence of brain-specific ataxin-3 microaggregates in two genetically distinct mouse models of SCA3. Selective neuropathological evaluation of SCA3 mice reveals that major neuronal loss and reactive glial proliferation are not shared features of phenotypically-manifesting SCA3 mice. Additional studies fail to provide evidence for loss-of-function of endogenous ataxin-3 in SCA3 mice. Our results suggest that neuronal dysfunction in SCA3 is mediated through a toxic gain-of-function mechanism by ataxin-3 microaggregates in the CNS. In Chapter 4, I discuss important areas for future research in polyQ disease. I describe studies that would help elucidate the structural nature of toxic soluble microaggregates, and their effects on other cellular proteins. I also consider how the results described in this thesis inform potential treatment strategies.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Properly folded proteins are necessary for all living organisms. Incorrectly folded proteins can lead to a variety of diseases such as Alzheimer’s Disease or Bovine Spongiform Encephalitis (Mad Cow Disease). Understanding the forces involved in protein folding is essential to the understanding and treatment of protein misfolding diseases. When proteins fold, a significant amount of surface area is buried in the protein interior. It has long been known that burial of hydrophobic surface area was important to the stability of the folded structure. However, the impact of burying polar surface area is not well understood. Theoretical results suggest that burying polar groups decreases the stability, but experimental evidence supports the belief that polar group burial increases the stability. Studies of tyrosine to phenylalanine mutations have shown the removal of the tyrosine OH group generally decreases stability. Through computational investigations into the effect of buried tyrosine on protein stability, favorable van der Waals interactions are shown to correlate with the change in stability caused by replacing the tyrosine with phenylalanine to remove the polar OH group. Two large-scale studies on nearly 1000 high-resolution x-ray structures are presented. The first investigates the electrostatic and van der Waals interactions, analyzing the energetics of burying various atom groups in the protein interior. The second large-scale study analyzes the packing differences in the interior of the protein and shows that hydrogen bonding increases packing, decreasing the volume of a hydrogen bonded backbone by about 1.5 Å3 per hydrogen bond. Finally, a structural comparison between RNase Sa and a variant in which five lysines replaced five acidic groups to reverse the net charge is presented. It is shown that these mutations have a marginal impact on the structure, with only small changes in some loop regions.

To understand protein stability and the mechanism of protein folding, it is essential that we gain a better understanding of the ensemble of conformations that make up the denatured state of a protein. The primary goal of the research described here was to see what we might learn about the denatured state using fluorescence. To this end, tryptophan was introduced at five sites in Ribonuclease Sa (RNase Sa): D1W, Y52W, Y55W, T76W, and Y81W. The fluorescent properties of the denatured states of these five proteins were studied and compared to the fluorescent properties of eight model compounds: N-acetyl-tryptophan-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and five pentapeptides based on the sequence around the original tryptophan substitutions in RNase Sa. Regardless of the denaturant, λmax for the proteins and model compounds differed very little, 349.3 ± 1.2 nm. However, significant differences were observed in the fluorescence intensity at λmax (IF), suggesting that IF is more sensitive to the immediate environment than λmax. The differences in IF are due in part to quenching by neighboring side chains. More importantly, IF was always significantly greater in the protein than in its corresponding pentapeptide, indicating that the protein exerts an effect on the tryptophan, which cannot be mimicked by the pentapeptide models. Acrylamide and iodide quenching experiments were also performed on the model compounds and proteins. Significant differences in the Stern-Volmer quenching constant (KSV) were also observed between the proteins and between the proteins and their corresponding pentapeptides. Importantly, the KSV for the protein was always less than in its corresponding pentapeptide. These data along with the IF data show that non-local structure in the unfolded state influences tryptophan fluorescence and accessibility. In summary, these and our other studies show that fluorescence can be used to gain a better understanding of the denatured states of proteins.

Proteins in nature fold to one dominant native structure. Despite being a heavily studied field, predicting the native structure from the amino acid sequence and modeling the folding process can still be considered unsolved problems. In this thesis I present a new approach to this problem with methods borrowed from theoretical physics. In the first part I show how it is possible to use a discrete Frenet frame to define the discrete curvature and torsion of the main chain of the protein. This method is then extended to the side chains as well. In particular I show how to use the discrete Frenet frame to produce a statistical distribution of angles that works in similar fashion as the commonly used Ramachandran plot and side chain rotamers. The discrete Frenet frame displays a gauge symmetry, in the choice of basis vectors on the normal plane, that is reminiscent of features of Abelian-Higgs theory. In the second part of the thesis I show how this similarity with Abelian-Higgs theory can be translated into an effective energy for a protein. The loops of the proteins are shown to correspond to solitons so that the whole protein can be constructed by gluing together any number of solitons. I present results of simulating proteins by minimizing the energy, starting from a real line or straight helix, where the correct native fold is attained. Finally the model is shown to display the same phase structure as real proteins.

Going down the folding funnel, proteins may sample a wide variety of conformations, some being outright detrimental to the organism. Yet, the vast majority of polypeptide molecules avoid such pitfalls. Not only do they reach the native minimum of the energy landscape; they do so via blazingly fast, biased, routes. This specificity and speed is remarkable, as the surrounding solution is filled to the brim with other molecules that could potentially interact with the protein and in doing so stabilise non-native, potentially toxic, conformations. How such incidents are avoided while maintaining native structure and function is not understood.  This doctoral thesis argues that protein structure and function can be separated in the folding code of natural protein sequences by use of multiple partly uncoupled factors that act in a concerted fashion. More specifically, we demonstrate that: i) Evolutionarily conserved functional and regulatory elements can be excised from a present day protein, leaving behind an independently folded protein scaffold. This suggests that the dichotomy between functional and structural elements can be preserved during the course of protein evolution. ii) The ubiquitous charges on soluble protein surfaces are not required for protein folding in biologically relevant timescales, but are critical to intermolecular interaction. Monomer folding can be driven by hydrophobicity and hydrogen bonding alone, while functional and structural intermolecular interaction depends on the relative positions of charges that are not required for the native bias inherent to the folding mechanism. It is possible that such uncoupling reduces the probability of evolutionary clashes between fold and function. Without such a balancing mechanism, functional evolution might pull the carpet from under the feet of structural integrity, and vice versa. These findings have implications for both de novo protein design and the molecular mechanisms behind diseases caused by protein misfolding. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Understanding structure and folding of a protein is the key to understanding its biological function and potential role in diseases. Despite the importance of protein folding, a molecular level understanding of this process is still lacking. Solution-state nuclear magnetic resonance (NMR) is a powerful technique to investigate protein structure, dynamics, and folding mechanisms, since it provides residue-specific information. One of the major contributions that govern protein structure appears to be the interaction with the solvent. The importance of these interactions is particularly apparent in membrane proteins, which exist in an amphiphilic environment. Here, individual peptide fragments taken from the disulfide bond forming protein B (DsbB) were investigated in various solvents. The alpha-helical structures that were obtained, suggest that DsbB follows the two-stage model for folding. However, side chains of polar residues showed different conformations compared to the X-ray structure of fulllength protein, implying that polar side-chains may re-orient upon helix packing in order to form the necessary tertiary interactions that stabilize the global fold of DsbB. Model peptides in general represent attractive systems for the investigation of non-covalent interactions important for protein folding, including those with the solvent. NMR structures of the water soluble peptide, BBA5, were obtained in the presence an organic co-solvent, methanol. These structures indicate that the addition of methanol stabilizes an alpha-helix segment, but disrupts a hydrophobic cluster forming a beta-hairpin. Since dynamic effects reduce the ability for experimental observation of individual, bound solvent molecules, results were compared with molecular dynamics simulations. This comparison indicates that the observed effects of NMR structures are due to preferred binding of methanol and reduction of peptide-water hydrogen bonding. NMR structures, such as those determined here, represent a distribution of conformations under equilibrium. The dynamic process of protein unfolding can nevertheless be accessed through denaturation. A method was developed to probe thermal denaturation by measuring the temperature dependence of NOE intensity. Applied to a model peptide, trpzip4, it was confirmed that the beta-hairpin structure of this peptide is stabilized by the hydrophobic cluster formed by tryptophan residues. Together, the peptides investigated here illustrate the important roles that solvent-peptide interactions and side chain-side chain hydrophobic interactions play in forming stable secondary and tertiary structures.