Systematic conformational search with constraint satisfaction

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2002.Includes bibliographical references (p. 170-177).Determining the conformations of biological molecules is a high scientific priority for biochemists and for the pharmaceutical industry. This thesis describes a systematic method for conformational search, an application of the method to determining the structure of the formyl-Met-Leu-Phe-OH (fMLF)peptide by solid-state NMR spectroscopy, and a separate project to determine the structure of a protein-DNA complex by X-ray crystallography. The purpose of the systematic search method is to enumerate all conformations of a molecule (at a given level of torsion angle resolution) that satisfy a set of local geometric constraints. Constraints would typically come from NMR experiments, but applications such as docking or homology modelling could also give rise to similar constraints. The molecule to be searched is partitioned into small subchains so that the set of possible conformations for the whole molecule may be constructed by merging the feasible conformations for the parts. However, instead of using a binary tree for straightforward divide-and-conquer, four innovations are introduced: (1) OMNIMERGE searches a subproblem for every possible subchain of the molecule. Searching every subchain provides the advantage that every possible merge is available; by choosing the most favorable merge for each subchain, the bottleneck subchain(s) and therefore the whole search may be completed more efficiently. (2) A cost function evaluates alternative divide-and-conquer trees, provided that a preliminary OMNIMERGE search of the molecule has been completed. Then dynamic programming determines the optimal partitioning or "merge-tree" for the molecule; this merge-tree can be used to improve the efficiency of future searches.(cont.) (3) PROPAGATION shares information by enforcing arc consistency between the solution sets of overlapping subchains. By filtering the solution set of each subchain, infeasible conformations are discarded rapidly. (4) An A* function prioritizes each subchain based on estimated future costs. Subchains with sufficiently low priority can be skipped, which improves efficiency. A common theme of these four ideas is to make good choices about how to break the large search problem into lower-dimensional subproblems. These novel algorithms were implemented and the effectiveness of each is demonstrated on a well-constrained peptide with 40 degrees of freedom.by Lisa Tucker-Kellogg.Ph.D

Supported Engineered Extracellular Matrices for 3D Cell Culture

In the current shift away from 2D tissue culture polystyrene and towards 3D cell culture models, several important design criteria have yet to be considered: (1) provision of large open areas where cells can create their own niche (2) fabrication of a scaffold that is chemically and mechanically tunable and (3) presentation of proteins that mimic native extracellular matrix (ECM). Polymer scaffolds fabricated by 3D jet writing provide extensive void space for maximum cell-cell and cell-ECM interactions. This work expands on such electrospinning technologies to establish a micromanufacturing process that modulates the flow of various polymer solutions through a manifold. The resulting scaffolds contain spatially distinct domains that can be customized to exhibit specific bulk or surface properties. Such tunability is not limited to the synthetic design space. We have discovered that hydrodynamically induced fibrillogenesis can yield remarkably stable networks of protein fibrils suspended across a support or scaffold that recapitulate important structural and functional hallmarks of cell-secreted ECM. These engineered networks of fibronectin serve as a breast cancer microenvironment, making it possible to culture an unfractionated patient sample (n=14), where less than 5% are cancer cells, into a self-selected composition of differentiated cancer cells, stem-like cancer cells, and various stromal cells. An average of 40% increase in the tumor-initiating population and at least a 7-fold increase in the cancer cell population was observed after six days (n=3). This user-defined 3D cell culture platform will enable investigation into the bidirectional relationship between cells and the ECM, not just for breast cancer but a variety of diseased or healthy tissue types.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140892/1/stacyram_1.pd

Sequence Determinants of the Individual and Collective Behaviour of Intrinsically Disordered Proteins

Intrinsically disordered proteins and protein regions (IDPs) represent around thirty percent of the eukaryotic proteome. IDPs do not fold into a set three dimensional structure, but instead exist in an ensemble of inter-converting states. Despite being disordered, IDPs are decidedly not random; well-defined - albeit transient - local and long-range interactions give rise to an ensemble with distinct statistical biases over many length-scales. Among a variety of cellular roles, IDPs drive and modulate the formation of phase separated intracellular condensates, non-stoichiometric assemblies of protein and nucleic acid that serve many functions. In this work, we have explored how the amino acid sequence of IDPs determines their conformational behaviour, and how sequence and single chain behaviour influence their collective behaviour in the context of phase separation. In part I, in a series of studies, we used simulation, theory, and statistical analysis coupled with a wide range of experimental approaches to uncover novel rules that further explore how primary sequence and local structure influence the global and local behaviour of disordered proteins, with direct implications for protein function and evolution. We found that amino acid sidechains counteract the intrinsic collapse of the peptide backbone, priming the backbone for interaction and providing a fully reconciliatory explanation for the mechanism of action associated with the denaturants urea and GdmCl. We discovered that proline can engender a conformational buffering effect in IDPs to counteract standard electrostatic effects, and that the patterning those proline residues can be a crucial determinant of the conformational ensemble. We developed a series of tools for analysing primary sequences on a proteome wide scale and used them to discover that different organisms can have substantially different average sequence properties. Finally, we determined that for the normally folded protein NTL9, the unfolded state under folding conditions is relatively expanded but has well defined native and non-native structural preferences. In part II, we identified a novel mode of phase separation in biology, and explored how this could be tuned through sequence design. We discovered that phase separated liquids can be many orders of magnitude more dilute than simple mean-field theories would predict, and developed an analytic framework to explain and understand this phenomenon. Finally, we designed, developed and implemented a novel lattice-based simulation engine (PIMMS) to provide sequence-specific insight into the determinants of conformational behaviour and phase separation. PIMMS allows us to accurately and rapidly generate sequence-specific conformational ensembles and run simulations of hundreds of polymers with the goal of allowing us to systematically elucidate the link between primary sequence of phase separation

Characterisation of the feline leukaemia virus fusion peptide: implications for the fusion mechanism

Membrane fusion, the merging of two initially distinct membranes to form one common lipid bilayer, is a fundamental mechanism of life. It occurs many times each day within every eukaryotic cell as part of essential daily homeostatic processes, as well as between individual cells, such as sperm and egg during fertilisation. The fusion mechanism is, however, also crucial to the development of many diseases. All enveloped viruses, and indeed many other obligate intracellular parasites, must fuse their own surrounding lipid bilayer with the membrane of their host's target cell in order to gain cell entry and thus the ability to replicate. These infections produce disease states, and possibly even death, in the host speciesDespite the clear importance of fusion, the precise molecular events that occur during this process are still not known. Fusion proteins of viruses have recently become popular tools for use in fusion studies. More specifically, several viruses have known fusion peptides, the sections of these proteins which confer their fusogenic activity. This thesis examines the structure and function of the putative fusion peptide of the retrovirus Feline leukaemia virus, (FeLV), using a variety of mainly biophysical techniques.The structural effects of the FeLV fusion peptide on lipid polymorphism were studied. Using differential scanning calorimetry, ³¹P nuclear magnetic resonance and time-resolved X-ray diffraction this peptide was found to induce changes in lipid conformation and motion similar to those of known fusogens: it favoured the formation of non-bilayer lipid conformations which have a relatively large negative curvature, namely the inverted hexagonal phase and isotropic lipid states. Moreover, using X-ray diffraction, a new lipid phase was observed in the presence of the FeLV peptideNeutron diffraction studies revealed a change in the packing of lipid molecules within a bilayer and also possible thinning ofthe bilayer, both ofwhich were induced by interaction with the FeLV fusion peptide.Fusogenic activity for this putative viral fusion peptide was demonstrated, using fusion assays, which measured the merging of lipid membranes in the presence ofthe FeLV fusion peptide.These findings are discussed in the light ofthe current concepts ofthe fusion mechanism. They add support to two currently favoured theories of fusion: precession by a fusion peptide as a means of inducing the initial destabilisation of a bilayer, and the formation ofhighly bent, high energy lipid intermediates, such as the 'modified stalk', in the multistep fusion pathway.Circular dichroism was employed to determine the secondary structure ofthe FeLV fusion peptide under a variety of experimental conditions. This peptide was observed to flip readily between a-helical and p sheet conformations. This suggests that structural plasticity may be an important dynamic property offusion peptides. Possible relationships between peptide structure and function are discusse

42nd Rocky Mountain Conference on Analytical Chemistry

Abstracts from the 42nd annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Broomfield, Colorado, July 30 - August 3, 2000

Monoclonal Antibodies

Monoclonal antibodies are established in clinical practice for the treatment of cancer, and autoimmune and infectious diseases. The first generation of antibodies has been dominated by classical IgG antibodies, however, in the last decade, the field has advanced, and, nowadays, a large proportion of antibodies in development have been engineered. This Special Issue on "Monoclonal Antibodies" includes original manuscripts and reviews covering various aspects related to the discovery, analytical characterization, manufacturing and development of therapeutic and engineered antibodies

49th Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 49th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-endorsed by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Breckenridge, Colorado, July 22-26, 2007

Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopy is a nondestructive technique that can be used to characterize a wide variety of systems. Sustained development of both methodology and instrumentation have allowed NMR to evolve as a powerful technology, with applications in pure sciences, medicine, drug development, and important branches of industry. NMR provides precise structural information down to each atom and bond in a molecule, and is the only method for the determination of structures of molecules in a solution. This book compiles a series of articles describing the application of NMR in a variety of interesting scientific challenges. The articles illustrate the versatility and flexibility of NMR