Conformational selection is the idea that proteins traverse positions on the conformational space represented by their potential energy landscape, and in particular positions considered as local energy minima. Conformational selection a useful concept in ligand binding studies and in exploring the behavior of protein structures within that energy landscape. Often, research that explores protein function requires the generation of conformational ensembles, or collections of protein conformations from a single structure. We describe a method of conformational ensemble generation that uses joint-constrained rigid-body dynamics (an approach that allows for explicit consideration of rigidity) and the elastic network model (providing structurally derived directional guides for the rigid-body model). We test our model on a selection of unbound proteins and examine the structural validity of the resulting ensembles, as well as the ability of such an approach to generate conformations with structural overlaps close to the ligand-bound versions of the proteins.

The recent determination of several crystal structures of voltage-gated ion channels has catalyzed computational efforts of studying these remarkable molecular machines that are able to conduct ions across biological membranes at extremely high rates without compromising the ion selectivity. Starting from the open crystal structures, we have studied the gating mechanism of these channels by molecular modeling techniques. Firstly, by applying a membrane potential, initial stages of the closing of the channel were captured, manifested in a secondary-structure change in the voltage-sensor. In a follow-up study, we found that the energetic cost of translocating this 310-helix conformation was significantly lower than in the original conformation. Thirdly, collaborators of ours identified new molecular constraints for different states along the gating pathway. We used those to build new protein models that were evaluated by simulations. All these results point to a gating mechanism where the S4 helix undergoes a secondary structure transformation during gating. These simulations also provide information about how the protein interacts with the surrounding membrane. In particular, we found that lipid molecules close to the protein diffuse together with it, forming a large dynamic lipid-protein cluster. This has important consequences for the understanding of protein-membrane interactions and for the theories of lateral diffusion of membrane proteins. Further, simulations of the simple ion channel antiamoebin were performed where different molecular models of the channel were evaluated by calculating ion conduction rates, which were compared to experimentally measured values. One of the models had a conductance consistent with the experimental data and was proposed to represent the biological active state of the channel. Finally, the underlying methods for simulating molecular systems were probed by implementing the CHARMM force field into the GROMACS simulation package. The implementation was verified and specific GROMACS-features were combined with CHARMM and evaluated on long timescales. The CHARMM interaction potential was found to sample relevant protein conformations indifferently of the model of solvent used. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

<p>Proteins are among the most complex entities known to science. Composed of just 20 fundamental building blocks arranged in simple linear strings, they nonetheless fold into a dizzying array of architectures that carry out the machinations of life at the molecular level.</p><p>Despite this central role in biology, we cannot reliably predict the structure of a protein from its sequence, and therefore rely on time-consuming and expensive experimental techniques to determine their structures. Although these methods can reveal equilibrium structures with great accuracy, they unfortunately mask much of the inherent molecular flexibility that enables proteins to dynamically perform biochemical tasks. As a result, much of the field of structural biology is mired in a static perspective; indeed, most attempts to naively model increased structural flexibility still end in failure.</p><p>This document details my work to validate alternative protein conformations beyond the primary or equilibrium conformation. The underlying hypothesis is that more realistic modeling of flexibility will enhance our understanding of how natural proteins function, and thereby improve our ability to design new proteins that perform desired novel functions.</p><p>During the course of my work, I used structure validation techniques to validate conformational alternatives in a variety of settings. First, I extended previous work introducing the backrub, a local, sidechain-coupled backbone motion, by demonstrating that backrubs also accompany sequence changes and therefore are useful for modeling conformational changes associated with mutations in protein design. Second, I extensively studied a new local backbone motion, helix shear, by documenting its occurrence in both crystal and NMR structures and showing its suitability for expanding conformational search space in protein design. Third, I integrated many types of local alternate conformations in an ultra-high-resolution crystal structure and discovered the combinatorial complexity that arises when adjacent flexible segments combine into networks. Fourth, I used structural bioinformatics techniques to construct smoothed, multi-dimensional torsional distributions that can be used to validate trial conformations or to propose new ones. Fifth, I participated in judging a structure prediction competition by using validation of geometrical and all-atom contact criteria to help define correctness across thousands of submitted conformations. Sixth, using similar tools plus collation of multiple comparable structures from the public database, I determined that low-energy states identified by the popular structure modeling suite Rosetta sometimes are valid conformations likely to be populated in the cell, but more often are invalid conformations attributable to artifacts in the physical/statistical hybrid energy function.</p><p>Unified by the theme of validating conformational alternatives by reference to high-quality experimental structures, my cumulative work advances our fundamental understanding of protein structural variability, and will benefit future endeavors to design useful proteins for biomedicine or industrial chemistry.</p> / Dissertation

In 1998 the genome sequence of Mycobacterium tuberculosis H37Rv was published1. M. tuberculosis is the primary causative agent of tuberculosis, a disease with a long history in humans, which still has a great impact on human mortality today. As part of the M. tuberculosis Structural Genomics Consortium we selected nine target genes (Rv0534c (menA); Rv0548c (menB); Rv0553 (menC); Rv0555 (menD); Rv0542c (menE); Rv3853 (menG); Rv0558 (ubiE); Rv0989c (grcC2) and Rv0990c) from M. tuberculosis, including all known members of the menaquinone biosynthesis pathway, for structural studies. All nine genes were taken through the structural genomics “pipeline”, either becoming stuck at various “bottlenecks” or continuing successfully to structure solution. At the initial bioinformatics analysis step, eight of the nine targeted genes were deemed suitable for further study. PCR amplification and cloning of these genes into several different expression vectors followed. Expression of the gene products for the seven successfully cloned genes was undertaken in an E. coli expression host, followed by experiments (refolding, lysis buffer and expression temperature screens) aimed at obtaining soluble protein in sufficient quantities for crystallisation. Of the seven proteins successfully overexpressed, five remain at this stage as they could not be obtained in soluble form. The remaining two, Rv3853 (MenG), solubilised by refolding, and MenB, solubilised by 24ºC expression, were purified and both successfully produced diffracting crystals. The crystal structure of Rv3853 was determined by isomorphous replacement (SIRAS) and refined at 1.9 Å resolution (R = 19.0% and Rfree = 22.0%). The structure of several different crystal forms of MenB, were determined by molecular replacement. Refinement of two of these structures, MenB_P43212 at 2.15Å resolution (R = 20.3% and Rfree = 23.1%) and MenB_C2-NCoA at 2.3 Å resolution (R = 19.7% and Rfree = 22.5%), has been completed. The structure of Rv3853, combined with the discovery that UbiE was more likely to catalyse the final, S-adenosylmethionine-dependent, methyltransfer step of menaquinone biosynthesis, led to the conclusion that Rv3853 had been misannotated as MenG. Combined with further bioinformatics analysis the Rv3853 structure has been useful in providing new ideas as to the real function of Rv3853. In contrast, the structure of MenB confirmed its place as a member of the crotonase superfamily although the C-terminus was located in a position not observed in other crotonase superfamily structures. Several flexible regions likely to be important in MenB function have been identified by examination of the various MenB structures / Author was the recipient of a University of Auckland Doctoral Scholarship and a Foundation of Research Science & Technology Top Achiever Doctoral Scholarship

In 1998 the genome sequence of Mycobacterium tuberculosis H37Rv was published1. M. tuberculosis is the primary causative agent of tuberculosis, a disease with a long history in humans, which still has a great impact on human mortality today. As part of the M. tuberculosis Structural Genomics Consortium we selected nine target genes (Rv0534c (menA); Rv0548c (menB); Rv0553 (menC); Rv0555 (menD); Rv0542c (menE); Rv3853 (menG); Rv0558 (ubiE); Rv0989c (grcC2) and Rv0990c) from M. tuberculosis, including all known members of the menaquinone biosynthesis pathway, for structural studies. All nine genes were taken through the structural genomics “pipeline”, either becoming stuck at various “bottlenecks” or continuing successfully to structure solution. At the initial bioinformatics analysis step, eight of the nine targeted genes were deemed suitable for further study. PCR amplification and cloning of these genes into several different expression vectors followed. Expression of the gene products for the seven successfully cloned genes was undertaken in an E. coli expression host, followed by experiments (refolding, lysis buffer and expression temperature screens) aimed at obtaining soluble protein in sufficient quantities for crystallisation. Of the seven proteins successfully overexpressed, five remain at this stage as they could not be obtained in soluble form. The remaining two, Rv3853 (MenG), solubilised by refolding, and MenB, solubilised by 24ºC expression, were purified and both successfully produced diffracting crystals. The crystal structure of Rv3853 was determined by isomorphous replacement (SIRAS) and refined at 1.9 Å resolution (R = 19.0% and Rfree = 22.0%). The structure of several different crystal forms of MenB, were determined by molecular replacement. Refinement of two of these structures, MenB_P43212 at 2.15Å resolution (R = 20.3% and Rfree = 23.1%) and MenB_C2-NCoA at 2.3 Å resolution (R = 19.7% and Rfree = 22.5%), has been completed. The structure of Rv3853, combined with the discovery that UbiE was more likely to catalyse the final, S-adenosylmethionine-dependent, methyltransfer step of menaquinone biosynthesis, led to the conclusion that Rv3853 had been misannotated as MenG. Combined with further bioinformatics analysis the Rv3853 structure has been useful in providing new ideas as to the real function of Rv3853. In contrast, the structure of MenB confirmed its place as a member of the crotonase superfamily although the C-terminus was located in a position not observed in other crotonase superfamily structures. Several flexible regions likely to be important in MenB function have been identified by examination of the various MenB structures / Author was the recipient of a University of Auckland Doctoral Scholarship and a Foundation of Research Science & Technology Top Achiever Doctoral Scholarship

In 1998 the genome sequence of Mycobacterium tuberculosis H37Rv was published1. M. tuberculosis is the primary causative agent of tuberculosis, a disease with a long history in humans, which still has a great impact on human mortality today. As part of the M. tuberculosis Structural Genomics Consortium we selected nine target genes (Rv0534c (menA); Rv0548c (menB); Rv0553 (menC); Rv0555 (menD); Rv0542c (menE); Rv3853 (menG); Rv0558 (ubiE); Rv0989c (grcC2) and Rv0990c) from M. tuberculosis, including all known members of the menaquinone biosynthesis pathway, for structural studies. All nine genes were taken through the structural genomics “pipeline”, either becoming stuck at various “bottlenecks” or continuing successfully to structure solution. At the initial bioinformatics analysis step, eight of the nine targeted genes were deemed suitable for further study. PCR amplification and cloning of these genes into several different expression vectors followed. Expression of the gene products for the seven successfully cloned genes was undertaken in an E. coli expression host, followed by experiments (refolding, lysis buffer and expression temperature screens) aimed at obtaining soluble protein in sufficient quantities for crystallisation. Of the seven proteins successfully overexpressed, five remain at this stage as they could not be obtained in soluble form. The remaining two, Rv3853 (MenG), solubilised by refolding, and MenB, solubilised by 24ºC expression, were purified and both successfully produced diffracting crystals. The crystal structure of Rv3853 was determined by isomorphous replacement (SIRAS) and refined at 1.9 Å resolution (R = 19.0% and Rfree = 22.0%). The structure of several different crystal forms of MenB, were determined by molecular replacement. Refinement of two of these structures, MenB_P43212 at 2.15Å resolution (R = 20.3% and Rfree = 23.1%) and MenB_C2-NCoA at 2.3 Å resolution (R = 19.7% and Rfree = 22.5%), has been completed. The structure of Rv3853, combined with the discovery that UbiE was more likely to catalyse the final, S-adenosylmethionine-dependent, methyltransfer step of menaquinone biosynthesis, led to the conclusion that Rv3853 had been misannotated as MenG. Combined with further bioinformatics analysis the Rv3853 structure has been useful in providing new ideas as to the real function of Rv3853. In contrast, the structure of MenB confirmed its place as a member of the crotonase superfamily although the C-terminus was located in a position not observed in other crotonase superfamily structures. Several flexible regions likely to be important in MenB function have been identified by examination of the various MenB structures / Author was the recipient of a University of Auckland Doctoral Scholarship and a Foundation of Research Science & Technology Top Achiever Doctoral Scholarship

In 1998 the genome sequence of Mycobacterium tuberculosis H37Rv was published1. M. tuberculosis is the primary causative agent of tuberculosis, a disease with a long history in humans, which still has a great impact on human mortality today. As part of the M. tuberculosis Structural Genomics Consortium we selected nine target genes (Rv0534c (menA); Rv0548c (menB); Rv0553 (menC); Rv0555 (menD); Rv0542c (menE); Rv3853 (menG); Rv0558 (ubiE); Rv0989c (grcC2) and Rv0990c) from M. tuberculosis, including all known members of the menaquinone biosynthesis pathway, for structural studies. All nine genes were taken through the structural genomics “pipeline”, either becoming stuck at various “bottlenecks” or continuing successfully to structure solution. At the initial bioinformatics analysis step, eight of the nine targeted genes were deemed suitable for further study. PCR amplification and cloning of these genes into several different expression vectors followed. Expression of the gene products for the seven successfully cloned genes was undertaken in an E. coli expression host, followed by experiments (refolding, lysis buffer and expression temperature screens) aimed at obtaining soluble protein in sufficient quantities for crystallisation. Of the seven proteins successfully overexpressed, five remain at this stage as they could not be obtained in soluble form. The remaining two, Rv3853 (MenG), solubilised by refolding, and MenB, solubilised by 24ºC expression, were purified and both successfully produced diffracting crystals. The crystal structure of Rv3853 was determined by isomorphous replacement (SIRAS) and refined at 1.9 Å resolution (R = 19.0% and Rfree = 22.0%). The structure of several different crystal forms of MenB, were determined by molecular replacement. Refinement of two of these structures, MenB_P43212 at 2.15Å resolution (R = 20.3% and Rfree = 23.1%) and MenB_C2-NCoA at 2.3 Å resolution (R = 19.7% and Rfree = 22.5%), has been completed. The structure of Rv3853, combined with the discovery that UbiE was more likely to catalyse the final, S-adenosylmethionine-dependent, methyltransfer step of menaquinone biosynthesis, led to the conclusion that Rv3853 had been misannotated as MenG. Combined with further bioinformatics analysis the Rv3853 structure has been useful in providing new ideas as to the real function of Rv3853. In contrast, the structure of MenB confirmed its place as a member of the crotonase superfamily although the C-terminus was located in a position not observed in other crotonase superfamily structures. Several flexible regions likely to be important in MenB function have been identified by examination of the various MenB structures / Author was the recipient of a University of Auckland Doctoral Scholarship and a Foundation of Research Science & Technology Top Achiever Doctoral Scholarship

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 6, 2008). Includes bibliographical references.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 203-215). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;