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CONFORMATIONAL DYNAMICS OF PROTEINS: INSIGHTS FROM STRUCTURAL AND COMPUTATIONAL STUDIES

LIU, LIN (2011) CONFORMATIONAL DYNAMICS OF PROTEINS: INSIGHTS FROM STRUCTURAL AND COMPUTATIONAL STUDIES. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    Proteins are not static; they undergo both random thermal fluctuations near a given equilibrium state, and transitions between different sub-states. These motions are usually intricately connected to the function of the protein. Therefore, understanding the dynamics of proteins is important to gain insights into the mechanisms of many biological phenomena. Only the combination of structure and dynamics does allow for describing a functional protein (or biological molecule) properly. Therefore, this thesis is centered on computational and structural studies of protein dynamics. I carried out full atomic simulations and coarse-grained analyses(using elastic network models) as computational approaches, and used NMR as well as X-ray crystallography on the experimental side. With regard to the understanding of the fluctuations accessible under equilibrium conditions, a detailed analysis of high-resolution structural data and computationally predicted dynamics was carried out for a designed sugar-binding protein. The mean-square deviations in the positions of residues derived from NMR models and those inferred from X-ray crystallographic B-factors for two different crystal forms were compared with the predictions based on the Gaussian network model (GNM) and the results from molecular dynamics (MD) simulations. The results highlighted the significance of considering ensembles of structures (or structural models) from experiments, in order to make an accurate assessment of the fluctuation dynamics of proteins under equilibrium conditions. Moreover, we analyzed the amplitudes, correlation times, and directions of residue motions in multiple MD runs of durations varying in the range 1 ns – 400 ns. Our data show that the distribution of residue fluctuations is insensitive to the simulation length, while the amplitudes increase with simulation time with a power law. Another area of interest concerned the phenomenon of “domain swapping”. We investigated the molecular basis of this unusual multimerization, using a broad range of approaches. A systematic analysis of a large set of domain-swapped structures was performed to this aim. Results suggest that almost any protein may be capable of undergoing domain swapping, and that domain swapping is solely a specialized form of oligomer assembly but is closely associated with the unfolding/folding process of proteins. We also use experimental 19F-NMR to study the thermodynamic and kinetic properties in CV-N domain swapping. The activation energy barrier for the passage between monomeric and domain swapped dimeric form is of similar magnitude to that for complete unfolding of the protein, indicating that the overall unfolding of the polypeptide is required for domain swapping. Crystal structures of a domain-swapped trimer and a tetramer of CV-N provide further insights into the potential mechanics of CV-N domain swapping.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmailORCID
    Committee ChairBahar, Ivetbahar@pitt.edu
    Committee CoChairGronenborn, Angelaamg100@pitt.edu
    Committee MemberRule, Gordonrule@andrew.cmu.edu
    Committee MemberWetzel, Ronaldrwetzel@pitt.edu
    Committee MemberZuckerman, Danielddmmzz@pitt.edu
    Title: CONFORMATIONAL DYNAMICS OF PROTEINS: INSIGHTS FROM STRUCTURAL AND COMPUTATIONAL STUDIES
    Status: Published
    Abstract: Proteins are not static; they undergo both random thermal fluctuations near a given equilibrium state, and transitions between different sub-states. These motions are usually intricately connected to the function of the protein. Therefore, understanding the dynamics of proteins is important to gain insights into the mechanisms of many biological phenomena. Only the combination of structure and dynamics does allow for describing a functional protein (or biological molecule) properly. Therefore, this thesis is centered on computational and structural studies of protein dynamics. I carried out full atomic simulations and coarse-grained analyses(using elastic network models) as computational approaches, and used NMR as well as X-ray crystallography on the experimental side. With regard to the understanding of the fluctuations accessible under equilibrium conditions, a detailed analysis of high-resolution structural data and computationally predicted dynamics was carried out for a designed sugar-binding protein. The mean-square deviations in the positions of residues derived from NMR models and those inferred from X-ray crystallographic B-factors for two different crystal forms were compared with the predictions based on the Gaussian network model (GNM) and the results from molecular dynamics (MD) simulations. The results highlighted the significance of considering ensembles of structures (or structural models) from experiments, in order to make an accurate assessment of the fluctuation dynamics of proteins under equilibrium conditions. Moreover, we analyzed the amplitudes, correlation times, and directions of residue motions in multiple MD runs of durations varying in the range 1 ns – 400 ns. Our data show that the distribution of residue fluctuations is insensitive to the simulation length, while the amplitudes increase with simulation time with a power law. Another area of interest concerned the phenomenon of “domain swapping”. We investigated the molecular basis of this unusual multimerization, using a broad range of approaches. A systematic analysis of a large set of domain-swapped structures was performed to this aim. Results suggest that almost any protein may be capable of undergoing domain swapping, and that domain swapping is solely a specialized form of oligomer assembly but is closely associated with the unfolding/folding process of proteins. We also use experimental 19F-NMR to study the thermodynamic and kinetic properties in CV-N domain swapping. The activation energy barrier for the passage between monomeric and domain swapped dimeric form is of similar magnitude to that for complete unfolding of the protein, indicating that the overall unfolding of the polypeptide is required for domain swapping. Crystal structures of a domain-swapped trimer and a tetramer of CV-N provide further insights into the potential mechanics of CV-N domain swapping.
    Date: 19 December 2011
    Date Type: Publication
    Defense Date: 26 September 2011
    Approval Date: 19 December 2011
    Submission Date: 02 November 2011
    Release Date: 19 December 2011
    Access Restriction: 3 year -- Restrict access to University of Pittsburgh for a period of 3 years.
    Patent pending: No
    Number of Pages: 148
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    Uncontrolled Keywords: conformational dynamics; domain swapping; structural biology; computational biology
    Schools and Programs: School of Medicine > Molecular Biophysics and Structural Biology
    Date Deposited: 19 Dec 2011 14:53
    Last Modified: 16 Jul 2014 17:02

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