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The Dynamic Nature of the Folded and Unfolded States of the Villin Headpiece Helical Subdomain

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The Dynamic Nature of the Folded and Unfolded States of the Villin Headpiece Helical Subdomain The Dynamic Nature of the Folded and Unfolded States of the Villin Headpiece Helical Subdomain A Dissertation Presented by Lauren Wickstrom to The Graduate School in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biochemistry and Structural Biology Stony Brook University May 2009 Stony Brook University The Graduate School Lauren Wickstrom We, the dissertation committee for the above candidate for the Doctor of Philosophy degree, Hereby recommend the acceptance of this dissertation. Carlos Simmerling, Ph. D., Advisor Department of Chemistry, Stony Brook University Daniel P. Raleigh, Ph. D., Advisor Department of Chemistry, Stony Brook University Steven O. Smith, Ph. D., Chair Department of Biochemistry and Cell Biology, Stony Brook University Robert C. Rizzo, Ph. D., Third Member Department of Applied Mathematics and Statistics, Stony Brook University Marivi Fernandez-Serra, Ph. D., Outside Member Department of Physics and Astronomy, Stony Brook University This dissertation is accepted by the Graduate School Lawrence Martin Dean of the Graduate School ii Abstract of the Dissertation The Dynamic Nature of the Folded and Unfolded States of the Villin Headpiece Helical Subdomain by Lauren Wickstrom Doctor of Philosophy in Biochemistry and Structural Biology Stony Brook University 2009 The symbiotic relationship between experiment and simulation is necessary for a complete understanding of biomolecular structure and dynamics. Computational approaches can provide structural populations and atomic detail, and describe motion not visible on the macroscopic level, which can be used to interpret the experimental average ensemble as well develop new experiments. In turn, simulations rely on experimental observables for validation of a particular model or method. In this work, both tools are used collaboratively to study the structure of the folded and the unfolded states of proteins. Solution NMR and X-ray structures of the folded state are widely used as a reference for simulations and experiments. Recent work has shown that the denatured state contains structure that is important for understanding protein stability and the folding pathway. This knowledge can be utilized to understand and treat protein misfolding diseases. One of the key model systems, the 36-residue villin headpiece helical subdomain (HP36), was chosen for these studies because of its simple topology, small size and fast iii folding properties. Structures of HP36 have been determined using X-ray crystallography and NMR spectroscopy, but the two structures exhibited clear differences. Molecular dynamics simulations and experimental double mutant cycles were used to show that the X-ray structure is the better representation of the folded state in solution. Previous experimental evidence has suggested that there is residual structure in the denatured state of HP36. Fragment analysis has shown that the three individual helices of HP36 lack significant structure compared to a larger fragment containing the first two helices (HP21). These techniques, however, are low resolution and are unable to quantify low levels of helical structure and whether it occurs in the same regions as HP36. Simulations were used to quantify the structure in all of the fragments. The HP21 ensemble contains less helical structure than predicted by NMR experimental observables possibly due to deficiencies in sampling and the force field. To address these limitations, simulation methodology and models were investigated. iv Table of Contents Table of Contents .............................................................................................................. v List of Figures.................................................................................................................... x List of Tables................................................................................................................ xxvii List of Publications ...................................................................................................... xxix Acknowledgements ....................................................................................................... xxx 1. Introduction ............................................................................................................ 1 1.1 Symbiotic Relationship between Experiments and Computational Studies ..... 1 1.2 The Importance of Understanding Folded and Unfolded State Structure........ 2 1.2.1 What is the protein folding problem?................................................................ 2 1.2.2 Importance of the folded state........................................................................... 3 1.3 Studying the Unfolded State .................................................................................. 4 1.3.1 Why is studying the unfolded state important? ................................................. 4 1.3.2 Difficulties in resolving the unfolded state ensemble experimentally .............. 4 1.4 How Can Simulations Help Us Study the Protein Folding Problem?................ 6 1.4.1 The limitations of simulations........................................................................... 6 1.5 Simulation Methodology........................................................................................ 7 1.5.1 Force field.......................................................................................................... 7 1.5.2 Solvation............................................................................................................ 8 1.5.3 Enhanced sampling with replica exchange molecular dynamics .................... 10 1.6 Model System Used to Study Protein Folding – Villin Headpiece Helical v Subdomain.......................................................................................................................12 1.7 Aims of this Thesis................................................................................................ 14 2. Reconciling the Solution and X-ray Structures of the Villin Headpiece Helical Subdomain: Molecular dynamics and Double Mutant Cycles Reveal a Stabilizing Cation-pi Interaction ...................................................................................................... 16 2.1 Introduction .......................................................................................................... 17 2.2 Methods ................................................................................................................. 22 2.2.1 Computational ................................................................................................. 22 2.2.2 Data analysis.................................................................................................... 24 2.3 Results.................................................................................................................... 24 2.3.1 Simulations of the NMR structure................................................................... 24 2.3.2 Structural similarities to the NMR Family ...................................................... 32 2.3.3 Specific sidechain interactions........................................................................ 34 2.3.4 Simulations of the X-ray structure .................................................................. 37 2.3.5 Experimental investigation of the putative sidechain interactions.................. 38 2.4 Discussion.............................................................................................................. 45 3. The Unfolded State of the Villin Headpiece Helical Subdomain: Computational Studies of the Role of Locally Stabilized Structure ........................... 51 3.1 Introduction .......................................................................................................... 52 3.2 Methods ................................................................................................................. 56 3.2.1 Explicit solvent simulations ............................................................................ 56 3.2.2 Implicit solvent simulations ............................................................................ 58 3.2.3 Data analysis.................................................................................................... 59 3.3 Results.................................................................................................................... 61 3.3.1 Measuring convergence of the REMD simulations......................................... 61 3.3.2 Comparison of structural ensembles obtained using explicit and implicit solvent models............................................................................................................... 62 3.3.3 Summary of data analysis approaches............................................................. 64 3.3.4 Simulations of Ala10 ........................................................................................ 65 3.3.5 Conformational preferences for fragment HP-1.............................................. 66 3.3.6 HP-2 shows no significant residual structure.................................................. 73 3.3.7 Analysis of HP-3 ............................................................................................. 75 vi 3.4 Discussion.............................................................................................................. 77 3.4.1 What may stabilize the high population of helical structure in HP-1?............ 77 3.4.2 Comparison with
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