Browsing by Subject "Protein Conformation"
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Item Enabling Structural Studies of the Yeast Prion Protein Within a Cellular Environment(2022-05) Costello, Whitney Nicole; Lin, Milo; Rizo-Rey, José; Diamond, Marc; Frederick, KendraMy motivation for the work in this thesis was to take steps towards bridging the gap in structural information between atomic models of metastable proteins in isolated and cellular environments. Most biophysical techniques are generally limited by either sample composition or resolution. One technique, Nuclear Magnetic Resonance (NMR) is not limited by sample composition and can provide atomic-level resolution. However, NMR is limited by sensitivity. Recent advancements in the field produced a sensitivity-enhanced solid-state NMR technique, namely Dynamic Nuclear Polarization (DNP) NMR. Using DNP NMR, I observed sensitivity enhancements of up to 90-fold increase in sensitivity, eliminating this barrier. Recent work on a metastable protein, Sup35, assembled in cellular lysates using DNP NMR demonstrates that the biological environment has a dramatic effect on the Sup35 protein structure. In this thesis, I sought to harness sensitivity gains from DNP NMR to identify strategies for the specific detection of isotopically labeled proteins at within a cellular lysate for structural analysis. First I present theoretical calculations, validated by experimental results, for the expected signal of detection ratio of an isotopically labeled protein within a cellular lysate. These results concluded that DNP NMR can specifically detect a 30 kDa, uniformly isotopically labeled protein at low micromolar concentrations. However, sensitivity is still a barrier to specifically detect proteins with lower molecular weights or non-uniform isotopic labeling. Therefore, I optimized sample preparation for maximum sensitivity for DNP NMR on cellular lysates. DNP sensitivity enhancement depends on sample composition. DNP NMR is performed at 100 K, and requires sample glassing, polarizing agent, and protonation for optimal DNP enhancement. Some of the first DNP NMR experiments on purified protein samples were optimized for a matrix of 60:30:10, d8-glycerol:D2O:H2O with 10 mM biradical. These matrix conditions became standard in the field, known as "DNP Juice". However, I found that these matrix conditions are not optimal for DNP NMR cellular lysate samples. In the presence of cellular lysate, sensitivity is improved by addition of lower cryoprotectant (15%) and biradical concentrations (5 mM). I also found that deuteration was unnecessary. Finally, I investigated methods to simplify DNP NMR spectra through segmental labeling of proteins. The strategies in this thesis benefit future research of structural studies on environmentally sensitive proteins, such as alpha-synuclein or tau, within their native environment at physiological concentrations.Item Improving Profile Similarity Search and Alignment of Protein Sequences(2015-11-20) Tong, Jing; Ranganathan, Rama; Otwinowski, Zbyszek; Borek, Dominika; Grishin, Nick V.Protein function prediction is one of the most important problems in the field of computational biology. The most reliable method to predict protein function is to detect homologs. Homologous proteins tend to possess conserved sequence motifs, the same structure folds, and similar functional sites. Current sequence-based homology search methods are still unable to detect many similarities evident from protein spatial structures. We present a new method, COMPADRE, to assess the relationship between the query sequence and a hit in the database by considering the similarity between the query and hit's known homologs. This method markedly boosts the homology detection precision rate. Successful homology-based protein function prediction is also determined by accurate alignment between a protein sequence and its homolog. Alignment errors are the main bottleneck for homology modeling when the query is distantly related to the template. Alignment methods often misalign secondary structural elements by a few residues. We present a refinement method, SFESA, to improve pairwise sequence alignments by evaluating alignment variants generated by local shifts of template-defined secondary structures. The potential values of these methods for structure/function predictions are illustrated by the detection of homology between evolutionary distant yet structurally similar protein domains.Item A New Approach to Optimize a Protein Energy Function on a Folding Pathway Using Gō-Like Potential and All-Atom, Ab Initio Monte Carlo Simulations(2016-01-19) Safronova, Aleksandra; Goldsmith, Elizabeth J.; Grishin, Nick V.; Otwinowski, Zbyszek; Rice, Luke M.Prediction of a protein structure is important for understanding the function of a protein. The process of protein structure prediction employs the approximation of a protein free energy that guides protein folding to the protein's native state. A function with a good approximation of the protein free energy should allow estimation of the structural distance of the evaluated candidate structure to the protein native state. Currently the energy optimization process relies on the correlation between the energy and the similarity to the native structure. The energy function is presented as a weighted sum of components which are designed by human experts with the use of statistical analysis of solved protein strictures. Values of the weights are derived through the procedure that maximizes the correlation between the energy and the similarity to the native structure measured by a root mean square deviation between coordinates of the protein backbone. Two major components are required for a successful ab initio modelling: (1) an effective energy function that discriminates the native protein structure out of all possible decoy structures; (2) an efficient sampling algorithm that quickly searches for the low-energy states. In this dissertation a new method for energy optimization is proposed. The method relies on a fast sampling algorithm and targets successful protein folding. The weights for energy components are optimized on a found with the Gō potential energy fast folding pathway. The Lennard-Jones potential, the Lazaridis-Karplus solvation potential, hydrogen bonding potential are used in the optimization algorithm. The optimized weights successfully predict all α and α/β proteins. The proposed strategy is conceptually different from the existing methods that optimize the energy on solved protein structures. The developed algorithm is a novel concept that allows the optimization of a more complex functional combination of the energy components that would improve the prediction quality.Item Optimization and Analysis of Weighted-Window Predictors of Structural Disorder in Proteins(2007-05-22) Holladay, Nathan Brent; Otwinowski, Zbyszek; Grishin, Nick V.; Pertsemlidis, Alexander; Rizo-Rey, JoséX-ray crystallographic protein structures often contain disordered regions that are observed as missing electron density. We have developed single sequence and profile-based weighted-window predictors of structural disorder in proteins, as well as a simple method for addressing disorder-prone chain termini in disorder prediction. Optimizing the parameters for these relatively simple predictors with crystallographic data using a simulated annealing type algorithm, we achieve performance similar to that of DISOPRED2. Optimized parameters from these disorder predictors provide information relating to physical processes underlying crystallographic disorder. Optimized score adjustment values suggest a simple, monotonic relationship between disorder and residue distance from termini that is nearly the same for amino- and carboxy-terminal positions. Residue disorder parameters are strongly associated with scales from certain experimental model systems that primarily reflect hydrophobic interactions. Our data do not suggest a strong association between crystallographic disorder and secondary structure beyond that explained by hydrophobicity. Our results lend support to the idea that while hydrophobic side chain interactions are primarily involved in determining stability of the folded conformation, hydrogen bonding and similar polar interactions are primarily involved in conformational and interaction specificity.