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Development of Fluorescence Probes of Protein Folding

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dc.contributor.advisor Raleigh, Daniel P en_US
dc.contributor.author Watson, Matthew Douglas en_US
dc.contributor.other Department of Chemistry en_US
dc.date.accessioned 2017-09-20T16:51:49Z
dc.date.available 2017-09-20T16:51:49Z
dc.date.issued 2017-05-01 en_US
dc.identifier.uri http://hdl.handle.net/11401/77061 en_US
dc.description 278 pg. en_US
dc.description.abstract Many proteins depend on a stable, well-defined three-dimensional structure to perform biological functions. Protein folding is the process through which a polypeptide chain rearranges to adopt the native structure encoded in its amino acid sequence. The high intrinsic time resolution and signal-to-noise make fluorescence spectroscopy an ideal approach for protein folding experiments. However, interpretation of intrinsic tryptophan fluorescence changes is complicated by multiple fluorescence quenching mechanisms and solvent interactions. This work describes the use of selenomethionine (M<sub>Se</sub>), the selenium analogue of methionine as a quencher of tryptophan and 4-cyanophenylalanine (F<sub>CN</sub>) fluorescence to follow protein and peptide folding. The introduction of a quencher simplifies the interpretation of fluorescence changes in both amino acids and allows for the examination of specific side chain interactions. The approach was extended to the study of protein-protein interactions by incorporation of F<sub>CN</sub> and M<sub>Se</sub> into the monomeric units of a heterodimeric coiled coil. The fluorescence signal intensity allows for the detection of coiled coil formation at lower protein concentrations than what is accessible by standard circular dichroism (CD) methods. In addition, it is shown that the fluorescence quenching system can also be used to rapidly and accurately determine the <i>K<sub>D</sub></i> of the coiled coil interaction. The structural revolution of the past several decades has generated a vast amount of data on protein structure but has had comparatively less impact on our understanding of the origins of protein stability. An analysis of published stability data was carried out examining length-dependent thermodynamic properties. A clear correlation with chain length is observed for Δ<i>H</i>, Δ<i>S</i> and Δ<i>C<sub>p</sub></i>. Although Δ<i>G°</i> at 298 K of individual proteins cannot be accurately determined using this model, predictions for the thermal stability of whole proteomes are possible. Existing datasets were significantly expanded and differences between proteins from mesophilic and thermophilic organisms were examined. The large dataset also permitted the reassessment of the existence of convergence temperatures in proteins and an analysis of thermodynamic mutation data was used to predict thermal shifts due to ligand binding. en_US
dc.description.sponsorship This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. en_US
dc.format Monograph en_US
dc.format.medium Electronic Resource en_US
dc.language.iso en_US en_US
dc.publisher The Graduate School, Stony Brook University: Stony Brook, NY. en_US
dc.subject.lcsh Biophysics -- Biochemistry en_US
dc.subject.other fluorescence quenching, p-cyanophenylalanine, protein folding, protein stability, selenomethionine, thermal shift en_US
dc.title Development of Fluorescence Probes of Protein Folding en_US
dc.type Dissertation en_US
dc.mimetype Application/PDF en_US
dc.contributor.committeemember Miller, Lisa M en_US
dc.contributor.committeemember Parker, Kathlyn A en_US
dc.contributor.committeemember Seeliger, Markus. en_US

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