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Protein folding and stability: distinguishing folded from unfolded state effects

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dc.contributor.advisor Raleigh, Daneil P en_US
dc.contributor.author Xiao, Shifeng en_US
dc.contributor.other Department of Chemistry en_US
dc.date.accessioned 2013-05-22T17:35:50Z
dc.date.accessioned 2015-04-24T14:47:38Z
dc.date.available 2013-05-22T17:35:50Z
dc.date.available 2015-04-24T14:47:38Z
dc.date.issued 2012-05-01
dc.identifier Xiao_grad.sunysb_0771E_10894 en_US
dc.identifier.uri http://hdl.handle.net/1951/59920 en_US
dc.identifier.uri http://hdl.handle.net/11401/71461 en_US
dc.description 197 pg. en_US
dc.description.abstract The villin headpiece subdomain (HP36) is one of the smallest naturally occurring protein domains that folds cooperatively in the absence of disulfide bonds or ligand binding. It has a simple topology consisting of three ? -helices that form a hydrophobic core. Kinetic studies have shown that the subdomain folds on the microsecond time scale, making it one of the fastest folding proteins. Its simple topology, small size, and rapid folding have made it a very popular model for computational, theoretical, and experimental studies. HP36 has a well packed hydrophobic core comprised in part of an unusual set of three closely packed phenylalanine residues F47, F51, F58. Aromatic aromatic interactions have been conjectured to play a critical role in specifying the subdomain fold and have been proposed to play a general role in stabilizing small proteins. The modest stability of HP36 has hindered studies of core packing since multiple mutations can lead to constructs which fail to fold and even single mutants can result in poorly folded variants. Using a hyperstable mutant of HP36 as the new background, generated by targeting surface residues, I show that aromatic aromatic interactions are not required for specifying the subdomain fold, although they have effects on the stability. Proline-aromatic interactions involving P62 and W64 have been proposed to play a critical role in specifying the subdomain fold by acting as gatekeeper residues, i.e. as residues absolutely essential for specifying the fold. Mutation studies based on the same new background reveal that proline-aromatic interactions are not required for specifying the subdomain fold. These studies argue against the concept of specific gatekeeper residues. The implications for protein folding are discussed. To probe unfolded state electrostatic interactions, the pH-dependent stability of wildtype HP36 and three mutants, K48M, K65M and K70M, all of which significantly increase the stability of the protein were examined. The increased stability of the K48M mutant is due to the removal of favorable electrostatic interactions in the unfolded state, the increased stability of the K65M mutant is due to the reduction of the desolvation penalty at the mutation site upon folding, while the increased stability of the K70M mutant is due to the introduction of a new hydrophobic interaction between the methionine and the hydrophobic core in the native state. The unfolded state electrostatic interactions were confirmed by double mutant thermodynamic cycle analysis and by using a method to estimate residue-specific unfolded state pKa values. The results demonstrate that electrostatic as well as hydrophobic interactions play an important role in the unfolded state, and illustrate an approach for distinguishing native state effects from unfolded state effects. This work also has interesting implications for studies which attempt to stabilize proteins by targeting surface electrostatics since it shows that the mechanism of stabilization may be much more complicated than generally anticipated. 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 Chemistry--Biophysics--Biochemistry en_US
dc.subject.other electrostatic interaction, protein folding, protein stability, unfolded state en_US
dc.title Protein folding and stability: distinguishing folded from unfolded state effects en_US
dc.type Dissertation en_US
dc.mimetype Application/PDF en_US
dc.contributor.committeemember London, Erwin en_US
dc.contributor.committeemember Tonge, Peter J en_US
dc.contributor.committeemember Seeliger, Markus A. en_US

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