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dc.contributor.advisorWebb, Lauren J.
dc.creatorSlocum, Joshua David
dc.date.accessioned2019-08-06T17:29:04Z
dc.date.available2019-08-06T17:29:04Z
dc.date.created2017-05
dc.date.submittedMay 2017
dc.identifier.urihttps://hdl.handle.net/2152/75383
dc.identifier.urihttp://dx.doi.org/10.26153/tsw/2488
dc.description.abstractThe nitrile stretching oscillation has been widely used as a probe of local environment to study dynamics, folding, and electrostatics in proteins. A popular model for interpreting nitrile frequencies is the vibrational Stark effect (VSE), which allows one to interpret changes in vibrational energy in terms of changes in force along the nitrile bond. In principle, this allows for the site-specific, directional measurement of electric fields in a complex protein environment. However, the interpretation of these frequencies in terms of electric fields is complicated by the fact that hydrogen bonding to the nitrile probe is known to cause frequency shifts that are not described by the VSE. To address this concern, we have biosynthetically incorporated para-cyanophenylalanine (pCNF) probes into green fluorescent protein (GFP) near the intrinsic fluorophore, whose sensitivity to electric fields has been well characterized. We observed that the vibrational and electronic probes of electrostatic environment have similar spectroscopic responses to a series of amino acid mutations, and that the intrinsic sensitivity of GFP emission energy to the mutations was unperturbed by the presence of the pCNF probes. Additionally, we compared the measured Stark effect shifts to pK [subscript a] changes of the GFP fluorophore and saw that these two orthogonal measurements of electrostatic environment were in agreement, which further corroborates the analysis of VSE shifts of nitriles in terms of electric fields. These studies provide confidence in the ability of nitrile frequencies to report faithfully on electric fields, even in the background of direct hydrogen bonding to the probes. Finally, based on our analysis of electrostatic forces near the GFP fluorophore, we designed and characterized an interesting GFP mutant that can be photoactivated by near-UV light. We obtained preliminary evidence in the form of mass spectrometry and kinetic analysis that support a novel charge transfer mechanism for this photoconversion, which could lead to the design of fluorescent proteins with enhanced properties. In summary, we have developed GFP as a robust model system for understanding and manipulating the finely tuned relationship between protein structure and function.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.subjectGreen fluorescent protein
dc.subjectElectrostatics
dc.subjectVibrational spectroscopy
dc.subjectpKa
dc.subjectCharge transfer
dc.titleMeasuring and manipulating electric fields near the green fluorescent protein fluorophore using vibrational Star effect spectroscopy of nitrile probes
dc.typeThesis
dc.date.updated2019-08-06T17:29:04Z
dc.contributor.committeeMemberBaiz, Carlos R
dc.contributor.committeeMemberMatouschek, Andreas T
dc.contributor.committeeMemberRen, Pengyu
dc.contributor.committeeMemberRoberts, Sean T
dc.description.departmentChemistry
thesis.degree.departmentChemistry
thesis.degree.disciplineChemistry
thesis.degree.grantorThe University of Texas at Austin
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy
dc.creator.orcid0000-0002-8705-3054
dc.type.materialtext


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