Undergraduate Honors Theses

Thesis Defended

Spring 2011

Document Type

Thesis

Department

Chemistry & Biochemistry

First Advisor

Dr. J. Mathias Weber

Second Advisor

Dr. Cortlandt G. Pierpont

Third Advisor

Dr. Henry c. Kapteyn

Abstract

Red‐emitting variants of Green Fluorescent Protein are widely used as biomarkers for monitoring cellular processes in vivo. The pressure dependence of fluorescence, which can provide information on protein stability, has been studied for green‐, blue‐, and yellow-emitting variants (1‐3), but this relationship has not yet been explored for red‐emitting variants. In the present work, the fluorescence of the red fluorescent proteins TagRFP‐S, TagRFP‐T, mCherry, mOrange2, mStrawberry, and mKO were studied as a function of pressure through the use of a diamond anvil cell. Variants TagRFP‐S, TagRFP‐T, mOrange2, and mStrawberry showed an initial increase in fluorescence intensity upon application of pressure above ambient conditions. This effect is probably due to a reduction in conformational flexibility of their chromophores. At higher pressures, the fluorescence intensity decreases dramatically for all proteins under study. This fluorescence depletion resulted from chromophore pocket deformation and pressure‐induced denaturation. Mild blue shifts in fluorescence peaks were seen in all of the proteins as pressure was increased, consistent with a change in Franck‐Condon factors due to limited flexibility of the chromophores. In two variants, mOrange2 and mStrawberry, an abrupt change in the fluorescence spectra occurs at higher pressures. In mStrawberry, the fluorescence spectrum shifts strongly to the blue, which may be caused by the protonation of the chromophoric phenoxide. In mOrange2, a pronounced red shift in the fluorescence spectrum may be the result of the decyclization of the dihydrooxazole ring in the chromophore. Pressure‐dependent fluorescence depletion was found to be partially reversible in mOrange2 and mStrawberry.

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