Using Photolabile Protecting Groups for the Rapid Triggering of Fast Biological Events

Citation

Hansen, Kirk Charles (2002) Using Photolabile Protecting Groups for the Rapid Triggering of Fast Biological Events. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/XP3F-G534. https://resolver.caltech.edu/CaltechTHESIS:02102012-145747544

Abstract

To circumvent problems associated with rapid mixing we have used photolabile protecting groups for rapid initiation of biochemical processes. Specifically we have utilized laser triggering to initiate electron transfer in quinol-oxidizing enzymes and to study conformational changes in proteins.

In order to probe the reaction chemistry of respiratory quinol-oxidizing enzymes on a rapid time scale, a photoreleasable quinol substrate was synthesized by coupling decylubiquinol with the water-soluble protecting group 3',5'-bis(carboxymethoxy)benzoin (BCMB). Photolysis of DQ-BCMB led to the release of the BCMB group in less than 10^(-6) s. The electron transfer from decylubiquinol to the respiratory enzymes Escherichia coli cytochrome bO_3 and mitochondrial cytochrome bc_1 was studied using "caged" decylubiquinol. Cytochrome bc_1 reacted with photoreleased decylubiquinol with distinct kinetic phases corresponding to rapid b-heme reduction and slower c-heme reduction. The discrimination of kinetic phases in the reaction of cytochrome bc_1 with ubiquinol substrates has provided a means of exploring the bifurcation of electron transfer that is central to the operation of the Q-cyde in this enzyme.

A general, rapid method for triggering protein unfolding and folding has been developed using the photolabile protecting group 3',5'-dimethoxybenzoin (DMB) and the photolabile linker 3'-(carboxymethoxy)benzoin (CMB) respectively. To study unfolding the DMB group was introduced in a site-specific manner to block a mutation known to destabilize the GCN4-p1 coiled-coil. Upon photolysis, the unfavorable interaction is unmasked and the peptide unfolds allowing for kinetic characterization.

The 35 amino-acid headpiece subdomain of the actin bundling protein Villin was constrained in a non-native state by cyc1ization of the N-terminus to a cysteine residue introduced in the middle of the protein. Since destabilization of the native state is accomplished by the conformational constraint of a head-to-side chain cyc1ization, the folding of the Villin headpiece can be initiated by rapid photolysis of the photo labile linker. The ensuing folding process was studied in real time, in the absence of added denaturants. This work demonstrates a general methodology for studying early events in protein folding, a process that is crucial for the understanding of how a protein reaches its native state.

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