Journal
JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY A-CHEMISTRY
Volume 340, Issue -, Pages 181-200Publisher
ELSEVIER SCIENCE SA
DOI: 10.1016/j.jphotochem.2017.01.027
Keywords
Pyrene; Squaraine; Co(III)hexamine; Serum albumin; Avidin; Lysozyme; Photocleavage; Flash photolysis; Circular dichroism; Fluorescence; Sequencing; Phenylalanine; SDS/PAGE; Steady state; Quantum yield; Docking
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Funding
- National Science Foundation [EAGER DMR-1441879]
- University of Connecticut OVPR
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A powerful strategy to photocleave the protein backbone at a single site, developed over nearly two decades, is described here. The remarkable ability to target one or even a couple of sites on a large protein with a small molecule, under photochemical control, is a considerable challenge. Systematic modification of the structure of the photoreagent provides valuable information on the binding site recognition as well as the mechanism of the photocleavage reaction. Some factors that impact the photocleavage reaction include the exact location of the probe binding site on the protein, conformations of the bound probe, protein size, functional groups present on the probe that interact with the protein surroundings either in a favorable or unfavorable manner, overall charge on the photoreagent, and photophysical/ photochemical properties of the probe. The protein photocleavage studies were preceded by detailed binding studies by a variety of spectroscopic methods. Methods as simple as absorption and fluorescence spectroscopies or more sophisticated circular dichroism spectroscopy were used. Conclusions that are most consistent with the binding data indicated a single binding site on most proteins, irrespective of the probe or the protein, with only one exception noted so far. Such high selectivity for targeting a binding site on a biomolecule by a small molecule is remarkable. Photocleavage studies and laser flash photolysis studies were used to probe the photochemical peptide bond cleavage reactions, which indicated the important role of aromatic cation radicals in the mechanistic path. Our work focused mainly on few generic proteins serum albumin, Lysozyme, and avidin; but may be extended to other proteins with appropriate modifications. This article strives to provide some guidance on such design strategies for other proteins or other photoreagents. Molecular modeling studies clearly supported our conclusions and the methods described here could be useful in a rational design of novel photoreagents to target desired sites on proteins in future studies. (C) 2017 Elsevier B.V. All rights reserved.
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