Multi-photon excitation of intrinsic protein fluorescence and its application to pharmaceutical drug screening.
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Buehler, Christof, Dreessen, Joerg, Mueller, Kurt, So, Peter T C, Schilb, Alain, Hassiepen, Ulrich, Stoeckli, Kurt and Auer, Manfred (2005) Multi-photon excitation of intrinsic protein fluorescence and its application to pharmaceutical drug screening. Assay and Drug Development Technologies, 3 (2). pp. 155-167. ISSN 1540-658X
Abstract
The majority of proteins contain intrinsic fluorophores as natural sensors of molecular structures, dynamics, and interactions. The intrinsic protein fluorescence signal allows for the label-free and, hence, undisturbed and rapid study of protein-ligand interactions. Ultraviolet-based drug screening is hampered by the background, photobleaching, light scattering, inner filter effects, and interfering assay compounds. Such problems can be overcome by means of molecular three-photon excitation (3PE) with infrared femtosecond light pulses since longer excitation wavelengths result in less Raleigh scattering, and the subfemtoliter (confocal-like) 3PE volume minimizes out-of-focus photobleaching, background generation, and inner filter effects. We demonstrate the general feasibility of 3PE for protein spectroscopy and illustrate the technique's excellent potential for high-throughput screening. By using the intrinsic fluorescence intensity of a protein-substrate, we were able to discriminate between ligands of different affinities in binding assays.
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| Date Deposited: | 14 Dec 2009 13:56 |
| Last Modified: | 31 Jan 2013 01:11 |
| URI: | https://oak.novartis.com/id/eprint/641 |