title: Multi-photon excitation of intrinsic protein fluorescence and its application to pharmaceutical drug screening.
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.
authors.affiliation: Discovery Technologies-Innovative Screening Technologies, Novartis Institutes for BioMedical Research GmbH & Co. KG, Vienna, Austria. christof.buehler@pharma.novartis.com
authors.forename: Christof
authors.initials: C
authors.lastname: Buehler
authors.forename: Joerg
authors.initials: J
authors.lastname: Dreessen
authors.forename: Kurt
authors.initials: K
authors.lastname: Mueller
authors.forename: Peter T C
authors.initials: PT
authors.lastname: So
authors.forename: Alain
authors.initials: A
authors.lastname: Schilb
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hassiepen
authors.forename: Kurt A
authors.initials: KA
authors.lastname: Stoeckli
authors.forename: Manfred
authors.initials: M
authors.lastname: Auer
mesh_terms: D017076:Computer-Aided Design; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005240:Feasibility Studies; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D010600:Pharmacology / Q000379:methods*; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*
chemical_list: D011506:Proteins