title: Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale.
abstract: Protein stability and ligand-binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5-20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000-fold to just 10(8) molecules. The stability of wild-type FKBP-12 and a destabilizing binding-pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP-12 and rapamycin originates from residue Phe99 in the binding site.
authors.affiliation: Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Gaudet
authors.forename: Nina
authors.initials: N
authors.lastname: Remtulla
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Jackson
authors.forename: Ewan R G
authors.initials: ER
authors.lastname: Main
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Bracewell
authors.forename: Gabriel
authors.initials: G
authors.lastname: Aeppli
authors.forename: Paul A
authors.initials: PA
authors.lastname: Dalby
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001665:Binding Sites; D002417:Cattle; D015195:Drug Design; D005453:Fluorescence; D044085:Microfluidics / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D020123:Sirolimus / Q000737:chemistry*; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
chemical_list: D000900:Anti-Bacterial Agents; D012710:Serum Albumin, Bovine; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus