Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale |
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Authors: | Matthieu Gaudet Nina Remtulla Sophie E Jackson Ewan R G Main Daniel G Bracewell Gabriel Aeppli Paul A Dalby |
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Institution: | 1. Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom;2. London Centre for Nanotechnology, University College London, London WC1H 0AH, United Kingdom;3. Department of Chemistry, Cambridge University, Cambridge CB2 1EW, United Kingdom;4. Department of Chemistry and Biochemistry, University of Sussex, Brighton BN1 9QG, United Kingdom;5. Department of Physics and Astronomy, University College London, London WC1E 6BT, United Kingdom |
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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 108 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. |
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Keywords: | drug screening FKBP‐12 microfluidics protein fluorescence Rapamycin |
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