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.

The Tribbles 2 (TRB2) pseudokinase binds to ATP and autophosphorylates in a metal-independent manner

“…In addition to detection of fluorescence from an extrinsic hydrophobic dye, the thermal stability of proteins can also be analysed by detecting the intrinsic fluorescence emitted by the indole ring of internal tryptophan residues as they become exposed to the solvent during denaturation [54]. We initially exploited this approach to measure intrinsic fluorescence changes associated with pseudokinase denaturation in the presence and absence of various ligands and cofactors.…”

“…This approach is automated and can measure multiple parameters simultaneously in a 96-well format, including temperature-induced aggregation effects [54]. Dye-binding analysis has been used previously to analyse over 30 pseudokinases and is recognized as a useful general approach for ligand screening [20,75,76].…”

Concurrent multiscale modelling of atomistic and hydrodynamic processes in liquids

“…The stochastic heat flow also has a zero average, while the covariance component is determined by the expression 6) or explicitly,q…”

Protein stability by number: high-throughput and statistical approaches to one of protein science's most difficult problems

“…Mayo and coworkers recently coupled this system with computational design of proteins libraries, enabling exhaustive characterization of computational predictions in a reasonable experimental timeframe [25**]. Dalby and coworkers have gone on to further miniaturize their method to nanoliter scale using microfluidics, which enables screening with very small amounts of proteins (perhaps only 10 8 molecules) [26]. This approaches a scale where concomitant miniaturization of protein production is a challenge for libraries, but it has great promise immediately for protein-ligand interactions.…”