Detecting protein denaturation and the interactions between proteins and drugs at the nanolitre scale

The way proteins change their structure in the presence of certain chemicals, acids or bases - protein denaturation - plays a key role in many important biological processes. And the way proteins interact with various simple molecules is essential to finding new drugs. Microfluidic systems are touted as promising systems for high-throughput analysis of very small sample volumes, with applications in drug discovery, medical diagnostics, drug formulation, biosensing and synthetic biology. Now, researchers have applied microfluidics in a new way to study protein denaturation and protein:drug interactions, using just nanolitres of sample.

In a recent paper in Protein Science, researchers from the UCL Department of Biochemical Engineering and the London Centre for Nanotechnology (Matthieu Gaudet, Paul Dalby, Daniel Bracewell and Gabriel Aeppli), in collaboration with Cambridge and Sussex Universities, describe a new nanolitre scale technique in micro-capillaries to measure intrinsic protein fluorescence and obtain accurate protein denaturation curves at equilibrium. Free energies of protein unfolding were determined for the first time by such a method at this scale, and used to determine the affinity of a drug, the immunosuppressant rapamycin, to a protein, the cellular immunophilin FKBP12. Also from a double mutant cycle analysis, which is a technique using combinations of two different mutations of a protein to study the molecular interactions within a given protein, the microcapillary technique was used to measure the interaction energy between rapamycin and the Phe99 residue of FKBP-12.

The technique the researchers describe in the recent article has significant future potential by coupling protein stability and drug affinity analysis to microfluidic devices for sample preparation. The technique could, for example, be used to create a high throughput, low cost platform for drug discovery, protein engineering and protein formulation applications in the pharmaceutical industry.

This work has been published in Protein Science (M. Gaudet et al, Protein Sci. 19, 1544-1554 (2010))

Journal link: Protein Sci. 2010 August; 19(8): 1544–1554.

Schematic of the microcapillary technique for label-free protein analysis.
Figure: Schematic of the microcapillary technique for label-free protein analysis.