Protein Characterization in 3D: Size, Folding, and Functional Assessment in a Unified Approach

Assessment of protein stability and function is key to the understanding of biological systems and plays an important role in the development of protein-based drugs. In this work, we introduce an integrated approach based on Taylor dispersion analysis (TDA), flow induced dispersion analysis (FIDA), and in-line intrinsic fluorescence which enables rapid and detailed assessment of protein stability and unfolding. We demonstrate that the new platform is able to efficiently characterize chemically induced protein unfolding of human serum albumin (HSA) in great detail. The combined platform enables local structural changes to be probed by monitoring changes in intrinsic fluorescence and loss of binding of a low-molecular weight ligand. Simultaneously, the size of the unfolding HSA is obtained by TDA on the same samples. The integration of the methodologies enables a fully automated characterization of HSA using only a few hundred nanoliters of sample. We envision that the presented methodology will find applications in fundamental biophysics and biology as well as in stability screens of protein-based drug candidates.