(493r) Shear-Induced Protein Conformation Changes Probed by Fluorescence Spectroscopy and Small-Angle Scattering

The investigation of the detailed structure of biologically and technologically important proteins and its stability or possible conformational changes in solution under different physiological conditions is a field of current research. Among these conditions, fluid shear can induce changes in the protein structure and can cause problems in the processing and handling of proteins in biotechnology applications, and can also change the protein activity and physiological role. Since relatively few models exist that investigate and quantify the role of fluid stresses in regulating the structure and function of proteins in solution, there is a need of finding new rapid and robust methods for this purpose.

In this work we examine the biophysical and biological features regulating the size and structure of the largest multimeric protein in blood, von Willebrand Factor (VWF) in solution, both under static and fluid shear conditions, by using two complementary techniques: small angle neutron scattering (SANS) and fluorescence methods. For our fluorescence studies we use the fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (bis-ANS) that partitions into the protein hydrophobic domains. VWF is particularly attractive for our study since previous biochemical studies suggest that shear-induced conformational changes in VWF may contribute to a variety of physiological and pathological processes in blood.

We report that the bis-ANS fluorescence intensity due to probe-multimeric VWF interaction increased, suggesting that new hydrophobic domains were exposed upon fluid shear application at shear rates greater than 2300-6000/s and at shear times greater than 1min. That was the case for multimeric but not recombinant dimeric VWF or the control protein bovine serum albumin, suggesting that the largest protein tested (MW~ 0.5-20MDa) was more susceptible to shear-induced conformation change. Some relaxation of the VWF structure was observed over the course of minutes following cessation of shear. SANS studies at higher resolution revealed structural changes in VWF at shear rates below 3000/s and at length scales less than 10nm. Overall, structural changes in VWF conformation reported here using bis-ANS binding and SANS likely correspond to functional changes in the protein found in the literature that are associated with atherothrombotic events. This study introduces the fluorescence bis-ANS binding and the SANS as new tools that may be useful in studies of shear-induced protein structure changes.