Application of fluorescence spectroscopy to quantify shear-induced protein conformation change.
Rapid and robust methods are required to quantify the effect of hydrodynamic shear on protein conformation change. We evaluated such strategies in this work and found that the binding of the fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to hydrophobic pockets in the blood protein von Willebrand factor (VWF) is enhanced upon the application of fluid shear to the isolated protein. Significant structural changes were observed when the protein was sheared at shear rates >or= 6000/s for approximately 3.5 min. The binding of bis-ANS to multimeric VWF, but not dimeric VWF or control protein bovine serum albumin, was enhanced upon fluid shear application. Thus, high-molecular-weight VWF is more susceptible to conformation change upon tensile loading. Although bis-ANS itself did not alter the conformation of VWF, it stabilized protein conformation once it bound the sheared molecule. Bis-ANS binding to VWF was reduced when the sheared protein was allowed to relax before dye addition. Taken together with functional data in the literature, our results suggest that shear-induced conformation changes in VWF reported by bis-ANS correlate well with the normal function of the protein under physiological/pathological fluid flow conditions. Further, this study introduces the fluorescent dye bis-ANS as a tool that may be useful in studies of shear-induced protein conformation change.
Themistou
Efrosyni
E
Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Buffalo, New York, USA.
Singh
Indrajeet
I
Shang
Chengwei
C
Balu-Iyer
Sathy V
SV
Alexandridis
Paschalis
P
Neelamegham
Sriram
S
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology*; D001703:Biophysics / Q000379:methods; D019281:Dimerization; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D008970:Molecular Weight; D011487:Protein Conformation; D016622:Silver Staining; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013314:Stress, Mechanical; D013718:Tensile Strength; D014841:von Willebrand Factor / Q000737:chemistry*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D014841:von Willebrand Factor; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate)