Protein sizes and stoichiometry in the chaperone SecB--RBPTI complex estimated by ANS fluorescence

Interaction of the SecB E. coli chaperone with model precursor protein, the reduced form of bovine pancreatic trypsin inhibitor (RBPTI), in aqueous solution was studied using ANS fluorescence. Binding of RBPTI by SecB-ANS led to an increase and blue shift in the ANS fluorescence. Two ANS emission centers exist in the proteins and their complex: the short-lived center (lifetime of approximately 1 nsec) where ANS is strongly quenched by polar amino acid residues and the long-lived center (6-12 nsec) where ANS emits from the hydrophobic pocket. Estimation of the volumes and diameters of RBPTI, SecB, and the SecB--RBPTI complex using fluorescence lifetime and polarization of ANS by a modified Levshin--Perrin equation was done. Effective diameter of the SecB equals to 33.4 A. The diameter of the SecB--RBPTI complex is 49.8 A. The volume of the complex equals to sum of volumes of one tetrameric SecB and four RBPTI molecules. The binding of RBPTI molecules into the tetramer SecB is not an insertion to a "pore" without increase of the protein sizes, but "paste" of four polypeptide molecules to four subunits of SecB.