CALORIMETRIC ANALYSES OF THE INTERACTION BETWEEN SECB AND ITS LIGANDS

SecB is a chaperone in Escherichia coli dedicated to export of protein s from the cytoplasm to the periplasm and outer membrane. It functions to bind and deliver precursors of exported proteins to the translocat ion apparatus before they fold into their native structures, thus main taining them in a competent state for translocation across the membran e. The natural ligands of SecB are precursor proteins containing leade r sequences. There are numerous reports in the literature indicating t hat SecB does not specifically recognize the leader peptides. However, two published investigations have concluded that the leader peptide i s the recognition element (Watanabe M, Blobel G. 1989. Cell 58:685-705 ; Watanabe M, Blobel G. 1995. Proc Natl Acad Sci USA 92:10133-10136). In this work we use titration calorimetry to show that SecB binds two physiological ligands, which contain leader sequences, with no higher affinity than the same molecules lacking their leader sequences. Indee d, for one ligand the presence of the leader sequence reduces the affi nity. Therefore, it can be concluded that the leader sequence provides no positive contribution to the binding energy.