(448h) A Zipper-Like Mechanism Steers the Recognition of Cyanobacterial Split Inteins

The formation of a peptide bond catalyzed by two halves of a split intein is called protein trans-splicing.  The potential to use this process synthetically offers many new applications, but little is known about the molecular recognition between the two polypeptide split-intein partners.  In this study, we investigated the association between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria:  Oscillatoria limnetica, Thermosynechococcus vulcanus and Nostoc species PCC7120.  Each split partner was attached to an affinity tag, making possible the direct immobilization of the protein using monoclonal antibodies. Atomic force microscopy (AFM) was then used in force mode to measure the binding forces between the different pairs.  The results show that native and mixed pairs exhibit similar binding forces within the error of the measurement technique (~52 pN).  Control experiments (e.g. using an engineered non interacting protein) were performed to support our finding.  In addition, these non-equilibrium measurements of the work of binding are compared with equilibrium measurements of the free energy of binding confirming the Jarzynski's inequality.  Using a bioinformatic sequence analysis and computational structural prediction, we have discovered the presence of highly conserved interspersed electrostatic and non-polar pairs in triplet interacting along the binding surface of the C- and N- terminal partners.  This complementary zipper explains the indistinguishable intermolecular forces between native and mixed pairs measured with AFM.