Tethering polypeptides through bifunctional PEG cross‐linking agents to probe protein function: Application to ATP synthase

Abstract

Chemical cross-linking mediated by short bifunctional reagents has been widely used for determining physical relationships among polypeptides in multi-subunit proteins, but less often for functional studies. Here we introduce the approach of tethering polypeptides by using bifunctional reagents containing a lengthy, flexible PEG linker as a form of cross-linking especially suited to functional analyses. The rotary molecular motor ATP synthase was used as a model subject. Single cysteine residues were introduced into selected positions of ATP synthase  subunit, a component of the rotor subcomplex of the enzyme, and the unrelated maltose binding protein (MBP), then the two purified recombinant proteins were cross-linked by means of a dimaleimido-PEG cross-linking agent.Following purification, the -PEG-MBP was incorporated into membrane-bound ATP synthase by reconstitution with -depleted F 1 -ATPase and membrane vesicles that had been stripped of endogenous F 1 . ATP synthase reconstituted using -PEG-MBP had reduced ATP hydrolytic activity that was uncoupled from the pumping of H + , indicating the physical blockage of rotation of the c 10 rotor by the conjugated MBP, whereas enzyme reconstituted with -PEG was normal. These results directly demonstrate the feasibility of studying mechanistic features of molecular motors through PEG-based conjugation of unrelated proteins. Since tethering polypeptides provides a means of maintaining proximity without directly specifying or modifying interactions, application of the general method to other types of protein functional studies is envisioned.3

General Strategy for Bioluminescence Sensing of Peptidase Activity In Vivo Based on Tumor-Targeting Probiotic

“…Herein, we introduced a background suppression technology that relies on highly efficient thiol-maleimide “click” reactions, − which should greatly reduce the impact of l -cysteine from the EcN-luc. We have used mPEG-maleimide (mPEG-MAL) to react specifically with sulfhydryl groups.…”

“…No significant cytotoxicity was observed after incubation with 2 mM mPEG-MAL for 5 min and subsequent addition of 2 mM GSH for 24 h. In general, cysteines in many proteins are buried rather than on the surface. The hydrophilic PEG further reduces the nonspecific interactions with protein surfaces, which results in negligible toxicity to mammalian cells. Notably, the concentration of GSH in the intracellular cytosol is at the mM level. , Thus, the addition of 2 mM GSH should not have an impact on cellular viability.…”