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Chemical crosslinking mediated by short bifunctional reagents has been widely used for determining physical relationships among polypeptides in multisubunit 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 crosslinking 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 epsilon subunit, a component of the rotor subcomplex of the enzyme, and the unrelated maltose binding protein (MBP), then the two purified recombinant proteins were crosslinked by means of a dimaleimido-PEG cross-linking agent. Following purification, the epsilon-PEG-MBP was incorporated into membrane-bound ATP synthase by reconstitution with epsilon-depleted F(1)-ATPase and membrane vesicles that had been stripped of endogenous F(1). ATP synthase reconstituted using epsilon-PEG-MBP had reduced ATP hydrolytic activity that was uncoupled from the pumping of H(+), indicating the physical blockage of rotation of the gammaepsilonc(10) rotor by the conjugated MBP, whereas enzyme reconstituted with epsilon-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.