Analysis of the role of interfacial tryptophan residues in controlling thetopology of membrane proteins

Tryptophans have a high affinity for the membrane-water interface and have been suggested to play a role in determining the topology of membrane prote ins. We investigated this potential role experimentally, using mutants of t he single-spanning Pf3 coat protein, whose transmembrane topologies are sen sitive to small changes in amino acid sequence. Mutants were constructed wi th varying numbers of tryptophans flanking the transmembrane region and tra nslocation was assessed by an in vitro translation/translocation system. Tr anslocation into Escherichia coli inner membrane vesicles could take place under a variety of experimental conditions, with co- or posttranslational a ssays and proton motive force-dependent or -independent mutants. It was fou nd that translocation can even occur in pure lipid vesicles, under which co nditions the tryptophans must directly interact with the lipids. However, u nder all these conditions tryptophans neither inhibited nor stimulated tran slocation, demonstrating that they do not affect topology and suggesting th at this may be universal for tryptophans in membrane proteins. In contrast, we could demonstrate that lysines clearly prefer to stay on the cis-side o f the membrane, in agreement with the positive-inside rule. A statistical a nalysis focusing on interfacially localized residues showed that in single- spanning membrane proteins lysines are indeed located on the inside, while tryptophans are preferentially localized at the outer interface. Since our experimental results show that the latter is not due to a topology-determin ing role, we propose instead that tryptophans fulfill a functional role as interfacially anchoring residues on the trans-side of the membrane.