title: Analysis of the role of interfacial tryptophan residues in controlling the topology of membrane proteins.
abstract: Tryptophans have a high affinity for the membrane-water interface and have been suggested to play a role in determining the topology of membrane proteins. We investigated this potential role experimentally, using mutants of the single-spanning Pf3 coat protein, whose transmembrane topologies are sensitive to small changes in amino acid sequence. Mutants were constructed with varying numbers of tryptophans flanking the transmembrane region and translocation was assessed by an in vitro translation/translocation system. Translocation into Escherichia coli inner membrane vesicles could take place under a variety of experimental conditions, with co- or posttranslational assays and proton motive force-dependent or -independent mutants. It was found that translocation can even occur in pure lipid vesicles, under which conditions the tryptophans must directly interact with the lipids. However, under all these conditions tryptophans neither inhibited nor stimulated translocation, demonstrating that they do not affect topology and suggesting that this may be universal for tryptophans in membrane proteins. In contrast, we could demonstrate that lysines clearly prefer to stay on the cis-side of the membrane, in agreement with the positive-inside rule. A statistical analysis 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-determining role, we propose instead that tryptophans fulfill a functional role as interfacially anchoring residues on the trans-side of the membrane.
authors.affiliation: Department of Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, The Netherlands. a.n.j.a.ridder@chem.uu.nl
authors.forename: A N
authors.initials: AN
authors.lastname: Ridder
authors.forename: S
authors.initials: S
authors.lastname: Morein
authors.forename: J G
authors.initials: JG
authors.lastname: Stam
authors.forename: A
authors.initials: A
authors.lastname: Kuhn
authors.forename: B
authors.initials: B
authors.lastname: de Kruijff
authors.forename: J A
authors.initials: JA
authors.lastname: Killian
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002213:Capsid / Q000737:chemistry* / Q000378:metabolism; D036022:Capsid Proteins; D002462:Cell Membrane / Q000737:chemistry; D004926:Escherichia coli; D008239:Lysine; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D017105:Pseudomonas Phages / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan
chemical_list: D036022:Capsid Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C084332:major coat protein, Pseudomonas phage Pf3; D014364:Tryptophan; D008239:Lysine