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.

The LysR-type transcriptional regulator CbbR controls the expression of the cbb and gap-pgk operons in Xanthobacter flavus, which encode the majority of the enzymes of the Calvin cycle required for autotrophic CO 2 fixation. The cbb operon promoter of this chemoautotrophic bacterium contains three potential CbbR binding sites, two of which partially overlap. Site-directed mutagenesis and subsequent analysis of DNA binding by CbbR and cbb promoter activity were used to show that the potential CbbR binding sequences are functional. Inverted repeat IR 1 is a high-affinity CbbR binding site. The main function of this repeat is to recruit CbbR to the cbb operon promoter. In addition, it is required for negative autoregulation of cbbR expression. IR 3 represents the main low-affinity binding site of CbbR. Binding to IR 3 occurs in a cooperative manner, since mutations preventing the binding of CbbR to IR 1 also prevent binding to the low-affinity site. Although mutations in IR 3 have a negative effect on the binding of CbbR to this site, they result in an increased promoter activity. This is most likely due to steric hindrance of RNA polymerase by CbbR since IR 3 partially overlaps with the ؊35 region of the cbb operon promoter. Mutations in IR 2 do not affect the DNA binding of CbbR in vitro but have a severe negative effect on the activity of the cbb operon promoter. This IR 2 binding site is therefore critical for transcriptional activation by CbbR.Xanthobacter flavus is a chemoautotrophic bacterium which uses the Calvin cycle to assimilate carbon dioxide (9, 12). The energy to drive carbon dioxide fixation is provided by the oxidation of compounds such as methanol, formate, and H 2 . The majority of the genes encoding the Calvin cycle enzymes constitute three transcriptional units: the cbb and gap-pgk operons and the tpi gene. The cbb operon encodes the key enzymes of the Calvin cycle, ribulose bisphosphate carboxylase/ oxygenase and phosphoribulokinase, and in addition a number of enzymes required for the regeneration of ribulose bisphosphate. Glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase are encoded by the gap-pgk operon and play a role in both the Calvin cycle and glycolysis. The tpi gene encodes triosephosphate isomerase (10,11,13,14,24). During heterotrophic growth on, for instance, succinate, the cbb operon is not expressed and the gap-pgk operon is transcribed at a low constitutive level. A transition from heterotrophic to autotrophic growth is accompanied by a rapid induction of the cbb operon and a superinduction of the gap-pgk operon (11,14). The first two genes of the cbb operon encode the CO 2 -fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO).The induction and superinduction of, respectively, the cbb and gap-pgk operons are completely dependent on the presence of the transcriptional regulator CbbR, which is encoded upstream and whose gene is transcribed divergently from the cbb operon (14,25). This transcriptional regulator is encountered in many photoautot...

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Transmembrane (TM) alpha-helical peptides with neutral flanking residues such as tryptophan form highly ordered striated domains when incorporated in gel-state 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers and inspected by atomic force microscopy (AFM) (1). In this study, we analyze the molecular organization of these striated domains using AFM, photo-cross-linking, fluorescence spectroscopy, nuclear magnetic resonance (NMR), and X-ray diffraction techniques on different functionalized TM peptides. The results demonstrate that the striated domains consist of linear arrays of single TM peptides with a dominantly antiparallel organization in which the peptides interact with each other and with lipids. The peptide arrays are regularly spaced by +/-8.5 nm and are separated by somewhat perturbed gel-state lipids with hexagonally organized acyl chains, which have lost their tilt. This system provides an example of how domains of peptides and lipids can be formed in membranes as a result of a combination of specific peptide-peptide and peptide-lipid interactions.