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Highlights • Homotypic TMD interfaces identified by different techniques share strong similarities. • The GxxxG motif is the feature most strongly associated with interfaces. • Other features include conservation, polarity, coevolution, and depth in the membrane • The role of each of each feature strongly depends on the individual protein. • Machine-learning helps predict interfaces from evolutionary sequence data, Interactions between their transmembrane domains (TMDs) frequently support the assembly of single-pass membrane proteins to non-covalent complexes. Yet, the TMD-TMD interactome remains largely uncharted. With a view to predicting homotypic TMD-TMD interfaces from primary structure, we performed a systematic analysis of their physical and evolutionary properties. To this end, we generated a dataset of 50 self-interacting TMDs. This dataset contains interfaces of nine TMDs from bitopic human proteins (Ire1, Armcx6, Tie1, ATP1B1, PTPRO, PTPRU, PTPRG, DDR1, and Siglec7) that were experimentally identified here and combined with literature data. We show that interfacial residues of these homotypic TMD-TMD interfaces tend to be more conserved, coevolved and polar than non-interfacial residues. Further, we suggest for the first time that interface positions are deficient in β-branched residues, and likely to be located deep in the hydrophobic core of the membrane. Overrepresentation of the GxxxG motif at interfaces is strong, but that of (small)xxx(small) motifs is weak. The multiplicity of these features and the individual character of TMD-TMD interfaces, as uncovered here, prompted us to train a machine learning algorithm. The resulting prediction method, THOIPA (www.thoipa.org), excels in the prediction of key interface residues from evolutionary sequence data.

Single-span membrane proteins take part in biological processes. In many cases their function has been shown to require dimerization via their transmembrane domains (TMDs). Research into the dimerization of TMDs has revealed sequence dependence, driven by a combination of amino acids. The helix interactions of multi-pass membrane proteins are well understood, as analysis of crystal structures shows that helix interfaces tend to contain small, polar and highly conserved residues. Unfortunately, for single-pass proteins structural data is scarce.The aim of this study is to characterize interfaces of TMDs that show strong self-interaction. In one study of this kind, we examine amino acid composition and conservation in order to identify patterns characteristic of interface residues. Our aim is to develop a method to predict the TMD interfaces from sequences alone.We used the ToxR assay in Escherichia coli to measure TMD homodimerization. In this assay, dimerization driven by TMDs results in the transcriptional activation of ctx promoter, inducing the expression of a reporter enzyme. The amount of reporter enzyme activity is a measure of the strength of the dimerization.Here, we present the scanning mutagenesis followed by ToxR analysis of 10 TMDs with strong self-interaction. Mutations were made to all amino acids within the TM helix. Some TMD interfaces were found to contain sequence patterns previously associated with parallel helix interactions, such as the GxxxG motif. However, in other cases motif searching would not have identified the interface, and the key residues in the interactions were diverse. The conservation of each amino acid in each TMD was calculated based on amino acid alignments against homologs. Preliminary analysis shows that amino acid conservation is a good indicator for the interface of some, but not all interacting TMDs.

Cytochrome P450 enzymes catalyse a wide variety of reactions, including the hydroxylation and epoxidation of C C bonds, and dealkylation reactions. There is high interest in these reactions for biotechnology and pharmaceutical processes. Many P450s require membrane surroundings and have substrates that do not cross biological membranes. To circumvent these obstacles, CYP106A2 from Bacillus megaterium was expressed on the outer membrane of Escherichia coli cells by Autodisplay. Exposure on the surface was confirmed by a protease accessibility test and flow cytometry after immunolabelling. HPLC assays showed that 0.5 ml of cells displaying the enzyme (OD 578 = 6) converted 9.13 μmol of deoxycorticosterone to 15β-OH-deoxycorticosterone within 1 h. Imipramine and abietic acid were also accepted as substrates. The number of active enzyme molecules per cell was calculated to be 20,000. Surprisingly, surface-exposed CYP106A2 was active in E. coli BL21 without the external addition of the heme group. However, when CYP106A2 was expressed on the surface of an E. coli strain lacking the TolC channel protein (JW5503), enzymatic activity was almost completely abolished. The activity of CYP106A2 on the surface of E. coli JW5503 could be restored by the external addition of the heme group. This suggests, as has been reported before, that E. coli uses a TolC-dependent mechanism to export heme into the growth media, where it can be scavenged by a surface-displayed apoenzyme. Our results indicate that Autodisplay enables the functional surface display of P450 enzymes and provides a new platform to access their synthetic potential.

Protein-protein interactions are the fundamental driving force of numerous cellular processes and cell signaling pathways. Characterizing whether proteins interact as dimers, trimers, or higher oligomers is essential to understanding these interactions. Several microscopy and advanced imaging techniques relying on Forster resonance energy transfer (FRET) between identical fluorophores (homo-FRET) have been developed to estimate protein stoichiometry. The increased FRET in oligomers is detected by measuring depolarization or emission time. Homo-FRET methods have a strong advantage in requiring only a single fluorophore, greatly simplifying sample preparation in comparison to conventional hetero-FRET methods. However, most homo-FRET methods require sophisticated imaging equipment, and both theoretical models and applications have been restricted to the study of membrane-bound proteins. Using a simple bulk homo-FRET and laser photobleaching approach, we demonstrate the feasibility of characterizing the oligomerization state of an interacting protein in-vitro. To simulate oligomers in a proof of concept, we constructed an extensive repertoire of fusion proteins with 1-6 consecutive green fluorescent protein (GFP) domains. We show how the resulting homo-FRET (measurable via steady-state anisotropy or fluorescence polarization) is proportional to the oligomerization state of proximal GFP domains. For the first time, this is demonstrated with soluble proteins. In both membrane and soluble proteins, oligomerization increases FRET and therefore anisotropy. However for soluble proteins oligomerization also slows fluorophore rotation, leading to a size-dependent decrease in anisotropy. Through gradual photobleaching of fluorophores these two effects can be distinguished, and the oligomerization state of a labeled protein of interest can be estimated. We therefore show how the theoretical framework developed for membrane proteins needs to be adjusted to account for this additional degree of freedom in soluble proteins. Overall, bulk homo-FRET and laser photobleaching is a promising method to determine the oligomerization state of a protein of interest, which can have a low concentration (0.1-0.5 µM) and needs only a single fluorescent label. The method requires only a photometer or microplate reader capable of measuring steady-state anisotropy.

Enhanced detoxification is the major mechanism responsible for pyrethroid resistance in Chinese populations of Helicoverpa armigera. Previous work has shown that enhanced oxidation contributes to resistance in the fenvalerate-selected Chinese strain, YGF. The current study provides evidence that enhanced hydrolysis by esterase isozymes also contributes to the resistance in this strain. The average esterase activity of third instar YGF larvae was 1.9-fold compared with that of a susceptible SCD strain. Much of this difference was attributed to isozymes at two zones which hydrolysed the model carboxylester substrate 1-naphthyl acetate and also a 1-naphthyl analogue of fenvalerate. A preparation enriched for enzymes migrating to one of these zones from YGF was shown to hydrolyse fenvalerate with a specific activity of about 2.9 nmol/min/mg. This material was also matched by mass spectrometry with four putative carboxylesterase genes, all of which clustered within a phylogenetic clade of secreted midgut esterases. Quantitative PCR on these four genes showed several-fold greater expression in tissues of YGF compared to SCD but no differences was found in the number of copies of the genes between the strains.

Tailoring surface properties of degradable polymer scaffolds is key to progress in various tissue engineering strategies. Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate- co -4-hydroxybutyrate) thin films were modified by low pressure ammonia plasma, low pressure water vapour plasma, or immersion in a sodium hydroxide solution to elaborate means to control the cell–matrix adhesion of human umbilical cord vein endothelial cells grown on these materials. Fibronectin (FN) heteroexchange and cell adhesion were correlated to the physicochemical characteristics of the modified polymer surfaces which were investigated by X-ray photoelectron spectroscopy (XPS), scanning force microscopy (SFM), electrokinetic measurements, and contact angle measurements. All treatments increased the hydrophilicity of the polymer samples, which could be accounted to newly created amine or carboxyl functionalities for ammonia plasma or water vapour plasma treatments, respectively, and ester hydrolysis for treatments with alkaline aqueous solutions. Main features of cell adhesion and FN reorganisation—evaluated after 1 h and after 5 days—could be attributed to the anchorage strength of pre-coated FN layers at the polymer surface, which was, in turn found to be triggered by the type of modification applied. In line with earlier studies referring to different materials cell adhesion and matrix reorganisation were shown to be sensitively controlled through the physicochemical profile of poly(hydroxybutyrate) surfaces.

Using genetic reporter assays, significant self-interaction has been discovered in over 50 transmembrane (TM) helices from single-pass membrane proteins. To understand the biological functions of these TM interactions, which include signal transfer across the membrane in cancer-related pathways, it is necessary to construct accurate structural models of the TM oligomer. However in most cases this is not possible because the oligomerization state (stoichiometry) is unknown. It is often assumed that the helices form a dimer, though biologically relevant TM oligomers include dimers, trimers, and pentamers, as exemplified by GpA, HLA II Ii, and phospholamban, respectively.To characterize the stoichiometry of interacting single-pass membrane proteins, we are establishing a novel method based on Forster resonance energy transfer between identical fluorophores (homo-FRET). In order to allow the rapid bacterial expression and the purification of a wide range of fluorophore-coupled TM helices, we have developed TM fusion proteins with green fluorescent protein (GFP) and affinity tags. The measured homoFRET (via steady-state anisotropy) is proportional to both the affinity of the helices and the oligomerization state. Using only a fluorescence microplate reader, we demonstrate significant homo-FRET-coupling for GFP fusion proteins. Theoretical studies have shown that gradual photobleaching should yield a pattern of polarization that depends on the oligomerization state. High-profile experimental studies have confirmed this in-vivo using advanced microscopy. We show for the first time that the “fingerprint”-like curves after laser photobleaching can be achieved in-vitro with relatively simple equipment, allowing us to differentiate between oligomerization states. This demonstrates the feasibility of a rapid homo-FRET assay to determine the stoichiometry of transmembrane proteins in liposomes.

Corrigendum to “Gene identification and proteomic analysis of the esterases of the cotton bollworm, Helicoverpa armigera” [Insect Biochem. Mol. Biol. 40 (2010) 1e16] Mark G. Teese , Peter M. Campbell , Colin Scott , Karl H.J. Gordon , Adam Southon , Daniel Hovan , Charles Robin , Robyn J. Russell , John G. Oakeshott a,* CSIRO Entomology, GPO Box 1700, Canberra ACT 2601, Australia Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia Department of Genetics, The University of Melbourne, Vic. 3010, Australia