The intestinal guanylyl cyclase-C (GC-C) was originally identified as an Escherichiacoli heat-stable enterotoxin (STa) receptor. STa stimulates GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin, causing severe diarrhea. To investigate the interactions of the endogenous and bacterial ligands with GC-C, we designed and characterized a soluble and properly folded fragment of the extracellular ligand-binding domain of GC-C. The membrane-bound guanylyl cyclases exhibit a single transmembrane spanning helix and a globularly folded extracellular ligand-binding domain that comprises about 410 of 1050 residues. Based on the crystal structure of the dimerized-binding domain of the guanylyl cyclase-coupled atrial natriuretic peptide receptor and a secondary structure-guided sequence alignment, we generated a model of the extracellular domain of GC-C comprised of two subdomains. Mapping of mutational and cross-link data onto this structural model restricts the ligand-binding region to the membrane proximal subdomain. We thus designed miniGC-C, a 197 amino acid fragment that mimics the ligand-binding membrane proximal subdomain. Cloning, expression and spectroscopic studies reveal miniGC-C to be a soluble and properly folded protein with a distinct secondary and tertiary structure. MiniGC-C binds STa with nanomolar affinity.

Libraries of random peptides displayed on the surface of filamentous phages are a valuable source for biospecific ligands. However, their successful use can be hindered by a disproportionate representation of different phage clones and fluctuation of their composition that arises during phage reproduction, which have potential to affect efficiency of selection of clones with an optimal binding. Therefore, there is a need to develop phage display libraries with extended and varied repertoires of displayed peptides. In this work, we compared the complexity, evolution and representation of two phage display libraries displaying foreign octamers and nonamers in 4000 copies as the N-terminal part of the major coat protein pVIII of phage fd–tet (landscape libraries). They were obtained by replacement of amino acids 2–4 and 2–5 of pVIII with random octa- and nonamers, respectively. Statistical analysis of the libraries revealed their dramatic censoring and evolution during amplification. Further, a survey of both libraries for clones that bind common selectors revealed the presence of different non-overlapping families of target-specific clones in each library justifying the concept that different landscape libraries cover different areas of a sequence space.

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO) contains three disulphide bridges linking residues C⁴¹XXC⁴⁴, C¹⁵⁵XXC¹⁵⁸, C¹⁷³XXXXC¹⁷⁸. To get information on the role played by these cross-links in determining the structural and functional properties of the protein, we performed site-directed mutagenesis on Cys residues and investigated the changes in folding, stability and functional features of the mutants and analysed the results with computational analysis. The reductase activity of SsPDO and its mutants was evaluated by insulin and thioredoxin reductase assays also coupled with peroxiredoxin Bcp1 of S. solfataricus. The three-dimensional model of SsPDO was constructed and correlated with circular dichroism data and functional results. Biochemical analysis indicated a key function for the redox site constituted by Cys155 and Cys158. To discriminate between the role of the two cysteine residues, each cysteine was mutagenised and the behaviour of the single mutants was investigated elucidating the basis of the electron-shuffling mechanism for SsPDO. Finally, cysteine pK values were calculated and the accessible surface for the cysteine side chains in the reduced form was measured, showing higher reactivity and solvent exposure for Cys155.

Computational prediction of signal peptides (SPs) and their cleavage sites is of great importance in computational biology; however, currently there is no available method capable of predicting reliably the SPs of archaea, due to the limited amount of experimentally verified proteins with SPs. We performed an extensive literature search in order to identify archaeal proteins having experimentally verified SP and managed to find 69 such proteins, the largest number ever reported. A detailed analysis of these sequences revealed some unique features of the SPs of archaea, such as the unique amino acid composition of the hydrophobic region with a higher than expected occurrence of isoleucine, and a cleavage site resembling more the sequences of gram-positives with almost equal amounts of alanine and valine at the position-3 before the cleavage site and a dominant alanine at position-1, followed in abundance by serine and glycine. Using these proteins as a training set, we trained a hidden Markov model method that predicts the presence of the SPs and their cleavage sites and also discriminates such proteins from cytoplasmic and transmembrane ones. The method performs satisfactorily, yielding a 35-fold cross-validation procedure, a sensitivity of 100% and specificity 98.41% with the Matthews’ correlation coefficient being equal to 0.964. This particular method is currently the only available method for the prediction of secretory SPs in archaea, and performs consistently and significantly better compared with other available predictors that were trained on sequences of eukaryotic or bacterial origin. Searching 48 completely sequenced archaeal genomes we identified 9437 putative SPs. The method, PRED-SIGNAL, and the results are freely available for academic users at http://bioinformatics.biol.uoa.gr/PRED-SIGNAL/ and we anticipate that it will be a valuable tool for the computational analysis of archaeal genomes.

Engineering of glycosidases with efficient transglycosidases activity is an alternative to glycosyltransferases or glycosynthases for the synthesis of oligosaccharides and glycoconjugates. However, the engineering of transglycosidases by directed evolution methodologies is hampered by the lack of efficient screening systems for sugar-transfer activity. We report here the development of digital imaging-based high-throughput screening methodology for the directed evolution of glycosidases into transgalactosidases. Using this methodology, we detected transglycosidase mutants in intact Escherichia coli cells by digital imaging monitoring of the activation of non- or low-hydrolytic mutants by an acceptor substrate. We screened several libraries of mutants of β-glycosidase from Thermus thermophilus using this methodology and found variants with up to a 70-fold overall increase in the transglycosidase/hydrolysis activity ratio. Using natural disaccharide acceptors, these transglycosidase mutants were able to synthesise trisaccharides, as a mixture of two regioisomers, with up to 76% yield.

Transcriptional activators that respond to ligands with no cellular targets are powerful tools that can confer regulated expression of a transgene in almost all biological systems. In this study, we altered the ligand-binding specificity of the human estrogen receptor (hER) so that it would recognize a non-steroidal synthetic compound with structural similarities to the phytoestrogen resveratrol. For this purpose, we performed iterative rounds of site-specific saturation mutagenesis of a fixed set of ligand-contacting residues and subsequent random mutagenesis of the entire ligand-binding domain. Selection of the receptor mutants and quantification of the interaction were carried out by exploiting a yeast two-hybrid system that reports the ligand-dependent interaction between hER and steroid receptor coactivator-1 (SRC-1). The screen was performed with a synthetic ligand (CV3320) that promoted growth of the reporter yeast strain to half maximal levels at a concentration of 3.7 µM. The optimized receptor mutant (L384F/L387M/Y537S) showed a 67-fold increased activity to the synthetic ligand CV3320 (half maximal yeast growth at 0.055 µM) and a 10-fold decreased activity to 17ß-estradiol (E2; half maximal yeast growth at 4 nM). The novel receptor-ligand pair partially fulfills the requirements for a specific ‘gene switch’ as it responds to concentrations of the synthetic ligand which do not activate the wildtype receptor. Due to its residual responsiveness to E2 at concentrations (4 nM) that might occur in vivo, further improvements have to be performed to render the system applicable in organisms with endogenous E2 synthesis.