Development of new hCaM-Alexa Fluor® biosensors for a wide range of ligands.

Eight new fluorescent biosensors of human calmodulin (hCaM) using Alexa Fluor® 350, 488, 532, and 555 dyes were constructed. These biosensors are thermodynamically stable, functional, and highly sensitive to ligands of the CaM. They resolve the problem of CaM ligands with similar spectroscopic properties to the intrinsic and extrinsic fluorophores of other biosensors previously reported. Additionally, they can be used in studies of protein-protein interaction through Förster resonance energy transfer (FRET). The variation in Tm (range 78.07-81.47 °C; 79.05 to WT) is no larger than two degrees in all cases in regards to CaM WT. The Kds calculated with all biosensors for CPZ and BIMI (a new inhibitor of CaM) are in the range of 0.45-1.86 and 0.69-1.54 µm respectively. All biosensors retain their ability to activate Calcineurin about 70%. Structural models built "in silico" show their possible conformation taking the fluorophores in protein thus we can predict system stability. Finally, these new biosensors represent a biotechnological development applied to an analytical problem, which aims to determine accurately the affinity of inhibitors of CaM without possible interference, to be put forward as possible drugs related to CaM.

Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1µM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins.

Identification and evaluation of inhibitors of the EhGEF1 protein from Entamoeba histolytica.

The Dbl family of guanine nucleotide exchange factors (GEFs) is made up of a vast array of members that participate in the activation of the Rho family of small GTPases. Dbl-family proteins promote the exchange of guanosine diphosphate/guanosine triphosphate (GDP/GTP) in their target molecules, resulting in the activation of a variety of signaling pathways involved in diverse cellular events, such as actin-cytoskeleton remodeling, cellular invasion, cell movement, and other functions. It has been reported that members of the Dbl family have important roles in several cellular events in Entamoeba histolytica. These include activation of the actin cytoskeleton, cytokinesis, capping, uroid formation, cellular proliferation, erythrophagocytosis, cell migration, and chemotaxis. Here, we report the identification and testing of inhibitors of the E. histolytica guanine nucleotide exchange factor 1 (EhGEF1) protein (the research compounds 2BYRF, 2BY05, 2BYT6, 2BYLX, and 2BYPD), which decreased the in vitro ability of the protein to exchange GDP/GTP at its target GTPases, EhRacG and EhRho1, by 14.9-85.2%. Interestingly, the drug 1,1'-(1,2-phenylene)-bis-(1H-pyrrole-2,5-dione), which completely inhibits the GEF activity of the Trio protein in human cells, decreases the GEF activity of the EhGEF1 protein on the EhRacG and EhRho1 GTPases by 55.7% and 3.2%, respectively. The identification and evaluation of such inhibitors opens up the possibility of obtaining a new pharmacological tool to study the function of amoeba GEF proteins, their roles in various Rho GTPase-mediated signaling pathways, and the repercussions of modulating their activities with respect to several mechanisms related to E. histolytica pathogenesis.

Entamoeba histolyticaEhGEF1 structure and mutational analysis: New specific residues critical for function.

This paper reports the EhGEF1-EhRacG and EhGEF1-EhRho1 molecular complexes from Entamoeba histolytica. The not conserved amino acids Gln201,Tyr299, Gln302, Lys312, Asn313, Phe314 and Ile324 were localized, by means of an in silico computational analysis, at the interface of the exposed face from the DH domain of EhGEF1, which are important to establish the contact with its target GTPases EhRacG and EhRho1. Functional studies of nucleotide exchange of Phe314Ala mutant showed a decrement of 80% on EhRacG GTPase; in contrast the Ile324Ala mutant exhibited a reduction of 77%, specifically on EhRho1; meanwhile the Gln302Ala mutant showed a reduction of approximately 50% on the exchange activity for both GTPases. Moreover, the functional studies of the protein EhGEF1 mutants in the conserved residues Thr194Ala, Asn366Ala and Glu367Ala indicated that contrary to what has been reported for other systems, the mutation of these residues did not alter considerably its catalytic activity.

ENDOR structural characterization of a catalytically competent acylenzyme reaction intermediate of wild-type TEM-1 beta-lactamase confirms glutamate-166 as the base catalyst.

The catalytically competent active-site structure of a true acylenzyme reaction intermediate of TEM-1 beta-lactamase formed with the kinetically specific spin-labeled substrate 6-N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carboxyl)-penicillanic acid isolated under cryoenzymologic conditions has been determined by angle-selected electron nuclear double resonance (ENDOR) spectroscopy. Cryoenzymologic experiments with use of the chromophoric substrate 6-N-[3-(2-furanyl)-propen-2-oyl]-penicillanic acid showed that the acylenzyme reaction intermediate could be stabilized in the -35 to -75 degrees C range with a half-life suitably long to allow freeze-quenching of the reaction species for ENDOR studies while a noncovalent Michaelis complex could be optically identified at temperatures only below -70 degrees C. The wild-type, Glu166Asn, Glu240Cys, and Met272Cys mutant forms of the mature enzyme were overexpressed in perdeuterated minimal medium to allow detection and assignment of proton resonances specific for the substrate and chemically modified amino acid residues in the active site. From analysis of the dependence of the ENDOR spectra on the setting of the static laboratory magnetic field H0, the dipolar contributions to the principal hyperfine coupling components were estimated to calculate the separations between the unpaired electron of the nitroxyl group and isotopically identified nuclei. These electron-nucleus distances were applied as constraints to assign the conformation of the substrate in the active site and of amino acid side chains by molecular modeling. Of special interest was that the ENDOR spectra revealed a water molecule sequestered in the active site of the acylenzyme of the wild-type protein that was not detected in the deacylation impaired Glu166Asn mutant. On the basis of the X-ray structure of the enzyme, the ENDOR distance constraints placed this water molecule within hydrogen-bonding distance to the carboxylate side chain of glutamate-166 as if it were poised for nucleophilic attack of the scissile ester bond. The ENDOR results provide experimental evidence of glutamate-166 in its functional role as the general base catalyst in the wild-type enzyme for hydrolytic breakdown of the acylenzyme reaction intermediate of TEM-1 beta-lactamase.

Overexpression and biosynthetic deuterium enrichment of TEM-1 beta-lactamase for structural characterization by magnetic resonance methods.

An expression system has been developed that allows high levels of production of TEM-1 beta-lactamase with ease of biosynthetic incorporation of nuclear isotopes. The gene for mature TEM-1 beta-lactamase fused to the leader sequence of the ompA protein was subcloned into the pET-24a(+) vector by introduction of an NdeI restriction site at the first codon of the fused genes and transformed into Escherichia coli BL21 (DE3) cells. With protein induction at 25 degrees C supported by LB medium supplemented with osmolytes (300 mM sucrose and 2.5 mM betaine), the extracellular, mature form of wild-type TEM-1 beta-lactamase was recovered at a level of 140 mg/L. The production level of E166N, E240C, E104C, and M272C mutants depended on the mutation but was invariably higher than reported by others for expression systems of the wild-type enzyme. Comparison of different carbon sources on the efficiency of biosynthetic incorporation of covalent deuterium showed maximal (90%) incorporation with minimal medium containing 99% (2)H(2)O and sodium d(3)-acetate (99 atom% (2)H). The yield of deuterium-enriched wild-type enzyme was 80 mg/L with yields for mutants proportionally reduced. The high level of protein deuteration achieved with this system allowed detection of the hyperfine coupling between the paramagnetic nitroxyl group of a spin-labeled penicillin substrate and hydrogens on the penicillin moiety in a cryokinetically isolated acylenzyme reaction intermediate because of the decrease in overlapping resonances of active site residues. The overexpression system is readily adaptable for other target proteins and facilitates studies requiring large quantities of protein in isotopically enriched forms.

Mutagenesis of beta-V198 in the F1-ATPase of yeast Saccharomyces cerevisiae and its role in binding nucleotide.

Residue beta-V198 of the yeast mitchondrial F1-ATPase abuts the P-loop motif and the side chain is within 3.8 A of the nucleotide as shown in the crystal structure of the bovine ATPase [J. P. Abrahams, A. G. W. Leslie, R. Lutter, and J. E. Walker (1984) Nature 370,621-628]. This study has made and analyzed 17 replacements of V198 to understand the importance of the side chain in the nucleotide binding site. In addition, a suppressor of V198S, beta-L390F, was studied in the presence of various replacements at position 198. In vivo and in vitro analyses indicate that the Val side chain is critical for forming a stable and active enzyme. Biochemical analysis of mitochondria isolated from the mutant strains indicates that amino acids with hydrophobic side chains are the most effective replacements. In addition, size is important, but a large side chain can be largely compensated for until the size reaches that of the Phe and Trp. A methyl group is the minimal side chain necessary for function, as the beta-subunit is not stable in vivo with Gly at position 198. These results indicate that V198 forms critical hydrophobic interactions with the adenine ring of the nucleotide.

Intragenic suppressors of P-loop mutations in the beta-subunit of the mitochondrial ATPase in the yeast Saccharomyces cerevisiae.

Three intragenic second-site suppressors, P353L, T237I, and L390F, were identified that suppressed two mutations in, and one adjacent to, the P-loop in the beta-subunit of the yeast F1-ATPase. The crystal structure of bovine F1-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628) shows that these suppressor residues are located in the nucleotide-binding domain. Specific hypotheses have been formulated that suggest the conformational coupling of the P-loop with the suppressor sites. P353L is in a "catch" region, which forms unique interactions with the gamma-subunit in the three different conformational states of the catalytic site. The identification of this suppressor mutation demonstrates genetically that the catch region is conformationally coupled to the P-loop. T237I is shown to interact with Lys-209, which occurs just after the P-loop. This suggests that this interaction changes the conformation of the P-loop to suppress the initial mutation. L390F interacts with Ala-181, which is adjacent to the P-loop. The mechanism of this suppression is suggested to occur through the interactions of L390F with Ala-181. These results identify critical interactions that modulate the structure of the P-loop and thus the biochemistry of the enzyme.