Improved Electrophoretic Separation to Assist the Monitoring of Bcl-xL Post-Translational Modifications.

Bcl-xL is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-xL shows unique eligibility to deamidation, a time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn→Asp/IsoAsp conversions or Glu→Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-xL generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-xL remains eligible for regulation by phosphorylation.

Establishment of a high throughput-screening system for nucleoside deoxyribosyltransferase II mutant enzymes with altered substrate specificity.

Nucleoside deoxyribosyltransferase II (NDT) catalyzes the transglycosylation reaction of the 2'-deoxyribose moiety between purine and/or pyrimidine bases and has been widely used in the synthesis of nucleoside analogs. The high specificity of NDT for 2'-deoxyribose limits its applications. Because 2'C- and/or 3'C-modified nucleosides have been widely used as antiviral or antitumour agents, improving the activity of NDT towards these modified nucleosides by protein engineering is an area of interest to the pharmaceutical industry. NDT engineering is hindered by a lack of effective screening methods. This study developed a high-throughput screening system, which was established by nucleoside deoxyribosyltransferase II-cytidine deaminase co-expression, indophenol colorimetric assay and whole-cell catalysis. A high-throughput screening system for NDT was established for the first time. This system can be applied to detect NDT-specific activity for a variety of cytidine analogs with glycosyl and base modifications, such as 5-aza-2'-deoxycytidine, 2',3'-dideoxycytidine, cytosine-ß-d-arabinofuranoside. In this study, we adopted the semi-rational design of NDT and constructed a mutant library of NDT from Lactobacillus helveticus (LhNDT) by site-saturation mutagenesis. Over 600 mutants were screened, and a variant with up to a 5.2-fold higher conversion rate of 2',3'-dideoxyinosine was obtained.

Proteome-wide onco-proteogenomic somatic variant identification in ER-positive breast cancer.

BACKGROUND: Recent advances in mass spectrometric instrumentation and bioinformatics have critically contributed to the field of proteogenomics. Nonetheless, whether that integrative approach has reached the point of maturity to effectively reveal the flow of genetic variants from DNA to proteins still remains elusive. The objective of this study was to detect somatically acquired protein variants in breast cancer specimens for which full genome and transcriptome data was already available (BASIS cohort). METHODS: LC-MS/MS shotgun proteomic results of 21 breast cancer tissues were coupled to DNA sequencing data to identify variants at the protein level and finally were used to associate protein expression with gene expression levels. RESULTS: Here we report the observation of three sequencing-predicted single amino acid somatic variants. The sensitivity of single amino acid variant (SAAV) detection based on DNA sequencing-predicted single nucleotide variants was 0.4%. This sensitivity was increased to 0.6% when all the predicted variants were filtered for MS "compatibility" and was further increased to 2.9% when only proteins with at least one wild type peptide detected were taken into account. A correlation of mRNA abundance and variant peptide detection revealed that transcripts for which variant proteins were detected ranked among the top 6.3% most abundant transcripts. The variants were detected in highly abundant proteins as well, thus establishing transcript and protein abundance and MS "compatibility" as the main factors affecting variant onco-proteogenomic identification. CONCLUSIONS: While proteomics fails to identify the vast majority of exome DNA variants in the resulting proteome, its ability to detect a small subset of SAAVs could prove valuable for precision medicine applications.

Design and assessment of an active anti-epidermal growth factor receptor (EGFR) single chain variable fragment (ScFv) with improved solubility.

ScFv is emerging as a therapeutic alternative to the full-length monoclonal antibodies due to its small size and low production cost, but its low solubility remains a limiting factor toward wider use. Here, we increased the solubility of an Anti-epidermal growth factor receptor ScFv (Anti-EGFR ScFv) by attaching, a short 12-residue solubility enhancing peptide (SEP) tag at its C terminus. We first estimated the solubility increase by running 500-ns Brownian dynamics (BD) simulations. We then experimentally evaluated the predictions by producing recombinant Anti-EGFR ScFv with and without a SEP tag (called C9R) in E. coli. At 20 °C, ∼85% of Anti-EGFR ScFv-C9R expressed in the soluble fraction, whereas all of the Anti-EGFR ScFv remained in the insoluble fraction. The total yield of Anti-EGFR ScFv-C9R was 17.15 mg which was ∼3 times higher than that of Anti-EGFR ScFv refolded from the insoluble fraction. Static and dynamic light scattering demonstrated the higher solubility of the purified Anti-EGFR ScFv-C9R, and Circular Dichroism (CD) indicated its high thermal stability, whereas the untagged protein aggregated at 37 °C and pH 6. Finally, the binding activity of Anti-EGFR ScFv-C9R to EGFR was confirmed by surface plasmon resonance (SPR). Altogether, these results illustrate the improved biophysical and biochemical characteristics of Anti-EGFR ScFv-C9R and emphasize the potentials of SEP-tags for enhancing the solubility of aggregation-prone antibody fragments.

A chemometric approach for characterization of serum transthyretin in familial amyloidotic polyneuropathy type I (FAP-I) by electrospray ionization-ion mobility mass spectrometry.

In this study, we describe a chemometric data analysis approach to assist in the interpretation of the complex datasets from the analysis of high-molecular mass oligomeric proteins by ion mobility mass spectrometry (IM-MS). The homotetrameric protein transthyretin (TTR) is involved in familial amyloidotic polyneuropathy type I (FAP-I). FAP-I is associated with a specific TTR mutant variant (TTR(Met30)) that can be easily detected analyzing the monomeric forms of the mutant protein. However, the mechanism of protein misfolding and aggregation onset, which could be triggered by structural changes in the native tetrameric protein, remains under investigation. Serum TTR from healthy controls and FAP-I patients was purified under non-denaturing conditions by conventional immunoprecipitation in solution and analyzed by IM-MS. IM-MS allowed separation and characterization of several tetrameric, trimeric and dimeric TTR gas ions due to their differential drift time. After an appropriate data pre-processing, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the complex datasets. A group of seven independent components being characterized by their ion mobility profiles and mass spectra were resolved to explain the observed data variance in control and patient samples. Then, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were considered for exploration and classification. Only four out of the seven resolved components were enough for an accurate differentiation. Furthermore, the specific TTR ions identified in the mass spectra of these components and the resolved ion mobility profiles provided a straightforward insight into the most relevant oligomeric TTR proteoforms for the disease.

Role of SH3b binding domain in a natural deletion mutant of Kayvirus endolysin LysF1 with a broad range of lytic activity.

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.

A ribonuclease inhibitor resistant dimer of human pancreatic ribonuclease displays specific antitumor activity.

Human pancreatic ribonuclease (HPR) and bovine seminal ribonuclease (BS-RNase) are members of the RNase A superfamily. HPR is monomeric, whereas BS-RNase is dimeric. BS-RNase has strong antitumor and cytotoxic activities. However, HPR lacks cytotoxic activity as it is inactivated by intracellular cytosolic ribonuclease inhibitor (RI). Earlier, an RI-resistant cytotoxic variant of HPR, termed HPR-KNE was generated which contained three residues Lys7, Asn71 and Glu111 of HPR, known to interact with RI, mutated to alanine. In this study, we have engineered HPR to develop two dimeric RI-resistant molecules having anti-tumor activity. By incorporating two cysteines in HPR and HPR-KNE, we generated disulfide linked dimeric HPR, and a dimer of HPR-KNE, termed as HPR-D and HPR-KNE-D respectively. HPR-KNE-D was resistant towards inhibition by RI, and was found to be highly toxic to a variety of cells. On J774A.1 cells HPR-KNE-D was >375-fold more cytotoxic than HPR, and 15-fold more toxic than HPR-D. Further, on U373 cells HPR-KNE-D was >65-fold more cytotoxic than HPR, and 9-fold more toxic than HPR-D. The study demonstrates that combining dimerization and RI-resistance results in providing potent anti-tumor activity to HPR. The cytotoxic variants of HPR will be useful in designing protein therapeutics with low immunogenicity.

Characterization of a thermostable mutant of Agaricus brasiliensis laccase created by phylogeny-based design.

Laccases are enzymes that oxidize various aromatic compounds, and therefore they have attracted much attention from the standpoints of medical and industrial applications. We previously isolated the cDNA that codes for a laccase isozyme (Lac2a) from the medicinal mushroom Agaricus brasiliensis (Matsumoto-Akanuma et al., Int. J. Med. Mushrooms, 16, 375-393, 2014). In this study, we first attempted heterologous expression of the wild-type laccase using a Pichia pastoris secretory expression system. However, the trial was unsuccessful most likely because the enzyme was too unstable and degraded immediately after production. Therefore, we improved the stability of the laccase by using a phylogeny-based design method. We created a mutant laccase in which sixteen original residues were replaced with those found in the phylogenetically inferred ancestral sequence. The resulting mutant protein was successfully produced using the P. pastoris secretory expression system and then purified. The designed laccase showed catalytic properties similar to those of other fungal laccases. Moreover, the laccase is highly thermally stable at acidic and neutral pH and is also stable at alkaline pH at moderate temperatures. We expect that the laccase will serve as a useful tool for enzymatic polymerization of di-phenolic compounds.

Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding.

The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein. To overcome this major bottleneck, a simple purification strategy for single tryptophan, single cysteine-containing mutant variants of the mouse prion protein is presented, with yields comparable to that of the wild type protein. The protein(s) obtained by this method are correctly folded, with a single reduced cysteine, and the native disulfide bond between residues C178 and C213 intact. The ß-sheet rich oligomers formed from these mutant variant protein(s) are identical to the wild type protein oligomer. The current strategy facilitates sample preparation for a number of high resolution spectroscopic measurements for the prion protein, which specifically require thiol labelling.

Somatic cancer mutations in the MLL1 histone methyltransferase modulate its enzymatic activity and dependence on the WDR5/RBBP5/ASH2L complex.

Somatic missense mutations in the mixed lineage leukemia 1 (MLL1) histone H3K4 methyltransferase are often observed in cancers. MLL1 forms a complex with WDR5, RBBP5, and ASH2L (WRA) which stimulates its activity. The MM-102 compound prevents the interaction between MLL1 and WDR5 and functions as an MLL1 inhibitor. We have studied the effects of four cancer mutations in the catalytic SET domain of MLL1 on the enzymatic activity of MLL1 and MLL1-WRA complexes. In addition, we studied the interaction of the MLL1 mutants with the WRA proteins and inhibition of MLL1-WRA complexes by MM-102. All four investigated mutations had strong effects on the activity of MLL1. R3903H was inactive and S3865F showed reduced activity both alone and in complex with WRA, but its activity was stimulated by the WRA complex. By contrast, R3864C and R3841W were both more active than wild-type MLL1, but still less active than the wild-type MLL1-WRA complex. Both mutants were not stimulated by complex formation with WRA, although no differences in the interaction with the complex proteins were observed. These results indicate that both mutants are in an active conformation even in the absence of the WRA complex and their normal control of activity by the WRA complex is altered. In agreement with this observation, the activity of R3864C and R3841W was not reduced by addition of the MM-102 inhibitor. We show that different cancer mutations in MLL1 lead to a loss or increase in activity, illustrating the complex and tumor-specific role of MLL1 in carcinogenesis. Our data exemplify that biochemical investigations of somatic tumor mutations are required to decipher their pathological role. Moreover, our data indicate that MM-102 may not be used as an MLL1 inhibitor if the R3864C and R3841W mutations are present. More generally, the efficacy of any enzyme inhibitor must be experimentally confirmed for mutant enzymes before an application can be considered.

Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum.

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a "down" state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the α-helices, devoid of ATP, enter the α3ß3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme's hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3ß3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the ßE-catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.

Identification of amino acids related to catalytic function of Sulfolobus solfataricus P1 carboxylesterase by site-directed mutagenesis and molecular modeling.

The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues. [BMB Reports 2016; 49(6): 349-354].

A novel viral RNA helicase with an independent translation enhancement activity.

RNA helicases have not been identified among negative sense RNA viruses. In this study, it is shown that Nonstructural protein (NSs) of Groundnut bud necrosis virus (GBNV) acts as a Mg(2+) - and ATP-dependent bipolar RNA helicase. Biophysical and biochemical analysis of the deletion mutants (NΔ124 NSs, CΔ80 NSs) revealed that both the N- and C-terminal residues are required for substrate binding, oligomerization and helicase activity, but are dispensable for ATPase activity. Interestingly, NSs could enhance the translation of RNA (~ 10-fold) independent of its helicase activity. This is the first report of a RNA helicase from negative strand RNA viruses.

Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example.

Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution.

Mechanistic insights into the phosphatidylinositol binding properties of the pleckstrin homology domain of lamellipodin.

Lamellipodin (Lpd) protein plays an important role in the formation of lamellipodial protrusion which is crucial in actin dynamics, cell polarity and motility. Lpd promotes actin polymerization with the help of members of the Ena/VASP family of actin regulators and tethering them to actin filaments. It is well documented that Lpd protein interacts with the membrane containing phosphatidylinositols through its pleckstrin homology (PH) domain and regulates several cellular functions and cell migration. However, the molecular mechanism that underlies how the PH domain of Lpd specifically gets recruited to phosphatidylinositols remains unclear. To understand their interaction properties, we quantitatively determined the binding parameters of the Lpd-PH domain employing a number of biophysical studies including surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET)-based competitive binding assay and monolayer penetration measurements. Our studies showed that the Lpd-PH domain strongly interacts with PI(3,4)P2 containing liposome without any membrane penetration. Mutational studies demonstrate that the presence of cationic residues within the phosphatidylinositol (PIP) binding site of the Lpd-PH domain is essential in membrane binding. The translocation patterns of the Lpd-PH domain and mutants in platelet-derived growth factor (PDGF) stimulated A549 cells are in good agreement with our in vitro binding measurements. Overall, these studies demonstrate an insight into how the Lpd-PH domain regulates cellular signals in a PI(3,4)P2 dependent manner.

Engineered hypoallergenic variants of osmotin demonstrate hypoallergenicity with in vitro and in vivo methods.

The molecular structure of protein and epitope mapping strategies are required to engineer the epitopes of a protein. In the present study, IgE binding regions of osmotin were identified and mutated to obtain hypoallergenic variant. The three dimensional (3-D) model of osmotin obtained by homology modeling comprised of a characteristic thaumatin-like fold. This model was used to predict IgE binding regions of osmotin. These regions were mutated and three mutant proteins with four mutations (Ma, Mb and Mc) and one with six mutations (Mabc) were expressed and purified to homogeneity. IgE binding of the mutant proteins was evaluated by in vitro studies using patients' sera. Ma, Mb and Mc demonstrated reduction in IgE binding of 73%, 83% and 77%, respectively, whereas Mabc showed complete abrogation of IgE binding. Ma, Mb and Mc showed inhibition of 48%, 44% and 38%, respectively to osmotin, while Mabc showed 24% inhibition at 10 µg with pooled patients' sera. Osmotin reached effective concentration at 50% inhibition (EC50) at 3 ng and none of the mutant proteins reach the EC50 value. The immunological response to mutant proteins was examined in mice. Blood, bronchoalveolar lavage fluid spleen and lung tissue were excised from mice for analysis. The mice treated with mutant proteins showed significant reduction in IgE and IgG1 levels as compared to mice given osmotin (p<0.001). Th2 cytokines level in splenocyte supernatant and BALF of mice given mutant proteins were significantly lower (p<0.001), accompanied with significant reduction in cellular infiltration in lungs (p<0.001). In conclusion, osmotin structure was predicted by homology modeling and IgE binding regions predicted were mutated to obtain a hypoallergenic protein.

Interaction of Kaposi's sarcoma-associated herpesvirus ORF6 protein with single-stranded DNA.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) ORF6 is homologous to the herpes simplex virus 1 (HSV-1) ICP8 and Epstein-Barr virus (EBV) BALF2 proteins. Here, we describe its single-stranded DNA (ssDNA) binding properties. Based on previous findings with ICP8 and BALF2, a 60-amino-acid C-terminal deletion mutant of Orf6 was generated, and the protein was purified to explore the function of the C terminus in ssDNA binding. We showed that full-length ORF6 binds cooperatively to M13 ssDNA, disrupting its secondary structure and extending it to a length equivalent to that of duplex M13 DNA. The width of the ORF6-ssDNA filament is 9 nm, and a 7.3-nm repeat can be distinguished along the filament axis. Fluorescence polarization analysis revealed that the wild-type and C-terminal mutant ORF6 proteins bind equally well to short ssDNA substrates, with dissociation constant (Kd) values of 2.2 × 10(-7)M and 1.5 × 10(-7)M, respectively. These values were confirmed by electrophoretic mobility shift assay (EMSA) analysis, which also suggested that binding by the full-length protein may involve both monomers and small multimers. While no significant difference in affinities of binding between full-length ORF6 and the C-terminal deletion mutant were observed with the short DNAs, binding of the C-terminal mutant protein to M13 ssDNA showed a clear lack of cooperativity as seen by electron microscopy (EM). Incubation of a duplex DNA containing a long single-stranded tail with double-helical ORF6 protein filaments revealed that the ssDNA segment can be enveloped within the protein filament without disrupting the filament structure. IMPORTANCE: This work describes the biochemical characterization of the single-stranded DNA binding protein of KSHV, ORF6, central to viral DNA replication in infected cells. A C-terminal deletion mutant protein was generated to aid in understanding the role of the C terminus in DNA binding. Here we analyze the binding of the wild-type and mutant proteins to short oligomeric and longer genomic ssDNA substrates. Although it is capable of interacting with the short substrates, the inability of mutant ORF6 to form oligomers in solution hindered it from fully covering the long genomic substrates. We previously showed that ORF6 forms long filaments in solution, and we showed here that these can absorb ssDNA without disruption of the filament structure. This work will provide an important basis for future studies by us and/or others.

A high throughput screening assay system for the identification of small molecule inhibitors of gsp.

Mis-sense mutations in the α-subunit of the G-protein, Gsα, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active Gsα and increased levels of cAMP. The aim of this study was to develop an assay system that would allow the identification of small molecule inhibitors specific for the mutant Gsα protein, the so-called gsp oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant Gsα proteins (R201C and R201H). Stable cell lines with equivalent transfected Gsα protein expression that had relatively lower (WT) or higher (R201C and R201H) cAMP levels were generated. These cell lines were used to develop a fluorescence resonance energy transfer (FRET)-based cAMP assay in 1536-well microplate format for high throughput screening of small molecule libraries. A small molecule library of 343,768 compounds was screened to identify modulators of gsp activity. A total of 1,356 compounds with inhibitory activity were initially identified and reconfirmed when tested in concentration dose responses. Six hundred eighty-six molecules were selected for further analysis after removing cytotoxic compounds and those that were active in forskolin-induced WT cells. These molecules were grouped by potency, efficacy, and structural similarities to yield 22 clusters with more than 5 of structurally similar members and 144 singleton molecules. Seven chemotypes of the major clusters were identified for further testing and analyses.

Recombinant production of the amino terminal cytoplasmic region of dengue virus non-structural protein 4A for structural studies.

BACKGROUND: Dengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus belonging to the Flaviviridae family. DENV causes dengue fever, currently the world's fastest-spreading tropical disease. Severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening. There is no specific treatment and no anti-DENV vaccines. Our recent data suggests that the amino terminal cytoplasmic region of the dengue virus non-structural protein 4A (NS4A) comprising amino acid residues 1 to 48 forms an amphipathic helix in the presence of membranes. Its amphipathic character was shown to be essential for viral replication. NMR-based structure-function analysis of the NS4A amino terminal region depends on its milligram-scale production and labeling with NMR active isotopes. METHODOLOGY/PRINCIPAL FINDINGS: This report describes the optimization of a uniform procedure for the expression and purification of the wild type NS4A(1-48) peptide and a peptide derived from a replication-deficient mutant NS4A(1-48; L6E, M10E) with disrupted amphipathic nature. A codon-optimized, synthetic gene for NS4A(1-48) was expressed as a fusion with a GST-GB1 dual tag in E. coli. Tobacco etch virus (TEV) protease mediated cleavage generated NS4A(1-48) peptides without any artificial overhang. Using the described protocol up to 4 milligrams of the wild type or up to 5 milligrams of the mutant peptide were obtained from a one-liter culture. Isotopic labeling of the peptides was achieved and initial NMR spectra were recorded. CONCLUSIONS/SIGNIFICANCE: Small molecules targeting amphipathic helices in the related Hepatitis C virus were shown to inhibit viral replication, representing a new class of antiviral drugs. These findings highlight the need for an efficient procedure that provides large quantities of the amphipathic helix containing NS4A peptides. The double tag strategy presented in this manuscript answers these needs yielding amounts that are sufficient for comprehensive biophysical and structural studies, which might reveal new drug targets.

Structure and biotechnological applications of odorant-binding proteins.

Odorant-binding proteins (OBPs) are small soluble polypeptides found in sensory organs of vertebrates and insects as well as in secretory glands and are dedicated to detection and release of chemical stimuli. OBPs of vertebrates belong to the family of lipocalin proteins, while those of insects are folded into α-helical domains. Both types of architectures are extremely stable to temperature, organic solvents and proteolytic digestion. These characteristics make OBPs suitable elements for fabricating biosensors to be used in the environment, as well as for other biotechnological applications. The affinity of OBPs for small volatile organic compounds is in the micromolar range, and they have broad specificity to a range of ligands. For biotechnological applications, OBPs can be expressed in bacterial systems at low cost and are easily purified. The large amount of information available on their structures and affinities to different molecules should allow the design of specific mutants with desired characteristics and represent a solid base for tailoring OBPs for different applications.