Identification of the native Torpedo californica nicotinic acetylcholine receptor's glycan composition after a multi-step sequential purification method using MALDI-ToF MS.

The Cys-loop pentameric ligand-gated ion channels comprise a dynamic group of proteins that have been extensively studied for decades, yielding a wealth of findings at both the structural and functional levels. The nicotinic acetylcholine receptor (nAChR) is no exception, as it is part of this large protein family involved in proper organismal function. Our efforts have successfully produced a highly pure nAChR in detergent complex (nAChR-DC), enabling more robust studies to be conducted on it, including beginning to experiment with high-throughput crystallization. Our homogeneous product has been identified and extensively characterized with 100% identity using Nano Lc MS/MS and MALDI ToF/ToF for each nAChR subunit. Additionally, the N-linked glycans in the Torpedo californica-nAChR (Tc-nAChR) subunits have been identified. To study this, the Tc-nAChR subunits were digested with PNGase F and the released glycans were analyzed by MALDI-ToF. The MS results showed the presence of high-mannose N-glycan in all native Tc-nAChR subunits. Specifically, the oligommanose population Man8-9GlcNac2 with peaks at m/z 1742 and 1904 ([M + Na]+ ions) were observed.

Novel hydrazone compounds with broad-spectrum antiplasmodial activity and synergistic interactions with antimalarial drugs.

The development of novel antiplasmodial compounds with broad-spectrum activity against different stages of Plasmodium parasites is crucial to prevent malaria disease and parasite transmission. This study evaluated the antiplasmodial activity of seven novel hydrazone compounds (referred to as CB compounds: CB-27, CB-41, CB-50, CB-53, CB-58, CB-59, and CB-61) against multiple stages of Plasmodium parasites. All CB compounds inhibited blood stage proliferation of drug-resistant or sensitive strains of Plasmodium falciparum in the low micromolar to nanomolar range. Interestingly, CB-41 exhibited prophylactic activity against hypnozoites and liver schizonts in Plasmodium cynomolgi, a primate model for Plasmodium vivax. Four CB compounds (CB-27, CB-41, CB-53, and CB-61) inhibited P. falciparum oocyst formation in mosquitoes, and five CB compounds (CB-27, CB-41, CB-53, CB-58, and CB-61) hindered the in vitro development of Plasmodium berghei ookinetes. The CB compounds did not inhibit the activation of P. berghei female and male gametocytes in vitro. Isobologram assays demonstrated synergistic interactions between CB-61 and the FDA-approved antimalarial drugs, clindamycin and halofantrine. Testing of six CB compounds showed no inhibition of Plasmodium glutathione S-transferase as a putative target and no cytotoxicity in HepG2 liver cells. CB compounds are promising candidates for further development as antimalarial drugs against multidrug-resistant parasites, which could also prevent malaria transmission.

Evaluation of the Antioxidant and Anti-Inflammatory Activities and Acute Toxicity of Caco Seed (Chrysobalanus icaco L.) in Murine Models.

Chysobalanus icaco L. (C. icaco) is a plant that is native to tropical America and Africa. It is also found in the southeast region of Mexico, where it is used as food and to treat certain diseases. This study aimed to carry out a phytochemical analysis of an aqueous extract of C. icaco seed (AECS), including its total phenol content (TPC), total flavonoid content (TFC), and condensed tannins (CT). It also aimed to examine the antioxidant and metal-ion-reducing potential of the AECS in vitro, as well as its toxicity and anti-inflammatory effect in mice. Antioxidant and metal-ion-reducing potential was examined by inhibiting DPPH, ABTS, and FRAP. The acute toxicity test involved a single administration of different doses of the AECS (0.5, 1, and 2 g/kg body weight). Finally, a single administration at doses of 150, 300, and 600 mg/kg of the AECS was used in the carrageenan-induced model of subplantar acute edema. The results showed that the AECS contained 124.14 ± 0.32 mg GAE, 1.65 ± 0.02 mg EQ, and 0.910 ± 0.01 mg of catechin equivalents/g dried extract (mg EC/g de extract) for TPC, TFC and CT, respectively. In the antioxidant potential assays, the values of the median inhibition concentration (IC50) of the AECS were determined with DPPH (0.050 mg/mL), ABTS (0.074 mg/mL), and FRAP (0.49 mg/mL). Acute toxicity testing of the AECS revealed no lethality, with a median lethal dose (LD50) value of >2 g/kg by the intragastric route. Finally, for inhibition of acute edema, the AECS decreased inflammation by 55%, similar to indomethacin (59%, p > 0.05). These results demonstrated that C. icaco seed could be considered a source of bioactive molecules for therapeutic purposes due to its antioxidant potential and anti-inflammatory activity derived from TPC, with no lethal effect from a single intragastric administration in mice.

PCR-assisted impedimetric biosensor for colibactin-encoding pks genomic island detection in E. coli samples.

A fast PCR-assisted impedimetric biosensor was developed for the selective detection of the clbN gene from the polyketide synthase (pks) genomic island in real Escherichia coli samples. This genomic island is responsible for the production of colibactin, a harmful genotoxin that has been associated with colorectal cancer. The experimental protocol consisted of immobilizing the designated forward primer onto an Au electrode surface to create the sensing probe, followed by PCR temperature cycling in blank, positive, and negative DNA controls. Target DNA identification was possible by monitoring changes in the system's charge transfer resistance values (Rct) before and after PCR treatment through electrochemical impedance spectroscopy (EIS) analysis. Custom-made, flexible gold electrodes were fabricated using chemical etching optical lithography. A PCR cycle study determined the optimum conditions to be at 6 cycles providing fast results while maintaining a good sensitivity. EIS data for the DNA recognition process demonstrated the successful distinction between target interaction resulting in an increase in resistance to charge transfer (Rct) percentage change of 176% for the positive DNA control vs. 21% and 20% for the negative and non-DNA-containing controls, respectively. Results showed effective fabrication of a fast, PCR-based electrochemical biosensor for the detection of pks genomic island with a calculated limit of detection of 17 ng/µL.

Structure, activity, and substrate selectivity of the Orf6 thioesterase from Photobacterium profundum.

Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles.

We have developed a pipeline to express, purify, and characterize HIV envelope protein (Env) gp145 from Chinese hamster ovary cells, to accelerate the production of a promising vaccine candidate. First in shake flasks, then in bioreactors, we optimized the growth conditions. By adjusting the pH to 6.8, we increased expression levels to 101 mg/L in a 50 L bioreactor, nearly twice the previously reported titer value. A battery of analytical methods was developed in accordance with current good manufacturing practices to ensure a quality biopharmaceutical. Imaged capillary isoelectric focusing verified proper glycosylation of gp145; dynamic light scattering confirmed the trimeric arrangement; and bio-layer interferometry and circular dichroism analysis demonstrated native-like properties (i.e., antibody binding and secondary structure). MALDI-TOF mass spectrometry was used as a multi-attribute platform for accurate mass determination, glycans analysis, and protein identification. Our robust analysis demonstrates that our gp145 product is very similar to a reference standard and emphasizes the importance of accurate characterization of a highly heterogeneous immunogen for the development of an effective vaccine. Finally, we present a novel guanosine microparticle with gp145 encapsulated and displayed on its surface. The unique properties of our gp145 microparticle make it amenable to use in future preclinical and clinical trials.

Home-Based Spirometry Telemonitoring After Allogeneic Hematopoietic Cell Transplantation: Mixed Methods Evaluation of Acceptability and Usability.

BACKGROUND: Home-based spirometry (HS) allows for the early detection of lung complications in recipients of an allogeneic hematopoietic cell transplant (AHCT). Although the usability and acceptability of HS are critical for adherence, patient-reported outcomes of HS use remain poorly understood in this setting. OBJECTIVE: The aim of this study is to design a longitudinal, mixed methods study to understand the usability and acceptability of HS among recipients of AHCT. METHODS: Study participants performed HS using a Bluetooth-capable spirometer that transmitted spirometry data to the study team in real time. In addition, participants completed usability questionnaires and in-depth interviews and reported their experiences with HS. Analysis of interview data was guided by the constructs of performance expectancy, effort expectancy, and social influence from the Unified Theory of Acceptance and Use of Technology model. RESULTS: Recipients of AHCT found HS to be highly acceptable despite modest technological barriers. On average, participants believed that the HS was helpful in managing symptoms related to AHCT (scores ranging from 2.22 to 2.68 on a scale of 0-4) and for early detection of health-related problems (score range: 2.88-3.12). Participants viewed HS favorably and were generally supportive of continued use. No significant barriers to implementation were identified from the patient's perspective. Age and gender were not associated with the patient perception of HS. CONCLUSIONS: Study participants found HS acceptable and easy to use. Some modifiable technical barriers to performing HS were identified; however, wider implementation of pulmonary screening is feasible from the patient's perspective.

Glutathione reductase-null malaria parasites have normal blood stage growth but arrest during development in the mosquito.

Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a gamma-glutamylcysteine synthetase (gamma-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for gamma-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and gamma-GCS by simultaneous disruption of gr and gamma-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.

A reversed phase HPLC method for the quantification of HIV gp145 glycoprotein levels from cell culture supernatants.

A reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly from culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent medium, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 × 50 mm, 3.5 µm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 × 12.5 mm, 5 µm), using 0.1% TFA and 2% n-propanol in LC-MS water as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile in LC-MS water as mobile phase B. The column temperature was 80 °C, the flow rate was 0.4 mL/min and the absorbance was monitored at 280 nm. The procedures and capabilities of the method were evaluated against the criteria for linearity, limit of detection (LOD), accuracy, repeatability, and robustness as defined by the International Conference on Harmonization (ICH) 2005 Q2(R1) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The method showed good linearity (R2 = 0.9996), a lower LOD of 2.4 µg/mL, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.

Soy and Frequent Dairy Consumption with Subsequent Equol Production Reveals Decreased Gut Health in a Cohort of Healthy Puerto Rican Women.

The U.S. Hispanic female population has one of the highest breast cancer (BC) incidence and mortality rates, while BC is the leading cause of cancer death in Puerto Rican women. Certain foods may predispose to carcinogenesis. Our previous studies indicate that consuming combined soy isoflavones (genistein, daidzein, and glycitein) promotes tumor metastasis possibly through increased protein synthesis activated by equol, a secondary dietary metabolite. Equol is a bacterial metabolite produced in about 20-60% of the population that harbor and exhibit specific gut microbiota capable of producing it from daidzein. The aim of the current study was to investigate the prevalence of equol production in Puerto Rican women and identify the equol producing microbiota in this understudied population. Herein, we conducted a cross-sectional characterization of equol production in a clinically based sample of eighty healthy 25-50 year old Puerto Rican women. Urine samples were collected and evaluated by GCMS for the presence of soy isoflavones and metabolites to determine the ratio of equol producers to equol non-producers. Furthermore, fecal samples were collected for gut microbiota characterization on a subset of women using next generation sequencing (NGS). We report that 25% of the participants were classified as equol producers. Importantly, the gut microbiota from equol non-producers demonstrated a higher diversity. Our results suggest that healthy women with soy and high dairy consumption with subsequent equol production may result in gut dysbiosis by having reduced quantities (diversity) of healthy bacterial biomarkers, which might be associated to increased diseased outcomes (e.g., cancer, and other diseases).

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: Effects on glycosylation and antigenicity.

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.

Structure-Based Screening of Plasmodium berghei Glutathione S-Transferase Identifies CB-27 as a Novel Antiplasmodial Compound.

Plasmodium falciparum parasites are increasingly drug-resistant, requiring the search for novel antimalarials with distinct modes of action. Enzymes in the glutathione pathway, including glutathione S-transferase (GST), show promise as novel antimalarial targets. This study aims to better understand the biological function of Plasmodium GST, assess its potential as a drug target, and identify novel antiplasmodial compounds using the rodent model P. berghei. By using reverse genetics, we provided evidence that GST is essential for survival of P. berghei intra-erythrocytic stages and is a valid target for drug development. A structural model of the P. berghei glutathione S-transferase (PbGST) protein was generated and used in a structure-based screening of 900,000 compounds from the ChemBridge Hit2Lead library. Forty compounds were identified as potential inhibitors and analyzed in parasite in vitro drug susceptibility assays. One compound, CB-27, exhibited antiplasmodial activity with an EC50 of 0.5 µM toward P. berghei and 0.9 µM toward P. falciparum multidrug-resistant Dd2 clone B2 parasites. Moreover, CB-27 showed a concentration-dependent inhibition of the PbGST enzyme without inhibiting the human ortholog. A shape similarity screening using CB-27 as query resulted in the identification of 24 novel chemical scaffolds, with six of them showing antiplasmodial activity ranging from EC50 of 0.6-4.9 µM. Pharmacokinetic and toxicity predictions suggest that the lead compounds have drug-likeness properties. The antiplasmodial potency, the absence of hemolytic activity, and the predicted drug-likeness properties position these compounds for lead optimization and further development as antimalarials.

Biotechnology and biochemistry of marine natural products.

Marine ecosystems are a source of biologically active compounds, many of which are currently in clinical use. With the goal of increasing the availability and the chemical diversity of these important compounds, more researchers are applying the tools of biotechnology to the discovery and production of marine natural products. This review summarizes the recent efforts made towards the characterization of the biochemical pathways that result in the production of marine natural products, with an emphasis on the work aimed at understanding the enzymatic activity involved in the biosynthesis of marine natural products.

Mental health services for children placed in foster care: an overview of current challenges.

Given the evidence from studies indicating that children in care have significant developmental, behavioral, and emotional problems, services for these children are an essential societal investment. Youth in foster care and adults who formerly were placed in care (foster care alumni) have disproportionately high rates of emotional and behavioral disorders. Among the areas of concern has been the lack of comprehensive mental health screening of all children entering out-of-home care, the need for more thorough identification of youth with emotional and behavioral disorders, and insufficient youth access to high-quality mental health services. In 2001, the American Academy of Child and Adolescent Psychiatry (AACAP) and the Child Welfare League of America (CWLA) formed a foster care mental health values subcommittee to establish guidelines on improving policy and practices in the various systems that serve foster care children (AACAP and CWLA, 2002). Because of the excellent quality and comprehensiveness of these statements, the Casey Clinical Foster Care Research and Development Project undertook consensus development work to enhance and build upon these statements. This article presents an overview of mental health functioning of youth and alumni of foster care, and outlines a project that developed consensus guidelines.

The Presence of Gut Microbial Genes Encoding Bacterial Genotoxins or Pro-Inflammatory Factors in Stool Samples from Individuals with Colorectal Neoplasia.

Gut bacterial toxins are thought to contribute to the development of colorectal cancer (CRC). This study examines the presence of specific gut bacterial toxin genes in stool samples from individuals with colorectal neoplasia (adenomas and/or CRC). The presence of bacterial genes encoding genotoxic or pro-inflammatory factors (pks, tcpC, gelE, cnf-1, AMmurB, and usp) was established by PCR of stool samples from individuals from mainland US (n = 30; controls = 10, adenoma = 10, CRC = 10) and from Puerto Rico (PR) (n = 33; controls = 13; adenomas = 8; CRC = 12). Logistic regression models and multinomial logistic regression models were used to estimate the magnitude of association. Distinct bacterial gene profiles were observed in each sample cohort. In individuals with CRC, AMmurB was detected more frequently in samples from the US and gelE in samples from PR. In samples from PR, individuals with ≥2 gut bacterial toxin genes in stool had higher odds of having colorectal neoplasia (OR = 11.0, 95%: CI 1.0⁻637.1): however, no significant association between bacterial genes and colorectal neoplasia was observed in the US cohort. Further analyses are warranted in a larger cohort to validate these preliminary findings, but these encouraging results highlight the importance of developing bacterial markers as tools for CRC diagnosis or risk stratification.

Hotspots of Sequence Variability in Gut Microbial Genes Encoding Pro-Inflammatory Factors Revealed by Oligotyping.

The gut microbiota has been implicated in a number of normal and disease biological processes. Recent studies have identified a subset of gut bacterial genes as potentially involved in inflammatory processes. In this work, we explore the sequence variability for some of these bacterial genes using a combination of deep sequencing and oligotyping, a data analysis application that identifies mutational hotspots in short stretches of DNA. The genes for pks island, tcpC and usp, all harbored by certain strains of E. coli and all implicated in inflammation, were amplified by PCR directly from stool samples and subjected to deep amplicon sequencing. For comparison, the same genes were amplified from individual bacterial clones. The amplicons for pks island and tcpC from stool samples showed minimal levels of heterogeneity comparable with the individual clones. The amplicons for usp from stool samples, by contrast, revealed the presence of five distinct oligotypes in two different regions. Of these, the oligotype GT was found to be present in the control uropathogenic clinical isolate and also detected in stool samples from individuals with colorectal cancer (CRC). Mutational hotspots were mapped onto the USP protein, revealing possible substitutions around Leu110, Glu114, and Arg115 in the middle of the pyocin domain (Gln110, Gln114, and Thr115 in most healthy samples), and also Arg218 in the middle of the nuclease domain (His218 in the uropathogenic strain). All of these results suggest that a level of variability within bacterial pro-inflammatory genes could explain differences in bacterial virulence and phenotype.

Artificial covalent linkage of bacterial acyl carrier proteins for fatty acid production.

Acyl carrier proteins (ACPs) are essential to the production of fatty acids. In some species of marine bacteria, ACPs are arranged into tandem repeats joined by peptide linkers, an arrangement that results in high fatty acid yields. By contrast, Escherichia coli, a relatively low producer of fatty acids, uses a single-domain ACP. In this work, we have engineered the native E. coli ACP into tandem di- and tri-domain constructs joined by a naturally occurring peptide linker from the PUFA synthase of Photobacterium profundum. The size of these tandem fused ACPs was determined by size exclusion chromatography to be higher (21 kDa, 36 kDa and 141 kDa) than expected based on the amino acid sequence (12 kDa, 24 kDa and 37 kDa, respectively) suggesting the formation of a flexible extended conformation. Structural studies using small-angle X-ray scattering (SAXS), confirmed this conformational flexibility. The thermal stability for the di- and tri-domain constructs was similar to that of the unfused ACP, indicating a lack of interaction between domains. Lastly, E. coli cultures harboring tandem ACPs produced up to 1.6 times more fatty acids than wild-type ACP, demonstrating the viability of ACP fusion as a method to enhance fatty acid yield in bacteria.

Antioxidant Activity of Collagen Extracts Obtained from the Skin and Gills of Oreochromis sp.

Efforts aimed at reduction of fishing waste generated during the evisceration and filleting are scarce. The fishing waste is used in the production of low value-added products, such as flours or silages. It is important to visualize an alternative and profitable use of this waste, as it constitutes a serious environmental problem. This research determined the antioxidant properties of collagenous extracts of skin and galls of Oreochromis sp. The raw materials were characterized by proximal chemical analysis. Three treatments were applied to extract the collagen: salt-soluble collagen, acid-soluble collagen (ASC), and pepsin-hydrolyzed collagen (PHC). The collagenous fractions were hydrolyzed (0.1% pepsin). The recovered collagen yield and antioxidant activity were determined to hydrolyzed collagen (HC) and nonhydrolyzed collagen (NHC). The ASC skin showed the highest extraction yield (3.02%). For galls, only the PHC extraction was feasible (0.16%). Antioxidant analysis of NHC did not reveal radical scavenging activities. HC displayed a 2,2-diphenyl-1-picrylhydrazyl %RSA of 22.58 (ASC skin) and 10.34% (PHC galls), and a 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] %RSA of 30.40% (PHC skin) and 29.43% (PHC galls), respectively. The ASC skin and PHC gall extracts exhibited 94.40% and 81.54% in ferric-reducing antioxidant power, and 43.63 and 38.08 µg ascorbic acid equivalents per milli liter for total antioxidant capacity, respectively. The collagen extracts showed %RSA and chelation of pro-oxidant metal ions. Different mechanism of antioxidant action was identified for the extracts: radical scavengers for HC and metal ion chelators for NHC. In conclusion, red tilapia skin collagen is recommended as an active ingredient of nutraceuticals, pharmaceuticals, or functional foods, for the identified bioactive properties.

Mutations in the fourth EGF-like domain affect thrombomodulin-induced changes in the active site of thrombin.

A number of alanine and more conservative mutants of residues in the fourth domain of thrombomodulin (TM) were prepared and assayed for protein C activation and for thrombin binding. Several of the alanine mutations appeared to cause misfolding or structural defects as assessed by poor expression and/or NMR HSQC experiments, while more conservative mutations at the same site appeared to allow correct folding and preserved activity. Several of the conservative mutants bound more weakly to thrombin despite the fact that the fourth domain does not directly contact thrombin in the crystal structure of the thrombin-TM complex. A few of the mutant TM fragments bound thrombin with an affinity similar to that of the wild type but exhibited decreases in k cat for protein C activation. These mutants were also less able to cause a change in the steady state fluorescence of fluorescein-EGR-chloromethylketone bound to the active site of thrombin. These results suggest that some residues within the fourth domain of TM may primarily interact with protein C but others are functionally important for altering the way TM interacts with thrombin. Residues in the fourth domain that primarily affect k cat for protein C activation may do this by changing the active site of thrombin.

Fused dimerization increases expression, solubility, and activity of bacterial dehydratase enzymes.

FabA and FabZ are the two dehydratase enzymes in Escherichia coli that catalyze the dehydration of acyl intermediates in the biosynthesis of fatty acids. Both enzymes form obligate dimers in which the active site contains key amino acids from both subunits. While FabA is a soluble protein that has been relatively straightforward to express and to purify from cultured E. coli, FabZ has shown to be mostly insoluble and only partially active. In an effort to increase the solubility and activity of both dehydratases, we made constructs consisting of two identical subunits of FabA or FabZ fused with a naturally occurring peptide linker, so as to force their dimerization. The fused dimer of FabZ (FabZ-FabZ) was expressed as a soluble enzyme with an ninefold higher activity in vitro than the unfused FabZ. This construct exemplifies a strategy for the improvement of enzymes from the fatty acid biosynthesis pathways, many of which function as dimers, catalyzing critical steps for the production of fatty acids.