Computational design of proteins stereochemically optimized in size, stability, and folding speed

Artificial proteins potentially barrier-free in the folding kinetics are approached computationally under the guidance of protein-folding theories. The smallest and fastest folding globular protein triple-helix-bundle (THB) is so modified as to minimize or eliminate its presumed barriers in folding speed. As the barriers may reside in the ordering of either secondary or tertiary structure, the elements of both secondary and tertiary structure in the protein are targeted for prenucleation with suitable stereochemically constrained amino acid residues. The required elements of topology and sequence for the THB are optimized independently; first the topology is optimized with simulated annealing in polypeptides of highly simplified alphabet; next, the sequence in side chains is optimized using the standard inverse design methods. The resultant three best-adapted THBs, variable in topology and distinctive in sequences, are assessed by comparing them with a few benchmark proteins. The results of mainly molecular dynamics (MD) comparisons, undertaken in explicit water at different temperatures, show that the designed sequences are favorably placed against the chosen benchmarks as THB proteins potentially thermostable in the native folds. Folding simulation experiments with MD establish that the designed sequences are rapid in the folding of individual helices, but not in the evolution of tertiary structure; energetic cum topological frustrations remain but could be the artifacts of the starting conformations that were chosen in the THBs in the folding simulations. Overall, a practical high-throughput approach for de novo protein design has been developed that may have fruitful application for any type of tertiary structure.     

Optimization of rifamycin B fermentation in shake flasks via a machine-learning-based approach

Rifamycin B is an important polyketide antibiotic used in the treatment of tuberculosis and leprosy. We present results on medium optimization for Rifamycin B production via a barbital insensitive mutant strain of Amycolatopsis mediterranei S699. Machine-learning approaches such as Genetic algorithm (GA), Neighborhood analysis (NA) and Decision Tree technique (DT) were explored for optimizing the medium composition. Genetic algorithm was applied as a global search algorithm while NA was used for a guided local search and to develop medium predictors. The fermentation medium for Rifamycin B consisted of nine components. A large number of distinct medium compositions are possible by variation of concentration of each component. This presents a large combinatorial search space. Optimization was achieved within five generations via GA as well as NA. These five generations consisted of 178 shake-flask experiments, which is a small fraction of the search space. We detected multiple optima in the form of 11 distinct medium combinations. These medium combinations provided over 600% improvement in Rifamycin B productivity. Genetic algorithm performed better in optimizing fermentation medium as compared to NA. The Decision Tree technique revealed the media-media interactions qualitatively in the form of sets of rules for medium composition that give high as well as low productivity.     

An optimized method for Aspergillus niger spore production on natural carrier substrates

Aspergillus niger spores have wide ranging applications in the fermentation industry as well as in wastewater treatment. We present an optimized method for production of A. niger spores on natural substrates such as rice, split pea, and millet. The specific productivity (number of spores per gram of dry substrate) was 31-fold greater and volumetric productivity was 750-fold greater compared to agar slopes. The important process variables were incubation temperature, moisture content, and inoculum quantity. We find that the optimal condition for total spore count is different from the viable spore count for millet. The optimum lies in a narrow region defined by the process parameters. Of the three substrates tested split pea gave the highest specific spore productivity of 3.1 x 10(10) spores per gram of dry substrate. This is the first report of systematic study on the effect of process parameters on spore viability. The method of A. niger spore production on natural substrate appears advantageous as compared to the currently practiced method in terms of scale-up, cost, and ease of operation.     

Transition state stabilization of subtilisins in organic media

Electrostatic forces are among the stabilizing interactions that contribute to the high degree of enzyme-transition state complementarity. The active-site polarity, which can differ substaintially from that of water, is thus an important determinant of transition state stabilization. Here we pose the question of whether the rate of an enzymatic reaction proceeding through a charged transition state can be increased by increasing the active-site polarity in an organic solvent. The active-site polarity of subtilisin has been reduced by dehydration and suspension in a nonpolar solvent (tetrahydrofuran), and then increased by adding water to the solvent. Enhancing the local polarity substantially increasing the rate of catalysis, implicating polarity as an important factor in stabilizing the charged tetrahedral transition state. Studies with subtilisins whose active sites have been modified by site-directed mutagenesis support the role of polarity in transition state stabilization. (c) 1994 John Wiley & Sons, Inc.     

Effect in rats of simultaneous prenatal exposure to ochratoxin A and aflatoxin B1. I. Maternal toxicity and fetal malformations

Ochratoxin A (OA) and Aflatoxin B1 (AFB1), the food borne mycotoxins are produced by several fungal species of the genera Aspergillus and Penicillium. To determine the teratogenic effects, these mycotoxins were administered orally either individually or in combination to the pregnant Wistar rats on days 6-15 of gestation. OA and AFB1 were dissolved in corn oil and different doses of OA (0.125, 0.25, 0.50, and 0.75 mg/kg), AFB1 (0.125, 0.25, 0.50, and 1.00 mg/kg), and a combination of OA+AFB1 (0.125+0.125; 0.25+0.50; 0.50+0.25 mg/kg) were given by gastric intubation to rats. During dosing period, the body weight and body weight gains significantly decreased at a higher dosage, in both individual and combined treatments. In all the combination treatments, the percent implants resorbed, fetal body weights, and crown-rump lengths were comparable to those of controls and with the individual mycotoxin treatment. The number of dead fetuses was significantly increased in the high OA combination (OA+AFB1 0.50+0.25) group as compared with the other two combinations. OA and AFB1 alone and in combination caused various gross, skeletal, and visceral anomalies. The occurrence was considerably less pronounced in fetuses of AFB1 and combination groups as compared with those of OA group fetuses. The exencephaly, incomplete closure of skull, wavy and fused ribs, agenesis of the ischium bone, and enlarged renal pelvis, recorded in OA treatment and ear abnormality and incomplete ossification of skull bones observed in AFB1 when given individually, were not seen in combination groups. However, new manifestations, such as gastroschisis and syndactyly were observed and the incidence of cardiac defects was increased in fetuses due to the combined treatment. The results of the present study indicated that there is some interaction between these mycotoxins that resulted in reduced teratogenic activity of OA in the presence of AFB1. Apparently, new manifestations observed in combination treatment points to the potential threat of teratogenicity in terms of public health hazards.     

Characterization and Application of a Robust Glucose Dehydrogenase from Paenibacillus pini for Cofactor Regeneration in Biocatalysis

Indian Journal of Microbiology - Glucose dehydrogenases are important auxiliary enzymes in biocatalysis, employed in the regeneration of reduced nicotinamide cofactors for oxidoreductase catalysed...     

Gene regulatory network modeling via global optimization of high-order dynamic Bayesian network

ABSTRACT: BACKGROUND: Dynamic Bayesian network (DBN) is among the mainstream approaches for modeling various biological networks, including the gene regulatory network (GRN). Most current methods for learning DBN employ either local search such as hill-climbing, or a meta stochastic global optimization framework such as genetic algorithm or simulated annealing, which are only able to locate sub-optimal solutions. Further, current DBN applications have essentially been limited to small sized networks. RESULTS: To overcome the above difficulties, we introduce here a deterministic global optimization based DBN approach for reverse engineering genetic networks from time course gene expression data. For such DBN models that consist only of inter time slice arcs, we show that there exists a polynomial time algorithm for learning the globally optimal network structure. The proposed approach, named GlobalMIT+, employs the recently proposed information theoretic scoring metric named mutual information test (MIT). GlobalMIT+ is able to learn high-order time delayed genetic interactions, which are common to most biological systems. Evaluation of the approach using both synthetic and real data sets, including a 733 cyanobacterial gene expression data set, shows significantly improved performance over other techniques. CONCLUSIONS: Our studies demonstrate that deterministic global optimization approaches can infer large scale genetic networks.     

Active-site titration of serine proteases in organic solvents

Calculation of kinetic constants of an enzymatic reaction in organic solvents requires knowledge of the functional active-site concentration in organic solvents, and this can be significantly different than that in water. An experimental method for active-site titration of serine proteases in organic media has been developed based on the kinetics of inhibition by phenylmethanesulfonyl fluoride (PMSF), a serine-specific inhibitor (or suicide substrate). This kinetic approach is fundamentally different from other techniques that require complete titration of all accessible enzyme active sites. This active site titration method was applied to subtilisins BPN' and Carlsberg and alpha-chymotrypsin and resulted in fractions of active sites that ranged from 8 to 62% (of the fraction active in water) depending on the enzyme, the method of enzyme preparation, and the organic solvent used. The active-site concentration of subtilisin BPN' and Carlsberg increased with increasing hydrophobicity of the solvent and with increasing solvent hydration in tetrahydrofuran. The dependence of the fraction of active sites on the nature of the organic solvent appears to be governed largely by solvent-induced inactivation caused by direct interaction of a hydrophilic solvent with the enzyme. (c) 1996 John Wiley & Sons, Inc.