The application of three approximate free energy calculations methods to structure based ligand design: trypsin and its complex with inhibitors.

Three approximate free energy calculation methods are examined and applied to an example ligand design problem. The first of the methods uses a single simulation to estimate the relative binding free energies for related ligands that are not simulated. The second method is similar, except that it uses only first derivatives of free energy with respect to atomic parameters (most often charge, van der Waals equilibrium distance, and van der Waals well depth) to calculate free energy differences. The last method PROFEC (Pictorial Representation of Free Energy Components), generates contour maps that show how binding free energy changes when additional particles are added near the ligand. These three methods are applied to a benzamidine/trypsin complex. They each reproduce the general trends in the binding free energies, indicating that they might be useful for suggesting how ligands could be modified to improve binding and, consequently, useful in structure-based drug design.

Can one predict protein stability? An attempt to do so for residue 133 of T4 lysozyme using a combination of free energy derivatives, PROFEC, and free energy perturbation methods.

Free energy derivatives, pictorial representation of free energy changes (PROFEC) and free energy perturbation methods were employed to suggest the modifications that may improve the stability of a mutant T4 lysozyme with a S-2-amino-3-cyclopentylpropanoic acid residue (Cpe) at position 133. The free energy derivatives and PROFEC methods were used to locate promising sites where modifications may be introduced. The effects of several candidate modifications on the enzyme's stability were analyzed by the free energy perturbation method. We found that this scheme is able to effectively suggest modifications that may increase the enzyme's stability. The modifications investigated are the introduction of a methyl, a tert-butyl or a trifluoromethyl group at the Cepsilon2 position and a cyclopropyl group between the Cdelta2 and Cepsilon2 position on the cyclopentyl ring. The stereochemistry of the introduced groups (in the alpha or beta configurations) was studied. Our calculations predict that the introduction of a methyl group in the alpha configuration or a cyclopropyl group in the beta configuration will increase the stability of the enzyme; the introduction of the two groups in the other configurations and the other modifications will decrease the stability of the enzyme. The results indicate that packing interactions can strongly influence the stability of the enzyme.

Analysis of the productivity of a continuous algal culture system.

We describe a first-principles analysis of a system for the continuous culture of the green alga Scenedesmus obliquus under light-limiting conditions. According to this analysis, the productivity of the algal culture is given by the relation Y = E(m)I(0)AK(1 - e(-alphacl)) - GRcV, where Y = yield (g cells/h), E(m) = 0.20 (the maximum attainable photosynthetic conversion on an energy basis), A = illuminated area (m(2)), K = 0.156[(g cells/h/W), the energy equivalent of the algae], I(0) = light intensity (W/m(2)), alpha = extinction coefficient (L/cm/g),c = cell concentration (g/L), I = light path (cm), R = respiration rate (g carbon/g cells/h), V = culture volume (L), and G = ratio of g cells to g carbon (2.04). This formula is completely determined and has no free adjustable parameters. Using parameter values determined independently, the model accurately predicted the relationship of productivity to cell density in the culture system.

Biomass recycle as a means to improve the energy efficiency of CELSS algal culture systems.

Algal cultures can be very rapid and efficient means to generate biomass and regenerate the atmosphere for closed environmental life support systems. However, as in the case of most higher plants, a significant fraction of the biomass produced by most algae cannot be directly converted to a useful food product by standard food technology procedures. This waste biomass will serve as an energy drain on the overall system unless it can be efficiently recycled without a significant loss of its energy content. We report experiments in which cultures of the algae Scenedesmus obliquus were grown in the light and at the expense of an added carbon source, which either replaced or supplemented the actinic light. As part of these experiments we tested hydrolyzed waste biomass from these same algae to determine whether the algae themselves could be made part of the biological recycling process. Results indicate that hydrolyzed algal (and plant) biomass can serve as carbon and energy sources for the growth of these algae, suggesting that the efficiency of the closed system could be significantly improved using this recycling process.

Do the higher oxidation states of the photosynthetic O2-evolving system contain bound H2O?

A modified mass spectrometer was used to determine whether the higher oxidation states of the photosynthetic O2-evolving system contain substrate water that is not freely exchangeable with the external medium. Our data indicated that the higher oxidation states contain no appreciable bound, non-exchangeable H2O. This suggests that H2O oxidation takes place via a rapid, concerted, all-or-none mechanism rather than by a mechanism involving stable, partially oxidized, H2O-derived intermediates. These findings set definite constraints on possible mechanisms of O2 evolution.

Algal culture studies related to a closed ecological life support system.

Long-term cultures of Scenedesmus obliquus were maintained in an annular air-lift column operated as a turbidostat. We observed a linear relationship between the dry weight of the cultured cells, their cell number, and their chlorophyll content over a broad range of cell density at constant illumination. Thus, the cells did not appear to be adapting to differences in growth rate or light intensity during these experiments. Productivity vs dry wt rose linearly until the cell density reached a level at which light became limiting; at this point approximately 89% of the photosynthetically active radiation (PAR) was being absorbed. The maximum dilution rate of the system corresponded to a doubling time of 13.8 hr, about half the maximum growth rate generally observed at this temperature. Productivity at the maximum was approximately 80% of the maximum theoretical productivity. The rather low incident intensities (approximately 10% of full sunlight) were a main contributing factor to the high light utilization efficiencies obtained in this system, since the cells were never driven into light saturation. In many respects, algae would be ideal plant components for a biologically-based closed life support system, since they are eminently suited to the closely coupled functions of food production and atmosphere regeneration. In this communication, we report some findings on the (steady-state) continuous culture of Scenedesmus obliquus, a physiologically well-characterized green alga with good growth characteristics.

Metabolism of biphenyl by Aspergillus toxicarius: induction of hydroxylating activity and accumulation of water-soluble conjugates.

Biphenyl metabolism in Aspergillus toxicarius occurs by successive hydroxylations in the 4- and 4'-positions, followed by conjugation with sulfate to produce 4-hydroxybiphenyl-O-sulfonic acid and 4,4'-dihydroxybiphenyl-O-sulfonic acid. The hydroxylation reactions normally occur only after a prolonged lag period after which the appearance of the monohydroxylated compound precedes the dihydroxylated compound. The accumulation of the monohydroxy compound is transient; therefore, it is an intermediate in the hydroxylating pathway. The onset of hydroxylating activity can be greatly accelerated when the culture is primed with the intermediate or product of the reaction (4-hydroxybiphenyl or 4,4'-dihydroxybiphenyl) at the time of biphenyl addition; a concentration of 0.05 mg 4,4'-dihydroxybiphenyl per ml produces optimal induction. Water-soluble conjugates of 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl were found in cultures of A. toxicarius grown in the presence of biphenyl plus inducer. The conjugate was shown to be the sulfate ester; no glucuronide or other conjugate species was found in any phase of the transformation. As with hydroxylating activity, the sulfotransferase activity appeared to be induced by the products of biphenyl metabolism.

Photosynthetic o(2) exchange kinetics in isolated soybean cells.

Light-dependent O(2) exchange was measured in intact, isolated soybean (Glycine max. var. Williams) cells using isotopically labeled O(2) and a mass spectrometer. The dependence of O(2) exchange on O(2) and CO(2) was investigated at high light in coupled and uncoupled cells. With coupled cells at high O(2), O(2) evolution followed similar kinetics at high and low CO(2). Steady-state rates of O(2) uptake were insignificant at high CO(2), but progressively increased with decreasing CO(2). At low CO(2), steady-state rates of O(2) uptake were 50% to 70% of the maximum CO(2)-supported rates of O(2) evolution. These high rates of O(2) uptake exceeded the maximum rate of O(2) reduction determined in uncoupled cells, suggesting the occurrence of another light-induced O(2)-uptake process (i.e. photorespiration).Rates of O(2) exchange in uncoupled cells were half-saturated at 7% to 8% O(2). Initial rates (during induction) of O(2) exchange in uninhibited cells were also half-saturated at 7% to 8% O(2). In contrast, steady-state rates of O(2) evolution and O(2) uptake (at low CO(2)) were half-saturated at 18% to 20% O(2). O(2) uptake was significantly suppressed in the presence of nitrate, suggesting that nitrate and/or nitrite can compete with O(2) for photoreductant.These results suggest that two mechanisms (O(2) reduction and photorespiration) are responsible for the light-dependent O(2) uptake observed in uninhibited cells under CO(2)-limiting conditions. The relative contribution of each process to the rate of O(2) uptake appears to be dependent on the O(2) level. At high O(2) concentrations (>/=40%), photorespiration is the major O(2)-consuming process. At lower (ambient) O(2) concentrations (

Light-driven Uptake of Oxygen, Carbon Dioxide, and Bicarbonate by the Green Alga Scenedesmus.

Mass spectrometric techniques were used to study several aspects of the competition between O(2) and species of inorganic carbon for photosynthetically generated reducing power in the green alga, Scenedesmus.In contrast to wild type, no appreciable light-driven O(2) uptake was observed in a mutant lacking photosystem I. It is concluded that the carbon cycle-independent reduction of O(2) occurs at the expense of photosystem I-generated reducing equivalents.The commonly observed differences between CO(2)-grown and air-grown Scenedesmus with respect to CO(2) uptake and glycolate formation cannot be ascribed to differences in their capacity for light-driven O(2) uptake. There were no intrinsic differences found in O(2) uptake capacity between the two physiological types under conditions in which CO(2) was saturating or CO(2) uptake was inhibited. It was only under CO(2)-limited conditions that pronounced differences between the two physiological types were observed. This fact suggests that differences in O(2) metabolism and sensitivity between the two types really reflect differences in their capacity to assimilate inorganic carbon; in this respect they are analogous to C(3) and C(4) plants.The hypothesis that air-grown Scenedesmus can assimilate HCO(3) (-) by directly monitoring the time course of dissolved CO(2), O(2) uptake, and O(2) evolution in illuminated algal suspensions at alkaline pH was tested. Inasmuch as the measuring technique employed was fast compared to the nonenzymic equilibration of the inorganic carbon species, it was possible to determine the degree to which the CO(2) concentration deviated from equilibrium (with the other inorganic carbon species) during the course of illumination. The observed kinetics in air-grown and CO(2)-grown algae in the presence and absence of carbonic anhydrase, and a comparison of these kinetics with theoretical (computer-generated) time courses, support the idea that air-adapted algae are able to assimilate HCO(3) (-) actively at a high rate. The data suggest that these algae preferentially assimilate CO(2) and supply the balance of their needs by taking up HCO(3) (-). Since (unlike C(4) plants) these algae have no special CO(2) pump, and thus have a relatively low affinity for CO(2), HCO(3) (-) assimilation is the major carbon uptake process at alkaline pH even when the total CO(2) is present in millimolar concentrations.

Rate-temperature curves as an unambiguous indicator of biological activity in soil.

Experiments are described in which we used a mass spectrometer to monitor O(2) uptake of enclosed soil samples as a function of temperature. We found that an Arrhenius plot of the rate of O(2) uptake showed pronounced local maxima attributable to biological activity, whereas similar plots of rates obtained with abiotic soils yielded straight lines. This procedure thus provides a basis for distinguishing biological from chemical activity for reactions, such as O(2) uptake, that can occur via either biological or chemical pathways.

Mass spectrometric determination of hydroxylamine photooxidation by illuminated chloroplasts.

A mass spectrometer with a special inlet was used to directly monitor the products evolved when hydroxylamine-treated chloroplasts were exposed to short saturating light flashes. We found that: 1. Molecular dinitrogen was the sole product of hydroxylamine photooxidation, and was formed in an amount equal to twice the O2 evolved during H2O photooxidation. 2. This reaction was driven by Photosystem II, and did not involve Photo-system I-generated superoxide or peroxide. 3. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, N2 was evolved only on the first flash. These results suggested that N2 was formed by the combination of two single-electron oxidation products of hydroxylamine.

Kinetics and Apparent K(m) of Oxygen Cycle under Conditions of Limiting Carbon Dioxide Fixation.

A mass spectrometer with a membrane inlet was used to monitor light-driven O(2) evolution, O(2) uptake, and CO(2) uptake in suspensions of algae (Scenedesmus obliquus). We observed the following. (a) The rate of O(2) uptake, which, in the presence of iodoacetamide, replaces the uptake of CO(2), showed a distinct plateau (V(max)) beyond approximately 30% O(2) and was half-maximal at approximately 8% O(2). We concluded that this light-driven O(2) uptake process, which does not involve carbon compounds, is saturated at lower O(2) concentrations than are photorespiration and glycolate formation. (b) In the absence of inhibitor, O(2) evolution was relatively unaffected by the presence or absence of CO(2). During the course of CO(2) depletion, electron flow to CO(2) was replaced by an equivalent flow to O(2). (c) There was a distinct delay between the cessation of CO(2) uptake and the increase in O(2) uptake. We ascribe this delay to the transient utilization of another electron acceptor-possibly bicarbonate or another bound form of CO(2).

Photoreduction of O(2) Primes and Replaces CO(2) Assimilation.

A mass spectrometer with a membrane inlet system was used to monitor directly gaseous components in a suspension of algae. Using labeled oxygen, we observed that during the first 20 seconds of illumination after a dark period, when no net O(2) evolution or CO(2) uptake was observed, O(2) evolution was normal but completely compensated by O(2) uptake. Similarly, when CO(2) uptake was totally or partially inhibited, O(2) evolution proceeded at a high (near maximal) rate. Under all conditions, O(2) uptake balanced that fraction of the O(2) evolution which could not be accounted for by CO(2) uptake.From these observations we concluded that O(2) and CO(2) are in direct competition for photosynthetically generated reducing power, with O(2) being the main electron acceptor during the induction process and under other conditions in which CO(2) reduction cannot keep pace with O(2) evolution. The high rate of the O(2) uptake reaction observed in the presence of iodoacetamide, KCN, or carbonyl cyanide p-trifluoromethyoxyphenylhydrazone, suggests that a special high capacity oxidase distinct from ribulose diphosphate oxygenase exists in whole cells. The rapid reduction of molecular O(2) after a period of darkness probably serves as a priming reaction for the photosynthetic apparatus. The high steady state rate of the O(2) cycle in the absence of CO(2) fixation suggests that the regulation of photosynthesis does not involve significant changes in the rate of photochemical electron transport.

An integrated multi-purpose biology instrument utilizing a single detector, the mass spectrometer.

A mass spectrometer is used to analyze the gas phase in a number of reaction vessels filled with Martian soil. By choosing appropriate incubation conditions this instrument can be used to perform a wide spectrum of experiments ranging from the observation of general indices of life, i.e. processes and patterns unexplainable by physico-chemical mechanisms, to assays utilizing isotopes which probe for specific metabolic processes. Of particular interest is the in situ incubation in which a Martian soil sample is maintained at a constant temperature and its gas phase composition analyzed with time. Properly interpreted, this is a very general life-detection probe which makes minimal assumption as to the nature of Martian biology. Other assays and measurements concerning the soil and the atmosphere compatible with this method are also described.