Evolution temperature adaptation

P. F. Wintrode and F. H. Arnold, Temperature adaptation of enzymes lessons from laboratory evolution, Adv, Protein Chem. 2000, 55, 161-225. 

TEMPERATURE ADAPTATION OF ENZYMES LESSONS FROM LABORATORY EVOLUTION... 

In this chapter, we will outline how evolutionary protein design methods are now being used to help uncover the molecular basis for temperature adaptation in enzymes. Before doing this, however, we will briefly review how temperature affects protein stability and enzyme activity. Then we will discuss some of the results of comparative studies of enzymes isolated from organisms adapted to different temperatures and the questions that can be addressed by laboratory evolution. 

To date, only a small number of directed evolution studies of temperature adaptation have been carried out. From this small data set we can nonetheless draw several interesting conclusions. 

In a recent paper [11] this approach has been generalized to deal with reactions at surfaces, notably dissociation of molecules. A lattice gas model is employed for homonuclear molecules with both atoms and molecules present on the surface, also accounting for lateral interactions between all species. In a series of model calculations equilibrium properties, such as heats of adsorption, are discussed, and the role of dissociation disequilibrium on the time evolution of an adsorbate during temperature-programmed desorption is examined. This approach is adaptable to more complicated systems, provided the individual species remain in local equilibrium, allowing of course for dissociation and reaction disequilibria. 

B. (3-Bromo-3,3-difluoropropyl)trimethylsilane. A 1-L, four-necked flask is equipped with a mechanical stirrer, thermometer, Claisen adapter, septum inlet, reflux condenser (the top of which is connected to a calcium chloride drying tube), and a solid addition funnel. The flask is charged with (1,3-dibromo-3,3-difluoropropyl)trimethylsilane (78.3 g, 0.25 mol), and anhydrous dimethyl sulfoxide (200 mL), and the solid addition funnel is charged with sodium borohydride (11.5 g, 0.30 mol) (Notes 7 and 8). The stirred solution is warmed to 80°C, and sodium borohydride is added at a rate sufficient to maintain a reaction temperature of 80-90°C (Note 9). Toward the end of the addition, an additional portion of dimethyl sulfoxide (200 mL) is added via syringe to lower the viscosity of the reaction mixture. After the addition is complete, the mixture is cooled in an ice-water bath, diluted with 100 mL of pentane, and cautiously quenched with 12 M hydrochloric acid until no further gas evolution occurs. The mixture is transferred to a separatory funnel and washed with three, 100-mL portions of 5% brine. The pentane extract is dried over calcium chloride and the solvent removed through a 15-cm Vigreux column. Further fractionation yields 41.5 g (72%) of 3-bromo-3,3-difluoropropyltrimethylsilane, bp 139-141 °C (Note 10). 

In the biochemical network, the processing elements do not learn from task examples, but the knowledge is already built in. For example, an enzyme recognizes a specific substrate and applies a specific rate for the catalytic reaction, as a function of the particular conditions, pH, temperature, and so on. Therefore, in such systems, adaptation is implemented by adjusting the catalytic characteristics according to environmental conditions and following laws already built in by evolution. 

A 250-ml. round-bottomed flask with a side arm is equipped with a distillation head and condenser. The receiving flask is attached to the condenser with an adapter, and the exit from the flask goes to a bubble counter containing high-boiling petroleum ether. A thermometer is inserted in the side arm of the distillation flask, and it reaches to the bottom. With exclusion of moisture, 147 g. (0.704 mole) of phosphorus pentachloride and 100 g. (0.704 mole) of -chloroanisole are added to the flask. The flask is heated in an oil bath the reaction begins when the inside temperature reaches 120° and occurs rapidly at 140°. The temperature is raised to 160° over a period of 2 hours, thereby distilling the phosphorus trichloride (Note 1). After the gas evolution subsides, the reaction mixture is heated to 175° for a short time. About 73-75 g. of phosphorus trichloride is collected. 

Rgure 2.39. Evolution of the wetting transition temperature T (c) for macroscopic interfaces and gelation temperature Tg(c) of emulsions of various droplet sizes. (Adapted from [113].)... 

Fig. 11. Evolution of the experimental room-temperature X-band CW-EPR spectra of mixtures of galacturonan, Cr(VI), and GSH taken at pH 7. The spectrum is adapted from ref. 108.

Traditionally, diazonium tetrafluoroborates are decomposed neat in the solid state. This solid, placed in a flask with large outlets and which must not be more than half full of the salt, is gently heated near its surface until decomposition starts. Often no more heat is required, the decomposition continuing spontaneously with evolution of dense vapors of boron trifluoride. The reaction medium is often brought to dull redness and the fluorinated product distills if sufficiently volatile.1,3 The filled reaction flask can also be immersed in a fluid brought to ca. 20 to 50 C above the decomposition temperature of the diazonium salt, previously determined in a capillary tube.1,3,200,201 In another procedure, the reaction flask can be heated to this temperature while empty, then the diazonium tetrafluoroborate is added little by little 200-201 This latter method has been adapted to perform the decomposition of diazonium tetrafluoroborates in a continuous way by two techniques ... 

Biogenic silicon (BSI) was determined, with minor modifications, by the method of DeMaster (17). As adapted, the technique involved time-course leaching of <20-mg samples of particulate matter in 30 mL of 1.0% Na2C03 in a water bath at 85 °C. Silica in leachates was quantified either colorimetrically (Technicon autoanalyzer procedure) or by nitrous oxide flame atomic absorption. A high-temperature catalytic-combustion technique (Perkin Elmer 240C) was used for particulate organic carbon determinations. Particulate inorganic (carbonate) carbon was measured on the same instrument by CO 2 evolution after treatment of the particles with phosphoric acid. 

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