The three-dimensional structure of the C-terminal 20 kDa portion of auxilin, which consists of the clathrin binding region and the C-terminal J-domain, has been determined by NMR. Auxilin is an Hsp40 family protein that catalytically supports the uncoating of clathrin-coated vesicles through recruitment of Hsc70 in an ATP hydrolysis-driven process. This 20 kDa auxilin construct contains the minimal sequential region required to uncoat clathrin-coated vesicles catalytically. The tertiary structure consists of six helices, where the first three are unique to auxilin and believed to be important in the catalytic uncoating of clathrin. The last three helices correspond to the canonical J-domain of Hsp40 proteins. The first helix, helix 1, which contains a conserved FEDLL motif believed to be necessary for clathrin binding, is transient and not packed against the rest of the structure. Helix 1 is joined to helix 2 by a flexible linker. Helix 2 packs loosely against the J-domain surface, whereas helix 3 packs tightly and makes critical contributions to the J-domain core. A long insert loop, also unique to the auxilin J-domain, is seen between helix 4 and helix 5. Comparison with a previously reported structure of auxilin containing only helices 3-6 shows a significant difference in the invariant HPD segment of the J-domain. The region where helix 1 is located corresponds to the expected region of the unstructured G/F-rich domain seen in DnaJ, i.e., the canonical N-terminal J-domain protein. In contrast, the location of helix 1 differs from the substrate binding regions of two other Hsp40 proteins, Escherichia coli Hsc20 and viral large T antigen. The variety of biological functions performed by Hsp40 proteins such as auxilin, as well as the observed differences in the structure and function of their substrate binding regions, supports the notion that Hsp40 proteins act as target-specific adaptors that recruit their more general Hsp70 partners to specific biological roles.

A new method is presented to precipitate proteins and amino acids from reverse micelles by dehydrating the micelles with molecular sieves. Nearly complete precipitation is demonstrated for alpha-chymotrypsin, cytochromec, and trytophan from 2-ethylhexyl sodium sulfosuccinate (AOT)/isooctane/water reverse micelle solutions. The products precipitate as a solid powder, which is relatively free of surfactant. The method does not require any manipulation of pH, ionic strength, temperature, pressure, or solvent composition, and is applicable over a broad range of these properties. This general approach is compared with other techniques. This general approach is compared with other techniques for the recovery of biomolecules from reverse micelles. (c) 1994 John Wiley & Sons, Inc.

Although there is great scientific and technological interest in examining the physiology and bioenergetics of microorganisms from extreme environments, difficulties encountered in their cultivation and lack of genetic systems hampers the investigation of these issues. As such, we have adapted methods for continuous cultivation of mesophilic organisms to extremes of temperature and pH to study extremophiles. Since the risk for contamination of extremophilic continuous cultures is relatively small, long-term, steady state experiments investigating physiological response to culture perturbations are possible. Experiments along these lines have provided insights into the significance of specific enzymes in the metabolism of particular substrates, in addition to providing a better understanding of stress response and unusual physiological characteristics of hyperthermophilic and extremely thermoacidophilic microorganisms. Several examples are provided here, including the thermal stress response of Metallosphaera sedula (T(opt) 74 °C) growing at pH 2.0, and the response of the heterotrophic hyperthermophiles Pyrococcus furiosus (T(opt) 98 °C), Thermococcus litoralis (T(opt) 88 °C) and T. maritima (T(opt) 80 °C) to changes in growth medium. Also discussed will be how the same experimental systems have been used to study exopolysaccharide production and biofilm formation by hyperthermophilic heterotrophs and facilitated the estimation of bioenergetic parameters for these organisms under a variety of growth conditions. Continuous culture, used in conjunction with genome sequence information, two-dimensional gel electrophoresis and differential gene expression, can provide important insights into the metabolism of high temperature extremophiles.

The response of an extremely thermoacidophilic archaeon, Metallosphaera sedula (growth temperature range, 50 to 79°C; optimum temperature, 74°C; optimum pH, 2.0), to thermal stress was investigated by using a 10-liter continuous cultivation system. M. sedula, growing at 74°C, pH 2.0, and a dilution rate of 0.04 hr ؊1 , was subjected to both abrupt and gradual temperature shifts in continuous culture to determine the responses of cell density levels and protein synthesis patterns. An abrupt temperature shift from 74 to 79°C resulted in little, if any, changes in cell density and a small increase in total protein per cell. When the culture temperature was shifted further to 80.5°C, cell density dropped to below 5 ؋ 10 6 cells/ml from 10 8 cells/ml, leading to washout of the culture. Operation at this temperature and slightly higher temperatures, however, could be achieved by exposing the culture to thermal stress more gradually (0.5°C increments). As a result, stable operation could be maintained at temperatures of up to 81°C, and the washout temperature could be increased to 82.5°C. Continuous culture operation at 81°C for 100 h (stressed phase) led to an approximately sevenfold lower steady-state cell density than that observed for operation at or below 79°C. However, sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis (both one and two dimensional) revealed significantly higher levels (sixfold increase) of a 66-kDa stress response protein (MseHSP60), immunologically related to Thermophilic Factor 55 from Sulfolobus shibatae (