Among its attributes, the mythical philosopher’s stone is supposedly capable of turning base metals to gold or silver. In an analogous fashion, we are finding that protein crystallization optimization using ionic liquids (ILs) often results in the conversion of base protein precipitate to crystals. Recombinant inorganic pyrophosphatases (8 of the 11 proteins) from pathogenic bacteria as well as several other proteins were tested for optimization by 23 ILs, plus a dH2O control, at IL concentrations of 0.1, 0.2, and 0.4 M. The ILs were used as additives, and all proteins were crystallized in the presence of at least one IL. For 9 of the 11 proteins, precipitation conditions were converted to crystals with at least one IL. The ILs could be ranked in order of effectiveness, and it was found that ~83% of the precipitation-derived crystallization conditions could be obtained with a suite of just eight ILs, with the top two ILs accounting for ~50% of the hits. Structural trends were found in the effectiveness of the ILs, with shorter-alkyl-chain ILs being more effective. The two top ILs, accounting for ~50% of the unique crystallization results, were choline dihydrogen phosphate and 1-butyl-3-methylimidazolium tetrafluoroborate. Curiously, however, a butyl group was present on the cation of four of the top eight ILs.
A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins -lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using -lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10Â macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.
Background. The purpose of this study was to establish a reproductive schedule and examine reproductive traits that shape fecundity of the Bigeye Chub, Hybopsis amblops Cyprinidae, in the Flint River system of north Alabama. Methods. Life history traits associated with reproduction, growth, and maturation were assessed. Fish collections were made monthly from August, 2013, through July, 2014. Results. The Bigeye Chub in Alabama primarily spawns in April and May as indicated by gonadosomatic index (GSI), ovarian condition and clutch size. Average GSI values began to rise in February, peaked in April and May at over 13% for females and 1.6% for males, and showed a steep decline from May to June for both sexes. Average clutch size was highest in April at 812. Diameter of the most mature oocyte stage averaged 0.74 mm, relatively small compared to other cyprinids found in the Flint River. Discussion. The Bigeye Chub’s relatively large clutch size as a measure of fecundity places the species intermediate between opportunistic and periodic in the trilateral life history scheme of Winemiller and Rose. The species is apparently responding to a flow regime with a defined seasonality as well as predictability of flow and resources.
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