We report the engineering of a new reversibly switching chromogenic protein, Dathail. Dathail was evolved from the extremely thermostable fluorescent proteins thermal green protein (TGP) and eCGP123 ...using directed evolution and ratiometric sorting. Dathail has two spectrally distinct chromogenic states with low quantum yields, corresponding to absorbance in a ground state with a maximum at 389nm, and a photo-induced metastable state with a maximum at 497nm. In contrast to all previously described photoswitchable proteins, both spectral states of Dathail are non-fluorescent. The photo-induced chromogenic state of Dathail has a lifetime of ~50min at 293K and pH7.5 as measured by UV–Vis spectrophotometry, returning to the ground state through thermal relaxation. X-ray crystallography provided structural insights supporting a change in conformation and coordination in the chromophore pocket as being responsible for Dathail's photoswitching. Neutron crystallography, carried out for the first time on a protein from the green fluorescent protein family, showed a distribution of hydrogen atoms revealing protonation of the chromophore 4-hydroxybenzyl group in the ground state. The neutron structure also supports the hypothesis that the photo-induced proton transfer from the chromophore occurs through water-mediated proton relay into the bulk solvent. Beyond its spectroscopic curiosity, Dathail has several characteristics that are improvements for applications, including low background fluorescence, large spectral separation, rapid switching time, and the ability to switch many times. Therefore, Dathail is likely to be extremely useful in the quickly developing fields of imaging and biosensors, including photochromic Förster resonance energy transfer, high-resolution microscopy, and live tracking within the cell.
Display omitted
•Dathail will be useful in imaging and biosensors, particularly pcFRET, high-resolution microscopy, and live cell tracking.•Dathail is a reversibly switching chromoprotein with two spectrally distinct chromogenic states with low quantum yields.•X-ray crystallography shows changes in conformation and coordination of the chromophore during Dathail's photoswitching.•Crystallography suggests photo-induced proton transfer from the chromophore with water-mediated proton relay to bulk solvent.•The first time that neutron crystallography has been used for a protein in the GFP family provides surprising new insights.
Ketopantoate hydroxymethyltransferase (KPHMT) catalyzes the first committed step in the biosynthesis of pantothenate, which is a precursor to coenzyme A and is required for penicillin biosynthesis. ...The crystal structure of KPHMT from
Mycobacterium tuberculosis was determined by the single anomalous substitution (SAS) method at 2.8 Å resolution. KPHMT adopts a structure that is a variation on the (β/α) barrel fold, with a metal binding site proximal to the presumed catalytic site. The protein forms a decameric complex, with subunits in opposing pentameric rings held together by a swapping of their C-terminal α helices. The structure reveals KPHMT's membership in a small, recently discovered group of (β/α) barrel enzymes that employ domain swapping to form a variety of oligomeric assemblies. The apparent conservation of certain detailed structural characteristics suggests that KPHMT is distantly related by divergent evolution to enzymes in unrelated pathways, including isocitrate lyase and phosphoenolpyruvate mutase.
Background: Translation initiation factor 5A (IF-5A) is reported to be involved in the first step of peptide bond formation in translation, to be involved in cell-cycle regulation and to be a ...cofactor for the Rev and Rex transactivator proteins of human immunodeficiency virus-1 and T-cell leukemia virus I, respectively. IF-5A contains an unusual amino acid, hypusine (N-epsilon-(4-aminobutyl-2-hydroxy)lysine), that is required for its function. The first step in the post-translational modification of lysine to hypusine is catalyzed by the enzyme deoxyhypusine synthase, the structure of which has been published recently.
Results: IF-5A from the archebacterium
Pyrobaculum aerophilum has been heterologously expressed in
Escherichia coli with selenomethionine substitution. The crystal structure of IF-5A has been determined by multiwavelength anomalous diffraction and refined to 1.75 å. Unmodified
P. aerophilum IF-5A is found to be a
β structure with two domains and three separate hydrophobic cores.
Conclusions: The lysine (Lys42) that is post-translationally modified by deoxyhypusine synthase is found at one end of the IF-5A molecule in an turn between
β strands
β4 and
β5; this lysine residue is freely solvent accessible. The C-terminal domain is found to be homologous to the cold-shock protein CspA of
E. coli, which has a well characterized RNA-binding fold, suggesting that IF-5A is involved in RNA binding.
Recovery colleges: quality and outcomes Meddings, Sara; McGregor, Jane; Roeg, Waldo ...
Mental health and social inclusion,
11/2015, Letnik:
19, Številka:
4
Journal Article
Purpose
– The purpose of this paper is to review the available evidence regarding the effectiveness and cost effectiveness of Recovery Colleges. To make suggestions for future research.
...Design/methodology/approach
– Selective review of relevant published studies, including reports in the “grey” literature.
Findings
– Despite methodological limitations, it has been consistently found that attendance at Recovery Colleges is perceived to be useful and to help people progress towards their recovery goals. There is some evidence of reductions in service use (and therefore costs). In addition, there is evidence of beneficial effects for peer trainers and possible positive impact on staff attitudes.
Research limitations/implications
– The existing research highlights the need for further robust studies, using both qualitative and quantitative methods, to understand better the overall impact of Recovery Colleges and the underlying mechanisms of change.
Practical implications
– There is a need for further studies of the relationship between the “key defining features” and outcomes. This means the collection and pooling of systematic, “practice-based” evidence.
Social implications
– The introduction of an explicitly recovery educational (“learning”) model into mainstream mental health services seems to have a profound effect on reducing the power differences inherent in traditional professional/patient relationships. If this can be replicated across organisations it could facilitate the kind of fundamental cultural change necessary to give back recovery to the people who have always owned it.
Originality/value
– The information collected together in this paper is already publicly available, however it is difficult to find. The analysis and interpretation is original.
Purpose - The purpose of this paper is to review the available evidence regarding the effectiveness and cost effectiveness of Recovery Colleges. To make suggestions for future research. ...Design/methodology/approach - Selective review of relevant published studies, including reports in the "grey" literature. Findings - Despite methodological limitations, it has been consistently found that attendance at Recovery Colleges is perceived to be useful and to help people progress towards their recovery goals. There is some evidence of reductions in service use (and therefore costs). In addition, there is evidence of beneficial effects for peer trainers and possible positive impact on staff attitudes. Research limitations/implications - The existing research highlights the need for further robust studies, using both qualitative and quantitative methods, to understand better the overall impact of Recovery Colleges and the underlying mechanisms of change. Practical implications - There is a need for further studies of the relationship between the "key defining features" and outcomes. This means the collection and pooling of systematic, "practice-based" evidence. Social implications - The introduction of an explicitly recovery educational ("learning") model into mainstream mental health services seems to have a profound effect on reducing the power differences inherent in traditional professional/patient relationships. If this can be replicated across organisations it could facilitate the kind of fundamental cultural change necessary to give back recovery to the people who have always owned it. Originality/value - The information collected together in this paper is already publicly available, however it is difficult to find. The analysis and interpretation is original.
Recent advancements in liver transplantation have resulted in extended survival both for grafts and recipients. Such improvement, together with the shortage of donor organs has prompted expansion of ...the donor pool to include less than ideal donors, especially in life-threatening situations. The use of older liver donors has been associated with lower long-term survival. However, potential morbidity such as gallstone formation has not been explored. We analyzed bile composition in a child who developed cholesterol gallstones in the proximal bile duct two years after undergoing emergency liver transplantation with a liver from a 78-year-old donor. Oral administration of ursodeoxycholic acid (ursodiol) shifted the cholesterol composition of the bile from a supersaturated, potentially crystallized state to a liquid (micellar) state. Unlike cyclosporin A, FK506 showed an increase in the proportion of chenodeoxycholic acid and a decrease in the proportion of cholic acid, and thus may exhibit minimal or no hepatotoxic effect. Thus, in donor livers with factors known to be associated with cholesterol gallstone formation (such as age, sex, or obesity), one may consider analyzing the bile composition at the time of procurement. Depending on cholesterol and bile acid composition the use of FK506 with or without addition of ursodeoxycholic acid may be warranted.
PDF
