Structures of MauG in complex with quinol and quinone MADH

MauG has been cocrystallized with methylamine dehydrogenase (MADH) with its TTQ cofactor in the o-quinol (TTQ(OQ)) and quinone (TTQ(OX)) forms and the structures of the resulting complexes have been solved. The TTQ(OQ) structure crystallized in either space group P2(1) or C2, while the TTQ(OX) structure crystallized in space group P1. The previously solved structure of MauG in complex with MADH bearing an incompletely formed TTQ cofactor (preMADH) also crystallized in space group P1, although with different unit-cell parameters. Despite the changes in crystal form, the structures are virtually identical, with only very minor changes at the protein-protein interface. The relevance of these structures with respect to the measured changes in affinity between MauG and various forms of MADH is discussed

Structural, NMR Spectroscopic and Computational Investigation of Hemin Loading in the Hemophore HasAp from Pseudomonas aeruginosa

Heat shock protein 90 (Hsp90) inhibition by modulation of the N-or C-terminal binding site has become an attractive strategy for the development of anti-cancer chemotherapeutics. The first Hsp90 C-terminus inhibitor, novobiocin, manifested a relatively high IC50 value of ~700 μM. Therefore, investigation of the novobiocin scaffold has led to analogs with improved antiproliferative activity (nanomolar concentrations) against several cancer cell lines. During these studies, novobiocin analogs that do not inhibit Hsp90 were identified; however, these analogs demonstrated potent anti-proliferative activity. Compound 2, a novobiocin analog, was identified as a MAPK pathway signaling disruptor that lacked Hsp90 inhibitory activity. In addition, structural modifications of compound 2 were identified that segregated Hsp90 inhibition from MAPK signaling disruption. These studies indicate that compound 2 represents a novel scaffold for disruption of MAPK pathway signaling and may serve as a useful structure for the generation of new anti-cancer agents

Leadership, staffing and quality of care in nursing homes

<p>Abstract</p> <p>Background</p> <p>Leadership and staffing are recognised as important factors for quality of care. This study examines the effects of ward leaders' task- and relationship-oriented leadership styles, staffing levels, ratio of registered nurses and ratio of unlicensed staff on three independent measures of quality of care.</p> <p>Methods</p> <p>A cross-sectional survey of forty nursing home wards throughout Norway was used to collect the data. Five sources of data were utilised: self-report questionnaires to 444 employees, interviews with and questionnaires to 13 nursing home directors and 40 ward managers, telephone interviews with 378 relatives and 900 hours of field observations. Separate multi-level analyses were conducted for quality of care assessed by relatives, staff and field observations respectively.</p> <p>Results</p> <p>Task-oriented leadership style had a significant positive relationship with two of the three quality of care indexes. In contrast, relationship-oriented leadership style was not significantly related to any of the indexes. The lack of significant effect for relationship-oriented leadership style was due to a strong correlation between the two leadership styles (<it>r </it>= 0.78). Staffing levels and ratio of registered nurses were not significantly related to any of the quality of care indexes. The ratio of unlicensed staff, however, showed a significant negative relationship to quality as assessed by relatives and field observations, but not to quality as assessed by staff.</p> <p>Conclusions</p> <p>Leaders in nursing homes should focus on active leadership and particularly task-oriented behaviour like structure, coordination, clarifying of staff roles and monitoring of operations to increase quality of care. Furthermore, nursing homes should minimize use of unlicensed staff and address factors related to high ratios of unlicensed staff, like low staff stability. The study indicates, however, that the relationship between staffing levels, ratio of registered nurses and quality of care is complex. Increasing staffing levels or the ratio of registered nurses alone is not likely sufficient for increasing quality of care.</p

Structure and Function of the Zinc Binding Protein ZrgA from <i>Vibrio cholerae</i>

ATP binding cassette (ABC) transporters are the primary means by which bacteria acquire trace elements from the environment. They rely on solute binding proteins (SBPs) to bind the relevant substrate and deliver it to the integral membrane permease for ATP-powered import into the cytoplasm. SBPs of cluster A-I are known to facilitate the transport of essential metals zinc, manganese, and iron, and many have been characterized to date. A group of ABC transporter operons dubbed zinc-regulated genes (zrg) have recently been shown to transport zinc with putative SBPs (zrgA) bearing no homology to the classical cluster A-I family, and a recent crystal structure of a representative protein from Pseudomonas aeruginosa shows no structural similarity to classical SBPs. Thus, the ZrgA proteins appear to represent a newly discovered family of zinc SBPs widespread among Gram-negative bacteria, including human pathogens. Here, we have determined the crystal structure of ZrgA from Vibrio cholerae and characterized its zinc binding in vitro and function in vivo. We also assessed the role of a histidine-rich sequence that appears to be a hallmark of ZrgA proteins that is particularly long in V. cholerae ZrgA. The results show that the zrgA gene is critical to the function of the operon, consistent with a function as an SBP in this system. Further, the His-rich region is not essential to the function of ZrgA, but it does provide additional zinc binding sites in vitro. The structure and zinc binding data for ZrgA reveal interesting differences between it and its homologue from P. aeruginosa, illustrating diversity within this little-studied protein family

Site-Directed Mutagenesis Of Gln103 Reveals The Influence Of This Residue On The Redox Properties And Stability Of Maug

The diheme enzyme MauG catalyzes a six-electron oxidation that is required for the posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived cofactor, tryptophan tryptophylquinone (TTQ). Crystallographic and computational studies have implicated Gln103 in stabilizing the FeIV=O moiety of the bis-FeIV state by hydrogen bonding. The role of Gln103 was probed by site-directed mutagenesis. Q103L and Q103E mutations resulted in no expression and very little expression of the protein, respectively. Q103A MauG exhibited oxidative damage when isolated. Q103N MauG was isolated at levels comparable to that of wild-type MauG and exhibited normal activity in catalyzing the biosynthesis of TTQ from preMADH. The crystal structure of the Q103N MauG-preMADH complex suggests that a water may mediate hydrogen bonding between the shorter Asn103 side chain and the FeIV=O moiety. The Q103N mutation caused the two redox potentials associated with the diferric/diferrous redox couple to become less negative, although the redox cooperativity of the hemes of MauG was retained. Upon addition of H2O2, Q103N MauG exhibits changes in the absorbance spectrum in the Soret and near-IR regions consistent with formation of the bis-FeIV redox state. However, the rate of spontaneous return of the spectrum in the Soret region was 4.5-fold greater for Q103N MauG than for wild-type MauG. In contrast, the rate of spontaneous decay of the absorbance at 950 nm, which is associated with charge-resonance stabilization of the high-valence state, was similar for wild-type MauG and Q103N MauG. This suggests that as a consequence of the mutation a different distribution of resonance structures stabilizes the bis-FeIV state. These results demonstrate that subtle changes in the structure of the side chain of residue 103 can significantly affect the overall protein stability of MauG and alter the redox properties of the hemes. © 2014 American Chemical Society