Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a non-heme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2. Here, using EPR, Mössbauer, and UV-Vis spectroscopies, we explored the binding mode of cysteamine and RGS5 to human and mouse ADO proteins in their physiologically relevant ferrous form. This characterization revealed that in the presence of nitric oxide as a spin probe and oxygen surrogate, both the small molecule and the peptide substrates coordinate to the iron center with their free thiols in a monodentate binding mode, in sharp contrast to binding behaviors observed in other thiol dioxygenases. We observed a substrate-bound B-type dinitrosyl iron center complex in ADO, suggesting the possibility of dioxygen binding to the iron ion in a side-on mode. Moreover, we observed a substrate-mediated reduction of the ferric to the ferrous oxidation state at the iron center. Subsequent MS analysis indicated corresponding disulfide formation of the substrates, suggesting that the presence of the substrate could reactivate ADO to defend against oxidative stress. The findings of this work contribute to the understanding of the substrate interaction in ADO and fill a gap in our knowledge of the substrate specificity of thiol dioxygenases.

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Conformational changes in the b2a2 and b6a6 loops in the alpha subunit of tryptophan synthase (aTS) are important for enzyme catalysis and coordinating substrate channeling with the beta subunit (bTS). It was previously shown that disrupting the hydrogen bond interactions between these loops through the T183V substitution on the b6a6 loop decreases catalytic efficiency and impairs substrate channeling. Results presented here also indicate that the T183V substitution decreases catalytic efficiency in Escherchia coli aTS in the absence of the bTS subunit. Nuclear magnetic resonance (NMR) experiments indicate that the T183V substitution leads to local changes in the structural dynamics of the b2a2 and b6a6 loops. We have also used NMR chemical shift covariance analyses (CHESCA) to map amino acid networks in the presence and absence of the T183V substitution. Under conditions of active catalytic turnover, the T183V substitution disrupts long-range networks connecting the catalytic residue Glu49 to the aTS-bTS binding interface, which might be important in the coordination of catalytic activities in the tryptophan synthase complex. The approach that we have developed here will likely find general utility in understanding long-range impacts on protein structure and dynamics of amino acid substitutions generated through protein engineering and directed evolution approaches, and provide insight into disease and drug-resistance mutations.

SUMMARY Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using NMR spectroscopy. The PIP-binding site was located on a highly dynamic α-helix that also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses.

b S Supporting Information ' BACKGROUND National awareness for science education reform has come to the forefront and grants have been solicited to explore and expand innovative approaches to teaching science. 1 Because of the nature of chemical research today, chemistry education has become increasingly multidisciplinary. However, starting with middle school and extending through high school, disciplines are separated into discrete subjects with little overlap. Discipline boundaries should be blurred because many of the concepts are inevitably intertwined.More attention needs to be given to underprivileged minorities and females in chemistry, as statistics show that these groups make up only a small percentage of the field. In terms of doctorate recipients in the physical sciences, 27.9% are female, while minorities account for much less, constituting 17.7% of recipients. 2 The fraction of individuals who are both female and part of a (racial or ethnic) minority group is only 4.9%. This problem can be addressed early on through science education at the level of KÀ12. Furthermore, researchers have stressed that high school and college collaborations in the form of outreach and mentoring programs have given science an "authentic voice". 3 Technology plays a significant role in promoting chemistry education, with positive outcomes from student attitudinal survey and standardized tests. 4 Kriftcher and colleagues have suggested that modern technology makes course content more interesting and directly motivates students to consider their futures in the field. 5 In Maine, schools were selected to test and explore the outcomes of a "laptop program" where all seventh grade students and teachers are provided with laptops to use in school and to take home. This program has resulted in students with increased focus, fewer discipline problems, lower detention rates, and increased enthusiasm. 6 Higher scores in mathematics, sciences, and visual and performing arts have also been observed. 6 Integrating technology with education, particularly computers, will likely play a major role in enhancing students' early learning experiences in grades KÀ12.