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Amelogenin constitutes ~90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest and most mineralized tissue in vertebrates—enamel. Most biophysical research with amelogenin uses recombinant protein expressed in Escherichia coli. In addition to providing copious amounts of protein, recombinant expression allows 13C‐ and 15N‐labeling for detailed structural studies using NMR spectroscopy. However, native amelogenin is phosphorylated at one position, Ser‐16 in murine amelogenin, and there is mounting evidence that Ser‐16 phosphorylation is important. Using a modified genetic code expansion protocol we have expressed and purified uniformly 13C‐, 15N‐labeled murine amelogenin (pS16M179) with ~95% of the protein being correctly phosphorylated. Homogeneous phosphorylation was achieved using commercially available, enriched, 13C‐, 15N‐labeled media, and protein expression was induced with isopropyl β‐D‐1‐thiogalactopyranoside at 310 K. Phosphoserine incorporation was verified from one‐dimensional 31P NMR spectra, comparison of 1H‐15N HSQC spectra, Phos‐tag SDS PAGE, and mass spectrometry. Phosphorus‐31 NMR spectra for pS16M179 under conditions known to trigger amelogenin self‐assembly into nanospheres confirm nanosphere models with buried N‐termini. Lambda phosphatase treatment of these nanospheres results in the dephosphorylation of pS16M179, confirming that smaller oligomers and monomers with exposed N‐termini are in equilibrium with nanospheres. Such 13C‐, 15N‐labeling of amelogenin with accurately encoded phosphoserine incorporation will accelerate biomineralization research to understand amelogenesis and stimulate the expanded use of genetic code expansion protocols to introduce phosphorylated amino acids into proteins. [ABSTRACT FROM AUTHOR]

Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the [C.sub.2]-[C.sub.9] alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for [C.sub.1]-[C.sub.5] alkanes and product inhibition profiles, sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by [[kappa].sub.cat]: [[kappa].sub.m] ratios). Relative rates of oxidation for [C.sub.1]-[C.sub.5] alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate [[kappa].sub.m] values. The low micromolar [[kappa].sub.m] for linear [C.sub.2]-[C.sub.5] alkanes and the millimolar [[kappa].sub.m] for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar [[kappa].sub.m] and [[kappa].sub.cat] values to those of methane. This inability to kinetically discriminate between the [C.sub.1] alkane and [C.sub.1] alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length [C.sub.2]-[C.sub.5] do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of [C.sub.3]-[C.sub.5] alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes [greater than or equal to] [C.sub.2], despite its high sequence homology with sMMO. These results represent, to the best of our knowledge, the first kinetic in vitro characterization of the closest known homologue of sMMO.

A mechanism of oxidation of a nucleotide, deoxyguanosine 5'-monophosphate by trans-d, l-1,2-diamoinocyclohexanetetrachloroplatinum to produce 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-monophosphate stoichiometrically is presented.

A detailed mechanism of the redox reaction between [Pt(sub IV)Cl(sub 4)(dach)] and 3'-d(guanosine monophosphate) (3-dGMP) is proposed. The Pt(super IV) complex blinds to N7 of guanine in cGMP, 9-Mxan, 5'-d[TTGTT]-3', and 5'-d[TTTTG]-3', but no subsequent transfer of electrons occurs in the reactions.