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Adenylylsulfate reductase (adenosine 5'-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-[Angstrom] resolution. Different from the [[alpha].sub.2][[beta].sub.2]-heterotetramer of the Archaeoglobusfulgidus, the overall structure of APSR from D. gigas comprises six [alpha][beta]-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the [alpha]-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the [alpha]-helix (amino acid 289 to 299) in the [alpha]-subunit. The positively charged residue Argl60 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the [beta]-subunit wraps around the [alpha]-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the ot- subunit from another [alpha][beta]-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the [alpha]-subunit and Asp159 in the C terminus of the [beta]-subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the [beta]-subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the [beta]-subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the [beta]-subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfate-reducing bacteria might self-regulate its activity through the C terminus of the [beta]-subunit. doi: 10.1128/JB.00583 -09

Whether turns play an active or passive role in protein folding remains a controversial issue at this juncture. Here we use a photolabile cage strategy in combination with laser-flash photolysis and photoacoustic calorimetry to study the effects of different turns on the kinetics of [beta]-hairpin refolding on a nanosecond time scale. This strategy opens up a temporal window to allow the observation of early kinetic events in the protein refolding process at ambient temperature and pH without interference from any denaturants. Our results provide direct evidence demonstrating that even a one-residue difference in the turn region can change the refolding kinetics of a peptide. This observation suggests an active role for turn formation in directing protein folding.

In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 [micro]M. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is [Tilde] 13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl [beta]-D-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an [alpha][beta][gamma] protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme.

It has been known that the structural transition from Pr[P.sup.C] to Pr[P.sup.Sc] leads to the prion formation. This putative conformational change challenges the central dogma of the protein folding theory--'one sequence, one structure.' Generally, scientists believe that there must be either a posttranslational modification or environmental factors involved in this event. However, all of the efforts to solve the mystery of the Pr[P.sup.C] to Pr[P.sup.Sc] transition have ended in vain so far. Here we provide evidence linking O-linked glycosylation to the structural transition based on prion peptide studies. We find that the O-linked [alpha]-GalNAc at Ser-135 suppresses the formation of amyloid fibril formation of the prion peptide at physiological salt concentrations, whereas the peptide with the same sugar at Ser-132 shows the opposite effect. Moreover, this effect is sugar specific. Replacing [alpha]-GalNAc with [beta]-GlcNAc does not yield the same effect.