Methanogens and anaerobic methane-oxidizing archaea (ANME) are important players in the global carbon cycle. Methyl-coenzyme M reductase (MCR) is a key enzyme in methane metabolism, catalyzing the last step in methanogenesis and the first step in anaerobic methane oxidation. Divergent mcr and mcr-like genes have recently been identified in uncultured archaeal lineages. However, the assembly and biochemistry of MCRs from uncultured archaea remain largely unknown. Here we present an approach to study MCRs from uncultured archaea by heterologous expression in a methanogen, Methanococcus maripaludis. Promoter, operon structure, and temperature were important determinants for MCR production. Both recombinant methanococcal and ANME-2 MCR assembled with the host MCR forming hybrid complexes, whereas tested ANME-1 MCR and ethyl-coenzyme M reductase only formed homogenous complexes. Together with structural modeling, this suggests that ANME-2 and methanogen MCRs are structurally similar and their reaction directions are likely regulated by thermodynamics rather than intrinsic structural differences.
Preparation of samples for nuclear magnetic resonance (NMR) characterization of larger proteins requires enrichment with less abundant, NMR-active, isotopes such as 13C and 15N. This is routine for proteins that can be expressed in bacterial culture where low-cost isotopically enriched metabolic substrates can be used. However, it can be expensive for glycosylated proteins expressed in mammalian culture where more costly isotopically enriched amino acids are usually used. We describe a simple, relatively inexpensive procedure in which standard commercial media is supplemented with 13C-enriched glucose to achieve labeling of all glycans plus all alanines of the N-terminal domain of the highly glycosylated protein, CEACAM1. We demonstrate an ability to detect partially occupied N-glycan sites, sites less susceptible to processing by an endoglycosidase, and some unexpected truncation of the amino acid sequence. The labeling of both the protein (through alanines) and the glycans in a single culture requiring no additional technical expertise past standard mammalian expression requirements is anticipated to have several applications, including structural and functional screening of the many glycosylated proteins important to human health.
Substitution of a glycine residue for the serine residue at position six in the nonapeptide bradykinin leads to a dramatic increase in the cis/trans ratio about the sixth peptide bond (~38% cis). Decreases in the cis/trans ratio determined by NMR in 1 M NaC104 and in 60% ,2CH30H have been correlated with decreases in the 219-nm CD band. Chemical shifts of the 20% 13C-labeled Gly6 residue exhibit a very different pattern from that observed in the dipeptide Gly-L-Pro; the cis-trans shift difference is ~0 ppm for the glycine carbonyl resonances and ~1.1 ppm for the glycine methylene resonances. In contrast, the cisand rrani-glycine carbonyl resonances are resolved in 60% methanol and in 1 M NaCKAi. A series of model peptides has been examined to determine the structural factors leading to the observed shift pattern. These studies indicate the importance of Phe8 both in determining the cis/trans ratio and in producing the observed chemical shifts. Glycine resonances in the tripeptide Gly-Pro-Phe are qualitatively similar to those observed in [Gly6]-bradykinin. The most probable explanation of these data is a favorable hydrophobic/solvent-excluding Pro-Phe association in the cis peptide, which leads to a rotation about the Pro C"carbonyl bond, placing the carbonyl oxygen in close proximity to the Gly6 methylene carbon. In the trans peptide, electrostatic repulsion between the Gly and Pro carbonyl oxygens opposes this effect. Significant differences in the cis/trans ratios between bradykinin and [Gly6]-bradykinin have prompted careful bioassay studies, which show the potency of [Gly6]-bradykinin to be only 50-70% that of bradykinin, although at saturating levels of peptide the responses are within experimental error. These data provide clues about the functional role of serine in bradykinin.
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