Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a β-helical arrangement, whereas others suggest a parallel in-register organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13C- and 15N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems.

Genome Sequence of Lactobacillus brevis Strain D6, Isolated from Smoked Fresh Cheese

The autochthonous Lactobacillus brevis strain D6, isolated from smoked fresh cheese, carries a 45-kDa S-layer protein. Strain D6 has shown adhesion to extracellular matrix proteins and to Caco-2 intestinal epithelial cells, as well as immunomodulatory potential and beneficial milk technological properties. Hence, it could be used as a potential probiotic starter culture for cheese production.

Characterization of PitA and PitB fromEscherichia coli

ABSTRACT Escherichia coli contains two major systems for transporting inorganic phosphate (Pi). The low-affinity Pi transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (pstSCAB) is induced at low external Pi concentrations by the phoregulon and is an ABC transporter. We isolated a third putative Pi transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional Pi transporter that may be repressed at low Pi levels by the pho regulon. While apitB + cosmid clone allowed growth on medium containing 500 μM Pi, E. coli with wild-type genomic pitB (pitAΔpstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitBΔpstC345 triple mutant. The mutation ΔpstC345 constitutively activates thepho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting thephoB-phoR operon allowed thepitB + pitAΔpstC345 strain to utilize Pi, with Pi uptake rates significantly higher than background levels. In addition, the apparent K m of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitAmutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.