Engineering topics
Publications and source records attributed to Robinson, Carol V..
Complementary XFEL methods reveal O 2 reaction intermediates and global protein dynamics during Isopenicillin N synthase catalysis.
Abstract not provided
F 1 F o ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. It comprises two molecular motors coupled together by a central and a peripheral stalk. Proton flow through the F o motor generates rotation of the central stalk, inducing conformational changes in the F 1 motor that catalyzes ATP production. Here we present nine cryo-EM structures of E. coli ATP synthase to 3.1–3.4 Å resolution, in four discrete rotational sub-states, which provide a comprehensive structural model for this widely studied bacterial molecular machine. We observe torsional flexing of the entire complex and a rotational sub-step of F o associated with long-range conformational changes that indicates how this flexibility accommodates the mismatch between the 3- and 10-fold symmetries of the F 1 and F o motors. We also identify density likely corresponding to lipid molecules that may contribute to the rotor/stator interaction within the F o motor.
The term proteoform, introduced in these pages in 2013, has rapidly gained acceptance in the proteomics community. The challenge and importance of comprehensively identifying proteoforms in complex samples has been recognized, and reports have begun to appear of new platforms towards that end. However, one interesting central ambiguity has emerged, namely determining precisely what is meant by a “proteoform identification”. At present, the only practical approaches for establishing the exact primary structure of a proteoform employ mass spectrometry (MS), and there is a wide range of MS results that provide “proteoform identifications”. This seemingly small matter has significant impact, as the ambiguity in knowing what is meant by an “identification” makes it difficult to compare results from different laboratories and approaches. This situation hinders the ability of the community to evaluate technological progress and to efficiently expand biological knowledge. To address this issue, we propose a five-level system for classifying proteoform identifications. Here, the classification scheme stems directly from a consideration of the four types of possible ambiguity possible for a proteoform identification, ranging from the most subtle (i.e., precise localization of a post-translational modification, or PTM) to the most dramatic (i.e., ambiguity in the gene of origin). The five classes then correspond to the level of ambiguity present in the identification, ranging from no ambiguity at all (Level 1), to ambiguity of all four types (Level 5). Details of the scheme are provided in Table 1 and Supplementary Table 1, with specific use cases and examples provided in Supplementary Figure 1.
The cell envelope of Mycobacterium tuberculosis is notable for the abundance of mycolic acids (MAs), essential to mycobacterial viability, and of other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, which contributes to virulence and antibiotic resistance. However, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell-wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA-containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan, but the exact function of MmpL3 remains elusive. Here, we report a crystal structure of Mycobacterium smegmatis MmpL3 at a resolution of 2.59 Å, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.
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