The membrane-integral glycerol 3-phosphate (G3P) acyltransferase PlsY catalyses the committed and essential step in bacterial phospholipid biosynthesis by acylation of G3P, forming lysophosphatidic acid. It contains no known acyltransferase motifs, lacks eukaryotic homologs, and uses the unusual acyl-phosphate as acyl donor, as opposed to acyl-CoA or acyl-carrier protein for other acyltransferases. Previous studies have identified several PlsY inhibitors as potential antimicrobials. Here we determine the crystal structure of PlsY at 1.48 Å resolution, revealing a seven-transmembrane helix fold. Four additional substrate- and product-bound structures uncover the atomic details of its relatively inflexible active site. Structure and mutagenesis suggest a different acylation mechanism of ‘substrate-assisted catalysis’ that, unlike other acyltransferases, does not require a proteinaceous catalytic base to complete. The structure data and a high-throughput enzymatic assay developed in this work should prove useful for virtual and experimental screening of inhibitors against this vital bacterial enzyme.

Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 °C (20-min heating). By contrast, the limit for the two popular GFPs is 64 °C and 74 °C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies.

Submonolayer thin films of a three-ring bent-core (or banana-shaped) compound, m-bis(4-n-octyloxystyryl)benzene (m-OSB), were vacuum-deposited on a mica surface, and a spontaneous transition from monolayer films to bilayer crystals was observed at room temperature, which was ascribed to the specific molecular shape and polar layered packing of the bent-core molecules [Tang et al. J. Phys. Chem. B 2004, 108 (34), 12921-12926]. The crystal nucleation and growth from the monolayer films as well as the melting phase transition from the bilayer crystals were investigated using atomic force microscopy (AFM). It was shown that after initial nucleation, the crystal growth was achieved through three pathways: direct absorption of molecules from monolayer films, molecular cluster diffusion, and quasi-Ostwald ripening. When annealing the bilayer crystals at elevated temperatures, morphological change from a bilayer to a monolayer was observed, and some new islands with fingerlike patterns were formed during this process, which resulted from a diffusion-controlled growth of the molten molecules. In general, the high-resolution AFM in combination with the molecularly thin m-OSB films provided us with direct visualization of nucleation, crystal growth, melting, and film morphology evolution on the mesoscopic scale, which are of fundamental interest from the theoretical viewpoint and are of central importance for the control of interfacial properties in practical applications.