Rationally seeded computational protein design of ɑ-helical barrels

Computational protein design is advancing rapidly. Here we describe efficient routes starting from validated parallel and antiparallel peptide assemblies to design two families of α-helical barrel proteins with central channels that bind small molecules. Computational designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides, and adjacent helices are connected by loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix–turn–helix–turn–helix motifs that are packed onto the barrels. Throughout these computational pipelines, residues that define open states of the barrels are maintained. This minimizes sequence sampling, accelerating the design process. For each of six targets, just two to six synthetic genes are made for expression in Escherichia coli. On average, 70% of these genes express to give soluble monomeric proteins that are fully characterized, including high-resolution structures for most targets that match the design models with high accuracy

Spatiotemporal control of chemical microenvironments of single cells

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Spatiotemporal control of chemical microenvironments of single cells

Abstract only

Involvement of the Arp2/3 Complex and Scar2 in Golgi Polarity in Scratch Wound Models

Cell motility and cell polarity are essential for morphogenesis, immune system function, and tissue repair. Many animal cells move by crawling, and one main driving force for movement is derived from the coordinated assembly and disassembly of actin filaments. As tissue culture cells migrate to close a scratch wound, this directional extension is accompanied by Golgi apparatus reorientation, to face the leading wound edge, giving the motile cell inherent polarity aligned relative to the wound edge and to the direction of cell migration. Cellular proteins essential for actin polymerization downstream of Rho family GTPases include the Arp2/3 complex as an actin nucleator and members of the Wiskott–Aldrich Syndrome protein (WASP) family as activators of the Arp2/3 complex. We therefore analyzed the involvement of the Arp2/3 complex and WASP-family proteins in in vitro wound healing assays using NIH 3T3 fibroblasts and astrocytes. In NIH 3T3 cells, we found that actin and Arp2/3 complex contributed to cell polarity establishment. Moreover, overexpression of N-terminal fragments of Scar2 (but not N-WASP or Scar1 or Scar3) interfere with NIH 3T3 Golgi polarization but not with cell migration. In contrast, actin, Arp2/3, and WASP-family proteins did not appear to be involved in Golgi polarization in astrocytes. Our results thus indicate that the requirement for Golgi polarity establishment is cell-type specific. Furthermore, in NIH 3T3 cells, Scar2 and the Arp2/3 complex appear to be involved in the establishment and maintenance of Golgi polarity during directed migration