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Rainey, Jan K.

Permanent URI for this collectionhttps://hdl.handle.net/10222/27736

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  • ItemOpen Access
    A rotatable flat coil for static solid-state nuclear magnetic resonance spectroscopy
    (2005-08) Rainey, JK; DeVries, JS; Sykes, BD
    No abstract available.
  • ItemOpen Access
    Recombinant Minimalist Spider Wrapping Silk Proteins Capable of Native-Like Fiber Formation
    (2012-11) Xu, Lingling; Rainey, Jan K.; Meng, Qing; Liu, Xiang-Qin
    Spider silks are desirable biomaterials characterized by high tensile strength, elasticity, and biocompatibility. Spiders produce different types of silks for different uses, although dragline silks have been the predominant focus of previous studies. Spider wrapping silk, made of the aciniform protein (AcSp1), has high toughness because of its combination of high elasticity and tensile strength. AcSp1 in Argiope trifasciata contains a 200-aa sequence motif that is repeated at least 14 times. Here, we produced in E. coli recombinant proteins consisting of only one to four of the 200-aa AcSp1 repeats, designated W-1 to W-4. We observed that purified W-2, W-3 and W-4 proteins could be induced to form silk-like fibers by shear forces in a physiological buffer. The fibers formed by W-4 were similar to 3.4 mu m in diameter and up to 10 cm long. They showed an average tensile strength of 115 MPa, elasticity of 37%, and toughness of 34 J cm(-3). The smaller W-2 protein formed fewer fibers and required a higher protein concentration to form fibers, whereas the smallest W-1 protein did not form silk-like fibers, indicating that a minimum of two of the 200-aa repeats was required for fiber formation. Microscopic examinations revealed structural features indicating an assembly of the proteins into spherical structures, fibrils, and silk-like fibers. CD and Raman spectral analysis of protein secondary structures suggested a transition from predominantly alpha-helical in solution to increasingly beta-sheet in fibers.
  • ItemOpen Access
    A Novel C-Terminal Region within the Multicargo Type III Secretion Chaperone CesT Contributes to Effector Secretion
    (2013-02) Ramu, Thangadurai; Prasad, Madhulika Esther; Connors, Erica; Mishra, Amit; Thomassin, Jenny-Lee; Leblanc, Jason; Rainey, Jan K.; Thomas, Nikhil A.
    The enteropathogenic Escherichia coli (EPEC) multicargo chaperone CesT interacts with at least 10 effector proteins and is central to pathogenesis. CesT has been implicated in coordinating effector hierarchy, although the mechanisms behind this regulation are poorly understood. To address this question, we set out to functionally characterize CesT with respect to roles in (i) effector binding, (ii) effector recruitment to the type III secretion system (T3SS), and (iii) effector translocation into host cells. A CesT variant expression library was screened in EPEC using a newly developed semi-high-throughput secretion assay. Among many deficient CesT variants, a predominant number were localized to a novel CesT C-terminal region. These CesT C-terminal variants exhibited normal effector binding yet reduced effector secretion levels. Structural correlation and thermal spectroscopy analyses of purified CesT variants implicated multiple surface-exposed residues, a terminal helix region, and a flexible C-terminal triple-serine stretch in effector secretion. Site-directed mutagenesis of the flexible CesT C-terminal triple-serine sequence produced differential effector secretion, implicating this region in secretion events. Infection assays further indicated that the C-terminal region of CesT was important for NleA translocation into host cells but was dispensable for Tir translocation. The findings implicate the CesT C terminus in effector secretion and contribute to a model for multiple-cargo chaperone function and effector translocation into host cells during infection.