The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins

The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NYCOMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline.     

Environmental variables affecting small-scale distributions of five rotifer species in Lancaster Lake,Michigan

The small-scale distributions of the rotifers Polyarthra vulgaris, Synchaeta stylata, Conochilus unicornis, Hexarthra mira, and Asplanchna priodonta were investigated in Lancaster Lake, Cheboygan County, Michigan, July 21, 1974. Measurements were taken for 13 depths (at 1-m intervals) and at 4 times of the day (1:00 AM, 7:00 AM, 1:00 PM, and 7:00 PM). In addition, the abundances of 6 crustacean, 2 planktonic dipteran, and 10 algal species, as well as temperature, light, oxygen, chlorophyll a, alkalinity, pH and free carbon dioxide were measured. Whereas abiotic factors appeared to control large scale occupation of the lake, and excluded most species from the deeper portions of the hypolimnion, small-scale distributional variation of the rotifers depended upon biotic interactions, particularly with the crustacean zooplankton.     

P2 pyridine N-oxide thrombin inhibitors: a novel peptidomimetic scaffold

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).     

Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus

We have recently used a green fluorescent protein (GFP) fusion to the gammab protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The gammab protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAbeta but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, betab (TGB1), betac (TGB3), and betad (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.