Chemokines regulate the chemotaxis, development, and differentiation of many cell types enabling the regulation of routine immunosurveillance and immunological adaptation. CC chemokine receptor 1 (CCR1) is the target of 11 chemokines. This promiscuity of receptorligand interactions and the potential for functional redundancy has led us to investigate the selective activation of CCR1-coupled pathways by known CCR1 agonists. Chemokines leukotactin-1, macrophage inflammatory protein (MIP)-1 § , monocyte chemotactic peptide (MCP)-3, RANTES, and MIP-1ˇall inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP-1ˇwas unable to signal via G 14 -, G 16 -or chimeric 16z44-coupled pathways. In a stable cell line expressing CCR1 and G § 14 , all of these five chemokines along with hemofiltrate CC chemokine (HCC)-1 and myeloid progenitor inhibitory factor (MPIF)-1 were able to stimulate G i/o -coupled pathways, but MIP-1ˇ, HCC-1 and MPIF-1 were unable to activate G 14 -mediated stimulation of phospholipase C g activity. In addition, MIP-1ˇwas unable to promote the phosphorylation of extracellular signalregulated kinase and c-Jun N-terminal kinase. This suggests that different chemokines are able to selectively activate CCR1-coupled pathways, probably because of different intrinsic ligand efficacies. CCR1 and G § 14 or G § 16 are co-expressed in several cell types and we hypothesize that selective activation of chemokine receptors provides a mechanism by which chemokines are able to fine-tune intracellular signaling pathways.

We report the design and engineering of a robust, reagentless fluorescent glucose biosensor based on the periplasmic glucose-binding protein obtained from Thermotoga maritima (tmGBP). The gene for this protein was cloned from genomic DNA and overexpressed in Escherichia coli, the identity of its cognate sugar was confirmed, ligand binding was studied, and the structure of its glucose complex was solved to 1.7 Å resolution by X-ray crystallography. TmGBP is specific for glucose and exhibits high thermostability (midpoint of thermal denaturation is 119 6 1°C and 144 6 2°C in the absence and presence of 1 mM glucose, respectively). A series of fluorescent conjugates was constructed by coupling single, environmentally sensitive fluorophores to unique cysteines introduced by site-specific mutagenesis at positions predicted to be responsive to ligand-induced conformational changes based on the structure. These conjugates were screened to identify engineered tmGBPs that function as reagentless fluorescent glucose biosensors. The Y13C d Cy5 conjugate is bright, gives a large response to glucose over concentration ranges appropriate for in vivo monitoring of blood glucose levels (1-30 mM), and can be immobilized in an orientation-specific manner in microtiter plates to give a reversible response to glucose. The immobilized protein retains its response after long-term storage at room temperature.Keywords: glucose-binding protein; structure; thermostable; fluorescent biosensor Bacterial periplasmic binding proteins (PBPs) are members of a superfamily that mediate chemotaxis and uptake of sugars, amino acids, oligopeptides, and metals (Ames 1986). The binding pocket of each PBP is located between two domains, which are linked by a hinge region (Quiocho and Ledvina 1996). Upon ligand binding, PBPs undergo a conformational change mediated by a hingebending event to adopt a closed form. Mesophilic PBPs have been successfully engineered as reagentless fluorescent (Gilardi et al. 1994;Tolosa et al. 1999 Abbreviations: PBP, periplasmic binding protein; tmGBP, Thermotoga maritima glucose-binding protein; ecRBP, Escherichia coli ribose-binding protein; ecGBP, E. coli glucose-binding protein; ttGBP, Thermus thermophilus glucose-binding protein; ecMBP, E. coli maltose-binding protein; ORF, open reading frame; CD, circular dichroism; GuHCl, guanidine hydrogen chloride; IANBD, N,N9-dimethyl-N-(iodoacetyl)-N9-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine; Zif, zinc finger domain; TCEP, Tris(2-carboxyethyl)-phosphine hydrochloride; IMAC, immobilized metal affinity chromatography; SV, Stern-Volmer.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi