Biological properties of chemokines are believed to be influenced by their association with glycosaminoglycans. Surface plasmon resonance kinetic analysis shows that the CXC chemokine stromal cell-derived factor-1␣ (SDF-1␣), which binds the CXCR4 receptor, associates with heparin with an affinity constant of 38.4 nM (k on ‫؍‬ 2.16 ؋ 10 6 M ؊1 s ؊1 and k off ‫؍‬ 0.083 ؋ s ؊1 ). A modified SDF-1␣ (SDF-1 3/6) was generated by combined substitution of the basic cluster of residues Lys 24 , His 25 , and Lys 27 by Ser. SDF-1 3/6 conserves the global native structure and functional properties of SDF-1␣, but it is unable to interact with sensor chip-immobilized heparin. The biological relevance of these in vitro findings was investigated. SDF-1␣ was unable to bind in a CXCR4-independent manner on epithelial cells that were treated with heparan sulfate (HS)-degrading enzymes or constitutively lack HS expression. The inability of SDF-1 3/6 to bind to cells underlines the importance of the identified basic cluster for the physiological interactions of SDF-1␣ with HS. Importantly, the amino-terminal domain of SDF-1␣ which is required for binding to, and activation of, CXCR4 remains exposed after binding to HS and is recognized by a neutralizing monoclonal antibody directed against the first residues of the chemokine. Overall, these findings indicate that the Lys 24 , His 25 , and Lys 27 cluster of residues forms, or is an essential part of, the HS-binding site which is distinct from that required for binding to, and signaling through, CXCR4.

CCR5 is a functional receptor for MIP-1␣, MIP-1␤, RANTES (regulated on activation normal T cell expressed), MCP-2, and MCP-4 and constitutes the main coreceptor for macrophage tropic human and simian immunodeficiency viruses. By using CCR5-CCR2b chimeras, we have shown previously that the second extracellular loop of CCR5 is the major determinant for chemokine binding specificity, whereas the amino-terminal domain plays a major role for human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus coreceptor function. In the present work, by using a panel of truncation and alanine-scanning mutants, we investigated the role of specific residues in the CCR5 amino-terminal domain for chemokine binding, functional response to chemokines, HIV-1 gp120 binding, and coreceptor function. Truncation of the amino-terminal domain resulted in a progressive decrease of the binding affinity for chemokines, which correlated with a similar drop in functional responsiveness. Mutants lacking residues 2-13 exhibited fairly weak responses to high concentrations (500 nM) of RANTES or MIP-1␤. Truncated mutants also exhibited a reduction in the binding affinity for R5 Env proteins and coreceptor activity. Deletion of 4 or 12 residues resulted in a 50 or 80% decrease in coreceptor function, respectively. Alaninescanning mutagenesis identified several charged and aromatic residues (Asp-2, Tyr-3, Tyr-10, Asp-11, and Glu-18) that played an important role in both chemokine and Env high affinity binding. The overlapping binding site of chemokines and gp120 on the CCR5 amino terminus, as well as the involvement of these residues in the epitopes of monoclonal antibodies, suggests that these regions are particularly exposed at the receptor surface.

The aim of this review is to give a compendium of colorimetric assays and spectrophotometric‐based quantification methods applicable to solid‐phase organic chemistry. Comprehensive experimental details for commonly employed color tests performed on solid support will be documented. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 71:110–118, 2000/2001.

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Analytical construct technology has been successfully applied to the single-bead analysis of a split-mix combinatorial library. Substrates can be released from the resin by conventional cleavage for biological screening. Alternatively, for the purpose of analysis and quality control, cleavage at an orthogonal construct linker produces an analytical fragment incorporating the substrate. This analytical fragmnent is highly sensitized to electrospray mass spectrometry (ESI-MS) and is easily identified by isotope labeling. The construct cleavage rendered readily visible even those compounds that clearly could not be seen by conventional cleavage and mass spectrometry analysis. A 1H NMR control experiment proved that the compounds cleaved conventionally were, however, present in the sample in good yield and purity. In view of the data obtained, we think that this is a significant and important step toward solving our current quality control problems.