A specific transition state for S-peptide combining with folded S-protein and then refolding

We measured the folding and unfolding kinetics of mutants for a simple protein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form ribonuclease S analogues: initially, S-peptide is disordered whereas S-protein is folded. The fluorescent probe provides a convenient spectroscopic probe for the reaction. The association rate constant, kon, and the dissociation rate constant, koff, were both determined for two sets of mutants. The dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows kon and koff to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding and refolding. The first set of mutants tests the role of the α-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the α-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log kon vs. log Kd for this series of mutants is linear over a very wide range, with a slope of −0.3, indicating that almost all of the molecules fold via a transition state involving the helix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, which are known to be important for binding S-peptide to S-protein and which also contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transition state. The results provide a remarkably clear description of a folding transition state.

Double-level “orthogonal” dynamic combinatorial libraries on transition metal template

Dynamic combinatorial libraries are mixtures of compounds that exist in a dynamic equilibrium and can be driven to compositional self adaptation via selective binding of a specific assembly of certain components to a molecular target. We present here an extension of this initial concept to dynamic libraries that consists of two levels, the first formed by the coordination of terpyridine-based ligands to the transition metal template, and the second, by the imine formation with the aldehyde substituents on the terpyridine moieties. Dialdehyde 7 has been synthesized, converted into a variety of ligands, oxime ethers L11–L33 and acyl hydrazones L44–L77, and subsequently into corresponding cobalt complexes. A typical complex, Co(L22)22+ is shown to engage in rapid exchange with a competing ligand L11 and with another complex, Co(L22)22+ in 30% acetonitrile/water at pH 7.0 and 25°C. The exchange in the corresponding Co(III) complexes is shown to be much slower. Imine exchange in the acyl hydrazone complexes (L44–L77) is strongly controlled by pH and temperature. The two types of exchange, ligand and imine, can thus be used as independent equilibrium processes controlled by different types of external intervention, i.e., via oxidation/reduction of the metal template and/or change in the pH/temperature of the medium. The resulting double-level dynamic libraries are therefore named orthogonal, in similarity with the orthogonal protecting groups in organic synthesis. Sample libraries of this type have been synthesized and showed the complete expected set of components in electrospray ionization MS.

Chemically distinct transition states govern rapid dissociation of single L-selectin bonds under force

Carbohydrate–protein bonds interrupt the rapid flow of leukocytes in the circulation by initiation of rolling and tethering at vessel walls. The cell surface carbohydrate ligands are glycosylated proteins like the mucin P-selectin glycoprotein ligand-1 (PSGL-1), which bind ubiquitously to the family of E-, P-, and L-selectin proteins in membranes of leukocytes and endothelium. The current view is that carbohydrate–selectin bonds dissociate a few times per second, and the unbinding rate increases weakly with force. However, such studies have provided little insight into how numerous hydrogen bonds, a Ca2+ metal ion bond, and other interactions contribute to the mechanical strength of these attachments. Decorating a force probe with very dilute ligands and controlling touch to achieve rare single-bond events, we have varied the unbinding rates of carbohydrate–selectin bonds by detachment with ramps of force/time from 10 to 100,000 pN/sec. Testing PSGL-1, its outer 19 aa (19FT), and sialyl LewisX (sLeX) against L-selectin in vitro on glass microspheres and in situ on neutrophils, we found that the unbinding rates followed the same dependence on force and increased by nearly 1,000-fold as rupture forces rose from a few to ≈200 pN. Plotted on a logarithmic scale of loading rate, the rupture forces reveal two prominent energy barriers along the unbinding pathway. Strengths above 75 pN arise from rapid detachment (<0.01 sec) impeded by an inner barrier that requires a Ca2+ bond between a single sLeX and the lectin domain. Strengths below 75 pN occur under slow detachment (>0.01 sec) impeded by the outer barrier, which appears to involve an array of weak (putatively hydrogen) bonds.