Peptidomimetic inhibitors targeting the CCR5-binding site on the human immunodeficiency virus type-1 gp120 glycoprotein complexed to CD4.

A helical tyrosine-sulfated epitope in CCR5 that is recognized by the human immunodeficiency virus type-1 envelope glycoprotein gp120 in its CD4-induced conformation can be mimicked structurally by a cyclic ß-hairpin peptide containing two sulfated tyrosine residues at positions i and i + 2 along one ß-strand.

Does chemical cross-linking with NHS esters reflect the chemical equilibrium of protein-protein noncovalent interactions in solution?

Chemical cross-linking in combination with mass spectrometry has emerged as a powerful tool to study noncovalent protein complexes. Nevertheless, there are still many questions to answer. Does the amount of detected cross-linked complex correlate with the amount of protein complex in solution? In which concentration and affinity range is specific cross-linking possible? To answer these questions, we performed systematic cross-linking studies with two complexes, using the N-hydroxysuccinimidyl ester disuccinimidyl suberate (DSS): (1) NCoA-1 and mutants of the interacting peptide STAT6Y, covering a K(D) range of 30 nM to >25 µM, and (2) α-thrombin and basic pancreatic trypsin inhibitor (BPTI), a system that shows a buffer-dependent K(D) value between 100 and 320 µM. Samples were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). For NCoA-1•STAT6Y, a good correlation between the amount of cross-linked species and the calculated fraction of complex present in solution was observed. Thus, chemical cross-linking in combination with MALDI-MS can be used to rank binding affinities. For the mid-affinity range up to about K(D) ≈ 25 µM, experiments with a nonbinding peptide and studies of the concentration dependence showed that only specific complexes undergo cross-linking with DSS. To study in which affinity range specific cross-linking can be applied, the weak α-thrombin•BPTI complex was investigated. We found that the detected complex is a nonspecifically cross-linked species. Consequently, based on the experimental approach used in this study, chemical cross-linking is not suitable for studying low-affinity complexes with K(D) >> 25 µM.

How to deal with weak interactions in noncovalent complexes analyzed by electrospray mass spectrometry: cyclopeptidic inhibitors of the nuclear receptor coactivator 1-STAT6.

Mass spectrometry, and especially electrospray ionization, is now an efficient tool to study noncovalent interactions between proteins and inhibitors. It is used here to study the interaction of some weak inhibitors with the NCoA-1/STAT6 protein with K(D) values in the microM range. High signal intensities corresponding to some nonspecific electrostatic interactions between NCoA-1 and the oppositely charged inhibitors were observed by nanoelectrospray mass spectrometry, due to the use of high ligand concentrations. Diverse strategies have already been developed to deal with nonspecific interactions, such as controlled dissociation in the gas phase, mathematical modeling, or the use of a reference protein to monitor the appearance of nonspecific complexes. We demonstrate here that this last methodology, validated only in the case of neutral sugar-protein interactions, i.e., where dipole-dipole interactions are crucial, is not relevant in the case of strong electrostatic interactions. Thus, we developed a novel strategy based on half-maximal inhibitory concentration (IC(50)) measurements in a competitive assay with readout by nanoelectrospray mass spectrometry. IC(50) values determined by MS were finally converted into dissociation constants that showed very good agreement with values determined in the liquid phase using a fluorescence polarization assay.

Molecular characterization of the NCoA-1-STAT 6 interaction.

Many protein-protein interactions involved in cell signalling, cell adhesion and regulation of transcription are mediated by short alpha-helical recognition motifs with the sequence Leu-Xaa-Xaa-Leu-Leu (LXXLL, where Xaa is any amino acid). Originally observed in cofactors that interact with hormone-activated nuclear receptors, LXXLL motifs are now known to occur in many transcription factors, including the STAT family, which transmit signals from activated cytokine receptors at the cell surface to target genes in the nucleus. STAT 6 becomes activated in response to IL-4 and IL-13, which regulate immune and anti-inflammatory responses. Structural studies have revealed how an LXXLL motif located in 2.5 turns of an alpha-helical peptide derived from STAT 6 provide contacts through the leucine side chains to the coactivator of transcription, NCoA-1. However, since many protein-protein interactions are mediated by LXXLL motifs, it is important to understand how specificity is achieved in this and other signalling pathways. Here, we show that energetically important contacts between STAT 6 and NCoA-1 are made in residues that flank the LXXLL motif, including the underlined residues in the sequence LLPPTEQDLTKLL. We also demonstrate how the affinity for NCoA-1 of peptides derived from this region of STAT 6 can be significantly improved by optimising knobs-into-holes contacts on the surface of the protein. The results provide important new insights into the origins of binding specificity, and might be of practical value in the design of novel small-molecule inhibitors of this important protein-protein interaction.

Quantitative single molecule measurements on the interaction forces of poly(L-glutamic acid) with calcite crystals.

The interaction between poly(L-glutamic acid) (PLE) and calcite crystals was studied with AFM-based single molecule force spectroscopy. Block copolymers of poly(ethylene oxide) (PEO) and PLE were synthesized and covalently attached to the tip of an AFM cantilever. In desorption measurements the molecules were allowed to adsorb on the calcite crystal faces and afterward successively desorbed. The corresponding desorption forces were detected with high precision, showing for example a force transition between the two blocks. Because of its importance in the crystallization process in biominerals, the PLE-calcite interaction was investigated as a function of the pH as well as the calcium concentration of the aqueous solution. The sensitivity of the technique was underlined by resolving different interaction forces for calcite (104) and calcite (100).

Bisoxazolines with one and two sidearms: stereodirecting ligands for copper-catalysed asymmetric allylic oxidations of alkenes.

A series of sidearm functionalized bisoxazoline ligands has been synthesized by reaction of the monolithiated methyl{bis(oxazolinyl)}methane with the appropriate electrophiles, and tested in the copper catalyzed asymmetric allylic oxidation of cyclohexene ("Kharasch-Sosnovski" reaction). The observed enantioselectivities were higher (up to 85% ee) than for the unfunctionalized bisoxazoline ("BOX") derivatives (ca. 60% ee). Regardless of the functional groups incorporated into the sidearm unit, the ee's obtained for the different derivatives were essentially indistinguishable. This implies that the sidearms do not interfere directly in this reaction and only play an indirect role by virtue of their steric demand. Three of the copper complexes have been characterized by X-ray diffraction, establishing a distorted octahedral coordination geometry around the copper atom in all three cases. In the elongated distorted CuN(2)O(4) octahedra, the two nitrogen atoms of the oxazolines and one oxygen atom of each acetate ligand occupy the 'equatorial' positions whereas the sidearms do not interact with the metal centres.

Simultaneous AFM manipulation and fluorescence imaging of single DNA strands.

We report combined atomic force and far-field fluorescence microscopic experiments which allow the simultaneous atomic force manipulation and optical observation of individual dye-labeled DNA molecules. A detailed understanding of the binding properties of DNA to different transparent surfaces is prerequisite for these investigations. Atomic force spectroscopy and fluorescence microscopy of single DNA strands yielded detailed insight into two different types of DNA binding onto transparent polylysine-coated and silanized glass surfaces. We subsequently demonstrate how the different binding can be exploited to perform two types of nanomanipulation experiments: On polylysine, strong electrostatic interactions over the whole length of the DNA strand enable the writing of micrometer-sized patterns. By contrast, the strong pointwise attachment of DNA to silanized surfaces allows horizontal stretching of single DNA strands to lengths exceeding 1.6 times the contour length of the DNA strand. With this new approach it is possible to directly observe the rupture of the strongly bonded DNA strand.

Highly stretched single polymers: atomic-force-microscope experiments versus ab-initio theory.

Experimental single-molecule stretching curves for three backbone architectures (single-stranded DNA, various types of peptides, polyvinylamine) are quantitatively compared with corresponding quantum-chemical (zero-temperature) ab-initio calculations in the high-force range of up to two nanonewtons. For high forces, quantitative agreement is obtained with the contour length of the polymers as the only fitting parameter. For smaller forces, the effects of chain fluctuations are accounted for by using recent theoretical results for the stretching response of a freely-rotating-chain model.

Single-molecule optomechanical cycle.

Light-powered molecular machines are conjectured to be essential constituents of future nanoscale devices. As a model for such systems, we have synthesized a polymer of bistable photosensitive azobenzenes. Individual polymers were investigated by single-molecule force spectroscopy in combination with optical excitation in total internal reflection. We were able to optically lengthen and contract individual polymers by switching the azo groups between their trans and cis configurations. The polymer was found to contract against an external force acting along the polymer backbone, thus delivering mechanical work. As a proof of principle, the polymer was operated in a periodic mode, demonstrating for the first time optomechanical energy conversion in a single-molecule device.

Long-Range Interaction Forces between Polymer-Supported Lipid Bilayer Membranes.

Much of the short-range forces and structures of softly supported DMPC bilayers has been described previously. However, one interesting feature of the measured force-distance profile that remained unexplained is the presence of a long-range exponentially decaying repulsive force that is not observed between rigidly supported bilayers on solid mica substrate surfaces. This observation is discussed in detail here based on recent static and dynamic surface force experiments. The repulsive forces in the intermediate distance regime (mica-mica separations from 15 to 40 nm) are shown to be due not to an electrostatic force between the bilayers but to compression (deswelling) of the underlying soft polyelectrolyte layer, which may be thought of as a model cytoskeleton. The experimental data can be fit by simple theoretical models of polymer interactions from which the elastic properties of the polymer layer can be deduced.