Ligand binding site identification by higher dimension molecular dynamics.

We propose a new molecular dynamics (MD) protocol to identify the binding site of a guest within a host. The method utilizes a four spatial (4D) dimension representation of the ligand allowing for rapid and efficient sampling within the receptor. We applied the method to two different model receptors characterized by diverse structural features of the binding site and different ligand binding affinities. The Abl kinase domain is comprised of a deep binding pocket and displays high affinity for the two chosen ligands examined here. The PDZ1 domain of PSD-95 has a shallow binding pocket that accommodates a peptide ligand involving far fewer interactions and a micromolar affinity. To ensure completely unbiased searching, the ligands were placed in the direct center of the protein receptors, away from the binding site, at the start of the 4D MD protocol. In both cases, the ligands were successfully docked into the binding site as identified in the published structures. The 4D MD protocol is able to overcome local energy barriers in locating the lowest energy binding pocket and will aid in the discovery of guest binding pockets in the absence of a priori knowledge of the site of interaction.

Structural analysis of the human cannabinoid receptor one carboxyl-terminus identifies two amphipathic helices.

Recent research has implicated the C-terminus of G-protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C-tail has proven a formidable task. Here, a peptide corresponding to the full-length C-tail of the human CB1 receptor (residues 400-472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an alpha-helical conformation in negatively charged and zwitterionic detergents (48-51% and 36-38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self-association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled ((15)N- and (13)C-) C-terminus in dodecylphosphocholine (DPC) identified two amphipathic alpha-helical domains. The first domain, S401-F412, corresponds to the helix 8 common to G protein-coupled receptors while the second domain, A440-M461, is a newly identified structural motif in the distal region of the carboxyl-terminus of the receptor. Molecular modeling of the C-tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions.

Carbohydrate surface attachment characterized by sum frequency generation spectroscopy.

Covalent surface attachment of carbohydrate moieties using maleimide-sulfhydril reaction was characterized by surface-selective vibrational sum-frequency generation (VSFG) spectroscopy. The comparative VSFG spectra of the precursor maleimide-terminated SAM and the product glucose adlayer reveal the high efficiency of the surface coupling reaction (>90%) and the details of the molecular organization of the formed carbohydrate adlayer. The glucose groups are orientationally well ordered, as judged by their sharp C-H stretch bands. The chemical structure of the linker can significantly affect the orientation of the carbohydrate moiety at the surface. Two alkanethiol linkers of different chain lengths (11 and 16 carbons) yield similar orientations of the glucose in the adlayer whereas the cysteine-containing linker produces markedly different relative peak intensities of the glucose C-H stretch bands in the VSFG spectra, suggesting a significantly different orientation with respect to the surface plane.

Molecular organization in SAMs used for neuronal cell growth.

The attachment of cells onto solid supports is fundamental in the development of advanced biosensors or biochips. In this work, we characterize cortical neuron adhesion, growth, and distribution of an adhesive layer, depending on the molecular structure and composition . Neuronal networks are successfully grown on amino-terminated alkanethiol self-assembled monolayer (SAM) on a gold substrate without adhesion protein interfaces. Neuron adhesion efficiency was studied for amino-terminated, carboxy-terminated, and 1:1 mixed alkanethiol SAMs deposited on gold substrates. Atomic force microscopy and X-ray photoelectron spectroscopy were used to measure the roughness of gold substrate and thickness of SAM monolayers. Conformational ordering and ionic content of SAMs were characterized by vibrational sum frequency generation (VSFG) spectroscopy. Only pure amino-terminated SAMs provide efficient neuronal cell attachment. Ordering of the terminal amino groups does not affect efficiency of neuron adhesion. VSFG analysis shows that ordering of the terminal groups improves with decreasing surface roughness; however the number of gauche defects in alkane chains is independent of surface roughness. We monitor partial dissociation of carboxy groups in mixed SAMs that implies formation of NH3+ neighbors and appearance of catanionic structure. Such catanionic environment proved inefficient for neuron adhesion. Surface roughness of metal within the 0.7-2 nm range has little effect on the efficiency of neuron adhesion. This approach can be used to create new methods that help map structure-property relationships of biohybrid systems.

Effect of nanoscale geometry on molecular conformation: vibrational sum-frequency generation of alkanethiols on gold nanoparticles.

Vibrational sum frequency generation (VSFG) spectroscopy was used to study the nanoscale geometric effects on molecular conformation of dodecanethiol ligand on gold nanoparticles of varying size between 1.8 and 23 nm. By analyzing the CH3 and CH2 stretch transitions of dodecanethiol using the spectroscopic propensity rules for the SFG process, we observe the increase of the gauche defects in the alkyl chain of the ligand on the nanoparticle surface when the curvature approaches the size of the molecule ( approximately 1.6 nm). In contrast, linear infrared absorption and Raman spectra, governed by different selection rules, do not allow observation of the size-dependent conformational changes. The results are understood in terms of the geometric packing effect, where the curvature of the nanoparticle surface results in the increased conical volume available for the alkyl chain.