Structure and Property Changes in Self-Assembled Lubricin Layers Induced by Calcium Ion Interactions.

Lubricin (LUB) is a "mucin-like" glycoprotein found in synovial fluids and coating the cartilage surfaces of articular joints, which is now generally accepted as one of the body's primary boundary lubricants and antiadhesive agents. LUB's superior lubrication and antiadhesion are believed to derive from its unique interfacial properties by which LUB molecules adhere to surfaces (and biomolecules, such as hyaluronic acid and collagen) through discrete interactions localized to its two terminal end domains. These regionally specific interactions lead to self-assembly behavior and the formation of a well-ordered "telechelic" polymer brush structure on most substrates. Despite its importance to biological lubrication, detailed knowledge on the LUB's self-assembled brush structure is insufficient and derived mostly from indirect and circumstantial evidence. Neutron reflectometry (NR) was used to directly probe the self-assembled LUB layers, confirming the polymer brush architecture and resolving the degree of hydration and level of surface coverage. While attempting to improve the LUB contrast in the NR measurements, the LUB layers were exposed to a 20 mM solution of CaCl2, which resulted in a significant change in the polymer brush structural parameters consisting of a partial denaturation of the surface-binding end-domain regions, partial dehydration of the internal mucin-domain "loop", and collapse of the outer mucin-domain surface region. A series of atomic force microscopy measurements investigating the LUB layer surface morphology, mechanical properties, and adhesion forces in phosphate-buffered saline and CaCl2 solutions reveal that the structural changes induced by calcium ion interactions also significantly alter key properties, which may have implications to LUB's efficacy as a boundary lubricant and wear protector in the presence of elevated calcium ion concentrations.

A mortise-tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes.

Bacterial autotransporters comprise a 12-stranded membrane-embedded ß-barrel domain, which must be folded in a process that entraps segments of an N-terminal passenger domain. This first stage of autotransporter folding determines whether subsequent translocation can deliver the N-terminal domain to its functional form on the bacterial cell surface. Here, paired glycine-aromatic 'mortise and tenon' motifs are shown to join neighbouring ß-strands in the C-terminal barrel domain, and mutations within these motifs slow the rate and extent of passenger domain translocation to the surface of bacterial cells. In line with this, biophysical studies of the autotransporter Pet show that the conserved residues significantly quicken completion of the folding reaction and promote stability of the autotransporter barrel domain. Comparative genomics demonstrate conservation of glycine-aromatic residue pairings through evolution as a previously unrecognized feature of all autotransporter proteins.

Screening of small molecule interactor library by using in-cell NMR spectroscopy (SMILI-NMR).

We developed an in-cell NMR assay for screening small molecule interactor libraries (SMILI-NMR) for compounds capable of disrupting or enhancing specific interactions between two or more components of a biomolecular complex. The method relies on the formation of a well-defined biocomplex and utilizes in-cell NMR spectroscopy to identify the molecular surfaces involved in the interaction at atomic scale resolution. Changes in the interaction surface caused by a small molecule interfering with complex formation are used as a read-out of the assay. The in-cell nature of the experimental protocol insures that the small molecule is capable of penetrating the cell membrane and specifically engaging the target molecule(s). Utility of the method was demonstrated by screening a small dipeptide library against the FKBP-FRB protein complex involved in cell cycle arrest. The dipeptide identified by SMILI-NMR showed biological activity in a functional assay in yeast.