Genetically-encoded protein photosensors, including the LOV (light, oxygen, voltage) domain, are promising tools for engineering optical control of cellular behavior. We are only beginning to understand how to couple these light detectors to effectors of choice. We report a method that increases the dynamic range of an artificial photoswitch based on the LOV2 domain of A. sativa phototropin1 (AsLOV2). This approach can potentially be used to improve many AsLOV2-based photoswitches.

Although the ␤-rich self-assemblies are a major structural class for polypeptides and the focus of intense research, little is known about their atomic structures and dynamics due to their insoluble and noncrystalline nature. We developed a protein engineering strategy that captures a self-assembly segment in a water-soluble molecule. A predefined number of self-assembling peptide units are linked, and the ␤-sheet ends are capped to prevent aggregation, which yields a mono-dispersed soluble protein. We tested this strategy by using Borrelia outer surface protein (OspA) whose single-layer ␤-sheet located between two globular domains consists of two ␤-hairpin units and thus can be considered as a prototype of self-assembly. We constructed self-assembly mimics of different sizes and determined their atomic structures using x-ray crystallography and NMR spectroscopy. Highly regular ␤-sheet geometries were maintained in these structures, and peptide units had a nearly identical conformation, supporting the concept that a peptide in the regular ␤-geometry is primed for self-assembly. However, we found small but significant differences in the relative orientation between adjacent peptide units in terms of ␤-sheet twist and bend, suggesting their inherent flexibility. Modeling shows how this conformational diversity, when propagated over a large number of peptide units, can lead to a substantial degree of nanoscale polymorphism of self-assemblies.␤-sheet ͉ ␤-strand interaction ͉ amyloid fibril ͉ nanomaterial ͉ protein engineering

Background: p120-catenin protein interactions regulate vascular permeability. Results: We identified p190RhoGAP-binding domain of p120-catenin and evaluated its functional significance. Conclusion: Binding of p190RhoGAP occurs at the amino acid 820 -843 domain of p120-catenin and promotes activation of Rac and down-regulation of Rho signaling, leading to increased endothelial barrier.Significance: These data demonstrate functional significance of uncoupling the p120-catenin-p190RhoGAP interaction in the context of agonist-induced endothelial permeability.

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Outer surface protein A(OspA) from Borrelia burgdorferi hasanunusual dumbbell-shapedstructure in which twog lobular domainsa re connected with a' 'single-layer'' b -sheet (SLB). The protein is highly soluble, and it hasb een recalcitrant to crystallization. Only OspA complexes withF ab fragments have been successfully crystallized.OspA contains alarge number of Lysand Glu residues, and these ''high entropy''residues maydisfavorcrystal packing because some of them wouldneed to be immobilized in formingacrystall attice.W er ationally designedatotal of 13 surface mutations in whichL ys andG lu residueswere replacedwith AlaorSer.W esuccessfully crystallized them utant OspA without abound Fabfragment ande xtended structure analysis to a1.15 A˚resolution. The newhigh-resolution structure revealed au niqueb ackboneh ydration pattern of the SLB segment in which water molecules fill the ''weak spots''o nb othf aces of the antiparallel b -sheet. These well-definedw ater moleculesp rovide additional structural links between adjacent b -strands, andt hust heym ay be important for maintaining the rigidity of the SLBthat inherently lacks tightpackingaffordedbyahydrophobic core. Thestructure alsorevealed newinformationonthe side-chain dynamics andonasolvent-accessible cavity in thecore of the C-terminal globular domain. This work demonstrates theu tility of extensive surface mutation in crystallizingr ecalcitrant proteinsa nd dramatically improving the resolution of crystal structures, and provides newi nsights into the stabilization mechanism of OspA.

High tidal volume mechanical ventilation and the resultant excessive mechanical forces experienced by lung vascular endothelium are known to lead to increased vascular endothelial leak, but the underlying molecular mechanisms remain incompletely understood. One reported mechanotransduction pathway of increased endothelial cell (EC) permeability caused by high magnitude cyclic stretch (18% CS) involves CS-induced activation of the focal adhesion associated signalosome, which triggers Rho GTPase signaling. This study identified an alternative pathway of CS-induced EC permeability. We show here that high magnitude cyclic stretch (18% CS) rapidly activates VEGF receptor 2 (VEGFR2) signaling by dissociating VEGFR2 from VE-cadherin at the cell junctions. This results in VEGFR2 activation, Src-dependent VE-cadherin tyrosine phosphorylation, and internalization leading to increased endothelial permeability. This process is also accompanied by CS-induced phosphorylation and internalization of PECAM1. Importantly, CSinduced endothelial barrier disruption was attenuated by VEGFR2 inhibition. 18% CS-induced EC permeability was linked to dissociation of cell junction scaffold afadin from the adherens junctions. Forced expression of recombinant afadin in pulmonary endothelium attenuated CS-induced VEGFR2 and VE-cadherin phosphorylation, preserved adherens junction integrity and VEGFR2⅐VE-cadherin complex, and suppressed CS-induced EC permeability. This study shows for the first time a mechanism whereby VEGFR2 activation mediates EC permeability induced by pathologically relevant cyclic stretch. In this mechanism, CS induces dissociation of the VEcadherin⅐VEGFR2 complex localized at the adherens juctions, causing activation of VEGFR2, VEGFR2-mediated Src-dependent phosphorylation of VE-cadherin, disassembly of adherens junctions, and EC barrier failure.

We investigated how the register between adjacent beta-strands is specified using a series of mutants of the single-layer beta-sheet (SLB) in Borrelia OspA. The single-layer architecture of this system eliminates structural restraints imposed by a hydrophobic core, enabling us to address this question. A critical turn (turn 9/10) in the SLB was replaced with a segment with an intentional structural mismatch. Its crystal structure revealed a one-residue insertion into the central beta-strand (strand 9) of the SLB. This insertion triggered a surprisingly large-scale structural rearrangement: (i) the central strand (strand 9) was shifted by one residue, causing the strand to flip with respect to the adjacent beta-strands and thus completely disrupting the native side-chain contacts; (ii) the three-residue turn located on the opposite end of the beta-strand (turn 8/9) was pushed into its preceding beta-strand (strand 8); (iii) the register between strands 8 and 9 was shifted by three residues. Replacing the original sequence for turn 8/9 with a stronger turn motif restored the original strand register but still with a flipped beta-strand 9. The stability differences of these distinct structures were surprisingly small, consistent with an energy landscape where multiple low-energy states with different beta-sheet configurations exist. The observed conformations can be rationalized in terms of maximizing the number of backbone H-bonds. These results suggest that adjacent beta-strands "stick" through the use of factors that are not highly sequence specific and that beta-strands could slide back and forth relatively easily in the absence of external elements such as turns and tertiary packing.

This is the first report of heat shock protein serving as a cell surface receptor of oxidized phospholipids. It also investigates downstream molecular mechanisms and shows that GRP78 is involved in assembly of S1PR1-Akt-Rac1 signalosome, which is critical for vasoprotective effects of oxidized phospholipids.