Scaffold proteins form a framework to organize signal transduction by binding multiple partners within a signaling pathway. This shapes the output of signal responses as well as providing specificity and localization. The Membrane Associated Guanylate Kinases (MAGuKs) are scaffold proteins at cellular junctions that localize cell surface receptors and link them to downstream signaling enzymes. Scaffold proteins often contain protein-binding domains that are connected in series by disordered linkers. The tertiary structure of the folded domains is well understood, but describing the dynamic inter-domain interactions (the superteritary structure) of such multidomain proteins remains a challenge to structural biology. We used 65 distance restraints from singlemolecule fluorescence resonance energy transfer (smFRET) to describe the superteritary structure of the canonical MAGuK scaffold protein PSD-95. By combining multiple fluorescence techniques, the conformational dynamics of PSD-95 could be characterized across the biologically relevant timescales for protein domain motions. Relying only on a qualitative interpretation of FRET data, we were able to distinguish stable interdomain interactions from freely orienting domains. This revealed that the five domains in PSD-95 partitioned into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GuK. We used our smFRET data for hybrid structural refinement to model the PDZ3-SH3-GuK supramodule and include explicit dye simulations to provide complete characterization of potential uncertainties inherent to quantitative interpretation of FRET as distance. Comparative structural analysis of synaptic MAGuK homologues showed a conservation of this supertertiary structure. Our approach represents a general solution to describing the supertertiary structure of multidomain proteins.intrinsic disorder | protein structure | single molecule fluorescence | fluorescence lifetime | fluorescence correlation spectroscopy N ature relies on scaffold proteins to provide the physical constraints necessary for efficient signal transduction. Scaffold proteins interact with multiple pathway components to hold the signal transduction machinery in close proximity. Scaffolds are often composed of modular, protein-binding domains linked together in series by intrinsically disordered linkers (1). The presence of disorder may be a defining feature for scaffolds and other proteins that interact with multiple binding partners (2).The high effective concentrations brought about by domain tethering can give rise to unexpected interactions between the protein-binding domains. In some cases, canonical protein-binding domains fold together into an inseparable structural supramodule (3). While much has been learned by studying truncated fragments, we need to put the pieces back together. Such questions are difficult to address because disorder presents a fundamental challenge to structural biology.The Membrane-Associated Guanylate Kinase (MAGuK) scaffold proteins regulate signaling at cellular junctions (4). ...

Electrochemical Machining (ECM) is a technique to shape metal parts by anodic dissolution in neutral solutions (e.g. aqueous NaNO3) at extremely large current densities of about 100 A cm-2. We developed a capillary based microcell to simulate this process in the laboratory and to simultaneously analyze the products by UV-Vis spectroscopy. In addition, a brief overview of electrochemical micro techniques is given

The fixation of cementless endoprostheses requires excellent fixation at the bone implant interface. Although the surface structures of these implants are designed to promote osteoblastic differentiation, poor bone quality may prevent or delay osseointegration. There is evidence that RGD peptides known as recognition motifs for various integrins, promote cellular adhesion, influence cellular proliferation, and differentiation of local cells. In this study, five different metal surfaces were analyzed: Sandblasted (TiSa) and polished (TiPol) Ti6Al4V, porocoated (CCPor) and polished (CCPol) cobalt chrome and polished stainless steel (SS) were coated by ethanol amine and poly(ethylene glycol) to attach covalently RGD peptides. Human mesenchymal stromal cells of healthy donors were cultivated onto prior functionalized metal surfaces for 14 days without osteogenic stimulation. Cell proliferation and differentiation were quantitatively evaluated for native (I), NaOH pre-activated (II), NaOH pre-activated, and PEG-coated (III) as well as for RGD (IV) coated surfaces. The RGD immobilization efficiency was analyzed by epi-fluorescence spectroscopy, cell morphology was documented by light and scanning electron microscopy. The RGD-binding efficiency was TiSa > TiPol > SS > CCPor > CCPol. RGD coated surfaces showed the highest average cell proliferation on CCPol > SS > CCPor > TiSa ≥ TiPol, whereas cellular differentiation mostly correlated with the observed proliferation results, such as CCPol > TiSa > SS > CCPor > TiPol. Considering statistical analyses (significance level of α = 0.05), the RGD-coating of all biometals in comparison and in respect of their particular controls showed no significant improvement in cellular proliferation and osteoblastic differentiation.

also discuss ligand migration pathways and protein dynamics upon CO photorelease in rHb1.

Recent advances in the computational description of proteins facilitate the atomic-level characterization of fast folding proteins using moleculardynamics simulations [1]. However, due to the femtosecond timestep of an MD simulation, 10^10 computationally expensive energy evaluations are required to reach timescales of the order of single folding events even for ultrafast-folding proteins. Stochastic simulation techniques can overcome this limiting factor by generating thermodynamic conformational ensembles without incorporating atomic vibrations, but most of these methods are inherently sequential. Here we investigate a parallel extension to the conventional Metropolis-Hastings algorithm, the Multiple-Try-Metropolis, enabling parallel simulation of reversible Markov-chains[2,3]. We successfully characterize the thermodynamic landscape of the Villin-headpiece-subdomain using a generalized MTM approach adapted to protein systems. We parameterized the polypeptide using the AMBER99SB-STAR-ILDN-forcefield with an implicit solvent model. In our simulations we could observe approximately two folding transitions per day on standard hardware, with a total of 109 transition events during 3*10^9 energy evaluations. The MTM approach thus facilitates the efficient thermodynamic characterization of proteins on common computer architectures. The comparison with experimental resolved folding times of 8ms translates to a time equivalent of 16ps per MTM step.

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difference between these two worlds is now simply the sample format, i.e., cuvette vs. microplate. It seems reasonable to assume that the cuvette with its straightforward implementation of right angle excitation-emission geometry offers significant advantages over the epi-illumination geometry and uncontained sample imposed by a microplate when it comes to data quality. We have implemented a prototype microplate reader equipped with a variety of pulsed laser sources for measurement of fluorescence spectra, fluorescence lifetimes, and anisotropy. The subject of this poster is benchmarking its performance relative to cuvette format. The plate reader employs direct waveform recording as an alternative to TCSPC; studies to compare the speed, accuracy, and precision of the two lifetime approaches are presented along with several examples of titration curves for rapid determination of binding affinities via time-resolved FRET.