Modeling Conformational Ensembles of Slow Functional Motions in Pin1-WW

Protein-protein interactions are often mediated by flexible loops that experience conformational dynamics on the microsecond to millisecond time scales. NMR relaxation studies can map these dynamics. However, defining the network of inter-converting conformers that underlie the relaxation data remains generally challenging. Here, we combine NMR relaxation experiments with simulation to visualize networks of inter-converting conformers. We demonstrate our approach with the apo Pin1-WW domain, for which NMR has revealed conformational dynamics of a flexible loop in the millisecond range. We sample and cluster the free energy landscape using Markov State Models (MSM) with major and minor exchange states with high correlation with the NMR relaxation data and low NOE violations. These MSM are hierarchical ensembles of slowly interconverting, metastable macrostates and rapidly interconverting microstates. We found a low population state that consists primarily of holo-like conformations and is a “hub” visited by most pathways between macrostates. These results suggest that conformational equilibria between holo-like and alternative conformers pre-exist in the intrinsic dynamics of apo Pin1-WW. Analysis using MutInf, a mutual information method for quantifying correlated motions, reveals that WW dynamics not only play a role in substrate recognition, but also may help couple the substrate binding site on the WW domain to the one on the catalytic domain. Our work represents an important step towards building networks of inter-converting conformational states and is generally applicable

J. Appl. Polym. Sci.

In a previous study, it was found that monodisperse polystyrene (PSt) hollow particles can be prepared under special conditions by combining a Shirasu Porous Glass (SPG) emulsification technique and subsequent suspension polymerization process. That is, a mixture of styrene (St), N,N-dimethylamino ethyl methacrylate (DMAEMA), hexadecane (HD), and initiator N,N'-azobis(2,4-dimethylvaleronitrile) (ADVN) was used as the dispersed phase in an aqueous phase containing poly(vinyl pyrrolidone) (PVP), sodium lauryl sulfate (SLS), and water-soluble inhibitor. The dispersed phase was created by pushing the oil phase through the uniform pores of an SPG membrane into the continuous phase to form uniform droplets. Then, the droplets were polymerized at 70degreesC. It has been puzzling that hollow microspheres were obtained only when sodium nitrite (NaNO2) was used as a water-soluble inhibitor, while one-hole particles were formed when hydroquinone (HQ) or diaminophenylene (DAP) was used. In this study, the mechanism of formation of the hollow microspheres was verified by measuring the variation of diameter, molecular weight distribution, and monomer conversion, and by observing morphological changes during the polymerization, as well as by changing the type and amount of hydrophilic monomer, and initiator. It was found that the diameter of the oil droplets decreased, and a large amount of secondary new particles formed immediately after polymerization started in the case of NaNO2, However, there was no such apparent behavior to be observed when HQ or DAP was used. It was determined that the hollow particles formed due to the rapid phase separation between PSt and HD, and as a consequence, a large amount of monomer diffused into the aqueous phase to form the secondary particles. Rapid phase separation confined the HD inside the droplets, a nonequilibrium morphology. On the other hand, one-hole particles, representing an equilibrium morphology, formed when the phase separation occurred slowly because a lot of monomer existed inside of the droplets to allow mobility of the PSt. The addition of DMAEMA allowed the hollow particles to be formed more easily by decreasing the interfacial tension between the copolymer and aqueous phase. (C) 2002 Wiley Periodicals, Inc.In a previous study, it was found that monodisperse polystyrene (PSt) hollow particles can be prepared under special conditions by combining a Shirasu Porous Glass (SPG) emulsification technique and subsequent suspension polymerization process. That is, a mixture of styrene (St), N,N-dimethylamino ethyl methacrylate (DMAEMA), hexadecane (HD), and initiator N,N'-azobis(2,4-dimethylvaleronitrile) (ADVN) was used as the dispersed phase in an aqueous phase containing poly(vinyl pyrrolidone) (PVP), sodium lauryl sulfate (SLS), and water-soluble inhibitor. The dispersed phase was created by pushing the oil phase through the uniform pores of an SPG membrane into the continuous phase to form uniform droplets. Then, the droplets were polymerized at 70degreesC. It has been puzzling that hollow microspheres were obtained only when sodium nitrite (NaNO2) was used as a water-soluble inhibitor, while one-hole particles were formed when hydroquinone (HQ) or diaminophenylene (DAP) was used. In this study, the mechanism of formation of the hollow microspheres was verified by measuring the variation of diameter, molecular weight distribution, and monomer conversion, and by observing morphological changes during the polymerization, as well as by changing the type and amount of hydrophilic monomer, and initiator. It was found that the diameter of the oil droplets decreased, and a large amount of secondary new particles formed immediately after polymerization started in the case of NaNO2, However, there was no such apparent behavior to be observed when HQ or DAP was used. It was determined that the hollow particles formed due to the rapid phase separation between PSt and HD, and as a consequence, a large amount of monomer diffused into the aqueous phase to form the secondary particles. Rapid phase separation confined the HD inside the droplets, a nonequilibrium morphology. On the other hand, one-hole particles, representing an equilibrium morphology, formed when the phase separation occurred slowly because a lot of monomer existed inside of the droplets to allow mobility of the PSt. The addition of DMAEMA allowed the hollow particles to be formed more easily by decreasing the interfacial tension between the copolymer and aqueous phase. (C) 2002 Wiley Periodicals, Inc

A single amino acid substitution in a WW-like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation.

Platelet-derived growth factor beta receptor (PDGFbetaR) is a transmembrane receptor tyrosine kinase involved in a variety of cellular functions. We have generated a constitutively activated murine PDGFbetaR containing a valine to alanine substitution at residue 536, located in the cytoplasmic juxtamembrane domain. When this mutant receptor (PR-V536A) was expressed in Ba/F3 cells, it allowed the cells to survive and proliferate in the absence of IL-3 or PDGF, and tyrosine phosphorylation of PR-V536A was increased markedly compared with that of the wild-type PDGFbetaR in the absence of ligand and similar to that observed in ligand-activated PDGFbetaR. PR-V536A displayed increased tyrosine kinase activity in vitro toward an exogenous substrate, and the tyrosine kinase activity of the receptor was required for the constitutive activation of the mutant. This valine to alanine substitution also activated a PDGFbetaR mutant unable to bind PDGF. Alanine substitutions at positions homologous to V536 of the murine PDGFbetaR also activated other members of the PDGF receptor subfamily. The amino acid sequence of this region revealed a strong similarity to WW domains present in other signal transduction proteins. Furthermore, GST fusion proteins containing the juxtamembrane region of the PDGFR specifically associated with peptides containing the WW domain consensus recognition sequence PPXY. The results suggest that the cytoplasmic juxtamembrane domain plays a role in the regulation of receptor activity and function, perhaps by participating in protein-protein interactions