PDZ affinity chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins

Protein Purification Using PDZ Affinity Chromatography

PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands

Analytic, Group-Theoretic Density Profiles for Confined, Correlated N-Body Systems

Confined quantum systems involving $N$ identical interacting particles are to be found in many areas of physics, including condensed matter, atomic and chemical physics. A beyond-mean-field perturbation method that is applicable, in principle, to weakly, intermediate, and strongly-interacting systems has been set forth by the authors in a previous series of papers. Dimensional perturbation theory was used, and in conjunction with group theory, an analytic beyond-mean-field correlated wave function at lowest order for a system under spherical confinement with a general two-body interaction was derived. In the present paper, we use this analytic wave function to derive the corresponding lowest-order, analytic density profile and apply it to the example of a Bose-Einstein condensate.Comment: 15 pages, 2 figures, accepted by Physics Review A. This document was submitted after responding to a reviewer's comment

Interaction Driven Quantum Hall Wedding cake-like Structures in Graphene Quantum Dots

Quantum-relativistic matter is ubiquitous in nature; however it is notoriously difficult to probe. The ease with which external electric and magnetic fields can be introduced in graphene opens a door to creating a table-top prototype of strongly confined relativistic matter. Here, through a detailed spectroscopic mapping, we provide a spatial visualization of the interplay between spatial and magnetic confinement in a circular graphene resonator. We directly observe the development of a multi-tiered "wedding cake"-like structure of concentric regions of compressible/incompressible quantum Hall states, a signature of electron interactions in the system. Solid-state experiments can therefore yield insights into the behaviour of quantum-relativistic matter under extreme conditions

Order statistics and the linear assignment problem

Under mild conditions on the distribution functionF, we analyze the asymptotic behavior in expectation of the smallest order statistic, both for the case thatF is defined on (–, +) and for the case thatF is defined on (0, ). These results yield asymptotic estimates of the expected optiml value of the linear assignment problem under the assumption that the cost coefficients are independent random variables with distribution functionF

Phosphorylation of synaptic GTPase-activating protein (synGAP) by polo-like kinase (Plk2) alters the ratio of its GAP activity toward HRas, Rap1 and Rap2 GTPases

SynGAP is a Ras and Rap GTPase-activating protein (GAP) found in high concentration in the postsynaptic density (PSD) fraction from mammalian forebrain where it binds to PDZ domains of PSD-95. Phosphorylation of pure recombinant synGAP by Ca^(2+)/calmodulin-dependent protein kinase II (CaMKII) shifts the balance of synGAP's GAP activity toward inactivation of Rap1; whereas phosphorylation by cyclin-dependent kinase 5 (CDK5) has the opposite effect, shifting the balance toward inactivation of HRas. These shifts in balance contribute to regulation of the numbers of surface AMPA receptors, which rise during synaptic potentiation (CaMKII) and fall during synaptic scaling (CDK5). Polo-like kinase 2 (Plk2/SNK), like CDK5, contributes to synaptic scaling. These two kinases act in concert to reduce the number of surface AMPA receptors following elevated neuronal activity by tagging spine-associated RapGAP protein (SPAR) for degradation, thus raising the level of activated Rap. Here we show that Plk2 also phosphorylates and regulates synGAP. Phosphorylation of synGAP by Plk2 stimulates its GAP activity toward HRas by 65%, and toward Rap1 by 16%. Simultaneous phosphorylation of synGAP by Plk2 and CDK5 at distinct sites produces an additive increase in GAP activity toward HRas (∼230%) and a smaller, non-additive increase in activity toward Rap1 (∼15%). Dual phosphorylation also produces an increase in GAP activity toward Rap2 (∼40–50%), an effect not produced by either kinase alone. As we previously observed for CDK5, addition of Ca^(2+)/CaM causes a substrate-directed doubling of the rate and stoichiometry of phosphorylation of synGAP by Plk2, targeting residues also phosphorylated by CaMKII. In summary, phosphorylation by Plk2, like CDK5, shifts the ratio of GAP activity of synGAP to produce a greater decrease in active Ras than in active Rap, which would produce a shift toward a decrease in the number of surface AMPA receptors in neuronal dendrites

Relation between inelastic electron tunneling and vibrational excitation of single adsorbates on metal surfaces

We analyse theoretically a relation between the vibrational generation rate of a single adsorbate by tunneling electrons and the inelastic tunneling (IET) current in scanning tunneling microscope, and the influence of the vibrational excitations on the rate of adsorbate motions. Special attention is paid to the effects of finite lifetime of the vibrational excitations. We show that in the vicinity and below the IET threshold the rate of adsorbate motion deviates from a simple power-law dependence on the bias voltage due to the effects of bath temperature and adsorbate vibrational lifetime broadenings. The temperature broadening appears to be confined near the threshold voltage within a narrow region of several $k_B T$, whereas the lifetime broadening manifests itself in a much wider region of applied voltages below the IET threshold.Comment: 8 pages including 4 figure

A Multicenter Examination and Strategic Revisions of the Yale Global Tic Severity Scale

Objective To examine the internal consistency and distribution of the Yale Global Tic Severity Scale (YGTSS) scores to inform modification of the measure. Methods This cross-sectional study included 617 participants with a tic disorder (516 children and 101 adults), who completed an age-appropriate diagnostic interview and the YGTSS to evaluate tic symptom severity. The distributions of scores on YGTSS dimensions were evaluated for normality and skewness. For dimensions that were skewed across motor and phonic tics, a modified Delphi consensus process was used to revise selected anchor points. Results Children and adults had similar clinical characteristics, including tic symptom severity. All participants were examined together. Strong internal consistency was identified for the YGTSS Motor Tic score (α = 0.80), YGTSS Phonic Tic score (α = 0.87), and YGTSS Total Tic score (α = 0.82). The YGTSS Total Tic and Impairment scores exhibited relatively normal distributions. Several subscales and individual item scales departed from a normal distribution. Higher scores were more often used on the Motor Tic Number, Frequency, and Intensity dimensions and the Phonic Tic Frequency dimension. By contrast, lower scores were more often used on Motor Tic Complexity and Interference, and Phonic Tic Number, Intensity, Complexity, and Interference. Conclusions The YGTSS exhibits good internal consistency across children and adults. The parallel findings across Motor and Phonic Frequency, Complexity, and Interference dimensions prompted minor revisions to the anchor point description to promote use of the full range of scores in each dimension. Specific minor revisions to the YGTSS Phonic Tic Symptom Checklist were also proposed

A model for regulation by SynGAP-α1 of binding of synaptic proteins to PDZ-domain 'Slots' in the postsynaptic density

SynGAP is a Ras/Rap GTPase-activating protein (GAP) that is a major constituent of postsynaptic densities (PSDs) from mammalian forebrain. Its α1 isoform binds to all three PDZ (PSD-95, Discs-large, ZO-1) domains of PSD-95, the principal PSD scaffold, and can occupy as many as 15% of these PDZ domains. We present evidence that synGAP-α1 regulates the composition of the PSD by restricting binding to the PDZ domains of PSD-95. We show that phosphorylation by Ca^(2+)/calmodulin-dependent protein kinase II (CaMKII) and Polo-like kinase-2 (PLK2) decreases its affinity for the PDZ domains by several fold, which would free PDZ domains for occupancy by other proteins. Finally, we show that three critical postsynaptic signaling proteins that bind to the PDZ domains of PSD-95 are present in higher concentration in PSDs isolated from mice with a heterozygous deletion of synGAP

Phosphorylation of Synaptic GTPase Activating Protein (synGAP) by Ca^(2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5) alters the ratio of its GAP activity toward Ras and Rap GTPases

SynGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentration in the postsynaptic density (PSD) fraction from mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca^(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N-terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phos-phorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAPs HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity, but CaMKII shifts the relative GAP activity toward inactivation of Rap1; whereas CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, S773 and S802. Phosphorylation at S773 inhibits r-synGAP activity, whereas phosphorylation at S802 increases it. However, the net effect of concurrent phosphorylation of both sites, S773 and S802, is an increase in GAP activity. SynGAP is phosphorylated at S773 and S802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons