Receptor-Galpha fusion proteins as a tool for ligand screening

We have prepared fusion proteins of muscarinic M1-M5 receptors with alpha subunits of G proteins Gi1, Gi2, Gs, G11, G16 and chimera of G protein alpha subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M2-Gi1alpha and M4-Gi1alpha, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [35S]GTPgammaS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of Gq/G11-coupled receptors such as M1-G11alpha, M3-G11alpha, and M5-G11alpha. The effect of agonists could be observed for fusion proteins with G16alpha of muscarinic M1, M2 and adrenergic beta2 receptors, but the extent of the effect was much less than that for fusion proteins with Gi1alpha of Gi/Go-coupled receptors. Fusion proteins of M1 receptors with Gi1alpha or chimera of G16alpha and Gi2alpha were also not effective in detecting the action of agonists.     

Ion concentration dynamics as a mechanism for neuronal bursting

We describe a simple conductance-based model neuron that includes intra- and extracellular ion concentration dynamics and show that this model exhibits periodic bursting. The bursting arises as the fast-spiking behavior of the neuron is modulated by the slow oscillatory behavior in the ion concentration variables and vice versa. By separating these time scales and studying the bifurcation structure of the neuron, we catalog several qualitatively different bursting profiles that are strikingly similar to those seen in experimental preparations. Our work suggests that ion concentration dynamics may play an important role in modulating neuronal excitability in real biological systems.     

Cholesterol as a co‐solvent and a ligand for membrane proteins

As of mid 2013 a Medline search on “cholesterol” yielded over 200,000 hits, reflecting the prominence of this lipid in numerous aspects of animal cell biology and physiology under conditions of health and disease. Aberrations in cholesterol homeostasis underlie both a number of rare genetic disorders and contribute to common sporadic and complex disorders including heart disease, stroke, type II diabetes, and Alzheimer's disease. The corresponding author of this review and his lab stumbled only recently into the sprawling area of cholesterol research when they discovered that the amyloid precursor protein (APP) binds cholesterol, a topic covered by the Hans Neurath Award lecture at the 2013 Protein Society Meeting. Here, we first provide a brief overview of cholesterol‐protein interactions and then offer our perspective on how and why binding of cholesterol to APP and its C99 domain (β‐CTF) promotes the amyloidogenic pathway, which is closely related to the etiology of Alzheimer's disease.     

Evidence for fast conformational change upon ligand dissociation in the HemAT class of bacterial oxygen sensors

Here we report the results of transient absorption and photoacoustic calorimetry studies of CO photodissociation from the heme domain of the bacterial oxygen sensor HemAT-Bs. The results indicate that CO photolysis is accompanied by an overall DeltaH of -19 kcal mol(-1) and DeltaV of +4 ml mol(-1) as well as a red-shifted kinetic difference spectrum all occurring in <50 ns. Analysis of the DeltaH/DeltaV reveals that a conformational change takes place with a DeltaH(conf) of -40 kcal mol(-1) and DeltaV(conf) of -22 ml mol(-1). These thermodynamic changes are consistent with an increase in the solvent accessible surface area of the protein upon ligand dissociation, as observed in the X-ray structure of the ferric CN-bound and CN free forms of HemAT-Bs.     

Wave-like dopamine dynamics as a mechanism for spatiotemporal credit assignment

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Evidence for methylation as a regulatory mechanism in HLA-DR x gene expression

We examined the possibility that one mechanism for controlling HLA-DR gene expression occurs through DNA hypomethylation. We employed the restriction enzyme Hpa II, which recognizes the sequence 5CCGG3 but not 5C^mCGG3, to study DNA methylation. We first compared a DR-positive B lymphoblastoid cell line (LCL) with an isogenic DR-negative T-LCL. Using a genomic probe for the DR gene, we showed that an Hpa II digestion of DNA from the B-LCL resulted in bands of lower molecular weight than that of the T-LCL. This indicates that the B-LCL DR gene is hypomethylated relative to the T-LCL gene. Demethylation of the gene from the B-LCL is incomplete, suggesting that complete demethylation is not required for its expression. We also examined somatic cell hybrids of T-LCL and B-LCL since the DR gene, which is inactive in the T-LCL, is expressed in the hybrids, providing a system to study DR gene induction. We examined the hybrid line 174 × CEM.T1, which contains and expresses solely the DR gene from the T-LCL parent since both copies of the DR gene from the B-LCL parent, 174, are deleted. The expressed DR gene from the hybrid was compared with the unexpressed gene from the T-LCL parental line, and again an association between DR gene expression and DNA hypomethylation was observed. In contrast to the DR gene from B-LCL, which is not completely demethylated, the DR gene in this hybrid line is not methylated at either of the Msp I sites covered by our probe. This suggests that different regulatory mechanisms operating through DNA methylation may be involved in the expression of DR genes from T-LCL and B-LCL. Examination of another hybrid line which has DR genes from both parental lines supports this contention. The implications of these findings are discussed.     

The biotin-thyroxin conjugate as a bifunctional ligand of binding proteins

The conjugate of the residue of vitamin H (biotin, Bt) with the hormone of thyroid gland thyroxin (T₄) was prepared by N-acylation of N-(3-aminopropyl)biotin amide with N-hydroxysuccinimide ester of N-acetyl thyroxin. The interactions of the Bt-T₄ conjugate with one or simultaneously with two binding proteins with affinity to Bt or T₄ in solution and on a solid phase were studied by electron spectroscopy, enzyme immunoassay, and computer modeling. Bt-T₄ was specifically fixed in the Bt-binding site of the streptavidin molecule via a large number of hydrogen bonds and hydrophobic interactions. The maximum of the streptavidin fluorescence shifted to a long-wave area and its intensity decreased as a result of complex formation. The degree of quenching of the protein emission was significantly higher than that of the streptavidin-Bt complex. Additional fluorescence quenching resulted from interactions which were sensitive to pH, ionic strength, and detergents and stabilized the position of the thyroxin part of the conjugate near Trp120 of streptavidin in its complex with Bt-T₄. The Bt-T₄ conjugate also formed a specific equimolar complex with T₄-binding human globulin (TBG) by the same mechanism as that for T₄. The Bt residue did not participate in the interactions which changed characteristics of the TBG fluorophores. The Bt-T₄ conjugate was bound to avidin on a solid phase in the solid phase enzyme immunoassay owing to its biotin function, whereas its thyroxin part was exposed to a solution and interacted with polyclonal antibodies to T₄. The intact T₄ competitively inhibited this interaction after its addition to the system. Bt-T₄ also exhibited its bifunctional activity in other immune analytic system. The conjugate bound streptavidin was labeled with Eu³⁺-chelate and subsequently formed a three component complex with participation of a monoclonal antibody to T₄ immobilized on a solid phase. Free T₄ inhibited the thyroxin function of the conjugate bound to the labeled streptavidin proportionally to its concentration in a sample of human blood serum. Parameters of the immunofluorescent analysis demonstrated that the streptavidin-Bt-T₄ complex was actively bound to the T₄-antibody, but had practically no interaction with serum T₄-binding proteins, including TBG. Probably, nonspecific interactions of the T₄ residue with streptavidin in its complex with Bt-T₄, along with steric factors, complicated penetration of thyroxin in this complex into active sites of TBG and other T₄-binding proteins of blood serum. The Bt-T₄ stable conjugate was synthesized according to a plain scheme and could be used as a bifunctional ligand of binding proteins in biochemical studies and immune analytical systems for medicinal diagnostics.     

Molecular dynamics simulations reveal multiple pathways of ligand dissociation from thyroid hormone receptors

Nuclear receptor (NR) ligands occupy a pocket that lies within the core of the NR ligand-binding domain (LBD), and most NR LBDs lack obvious entry/exit routes upon the protein surface. Thus, significant NR conformational rearrangements must accompany ligand binding and release. The precise nature of these processes, however, remains poorly understood. Here, we utilize locally enhanced sampling (LES) molecular dynamics computer simulations to predict molecular motions of x-ray structures of thyroid hormone receptor (TR) LBDs and determine events that permit ligand escape. We find that the natural ligand 3,5,3'-triiodo-L-thyronine (T(3)) dissociates from the TRalpha1 LBD along three competing pathways generated through i), opening of helix (H) 12; ii), separation of H8 and H11 and the Omega-loop between H2 and H3; and iii), opening of H2 and H3, and the intervening beta-strand. Similar pathways are involved in dissociation of T(3) and the TRbeta-selective ligand GC24 from TRbeta; the TR agonist IH5 from the alpha- and beta-TR forms; and Triac from two natural human TRbeta mutants, A317T and A234T, but are detected with different frequencies in simulations performed with the different structures. Path I was previously suggested to represent a major pathway for NR ligand dissociation. We propose here that Paths II and III are also likely ligand escape routes for TRs and other NRs. We also propose that different escape paths are preferred in different situations, implying that it will be possible to design NR ligands that only associate stably with their cognate receptors in specific cellular contexts.     

Carboxymethyl cellulose as a new heterobifunctional ligand carrier for affinity precipitation of proteins

Affinity precipitation is a technique that imparts selectivity to the widely used primary purification step of precipitation of proteins from crude extracts. Hetero-bifunctional affinity precipitation involves use of reversibly soluble/insoluble polymers that can be used as backbones to conjugate affinity ligands for specific separations. A variety of such polymers have been reported in literature. In this work we report development of carboxymethyl cellulose (CM cellulose) as a cheap, readily available and versatile reversibly soluble polymer system. Available CM cellulose as sodium salt could be quantitatively precipitated from its aqueous solution in presence of about 50 mM calcium and 7.2% w/v polyethylene glycol-4000, and could be resolubilised in the working buffer in absence of calcium, polyethylene glycol or both. Effectiveness of the CM cellulose-calcium-polyethylene glycol system was demonstrated by purifying lactate dehydrogenase from porcine muscle extractusing covalently conjugated Cibacron blue dye-ligand. By careful choice of conditions that suppressed non-specific interactions, the system was shown to be an effective affinity precipitation polymer system inspite of the polyelectrolytic nature of CM cellulose. Up to 23 fold purification of the enzyme from crude extarct was obtained in one single precipitation sequence. This revised version was published online in July 2006 with corrections to the Cover Date.     

Exploring ligand dissociation pathways from aminopeptidase N using random acceleration molecular dynamics simulation

Aminopeptidase N (APN) is a zinc-dependent ectopeptidase involved in cell proliferation, secretion, invasion, and angiogenesis, and is widely recognized as an important cancer target. However, the mechanisms whereby ligands leave the active site of APN remain unknown. Investigating ligand dissociation processes is quite difficult, both in classical simulation methods and in experimental approaches. In this study, random acceleration molecular dynamics (RAMD) simulation was used to investigate the potential dissociation pathways of ligand from APN. The results revealed three pathways (channels A, B and C) for ligand release. Channel A, which matches the hypothetical channel region, was the most preferred region for bestatin to dissociate from the enzyme, and is probably the major channel for the inner bound ligand. In addition, two alternative channels (channels B and C) were shown to be possible pathways for ligand egression. Meanwhile, we identified key residues controlling the dynamic features of APN channels. Identification of the dissociation routes will provide further mechanistic insights into APN, which will benefit the development of more promising APN inhibitors.

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Graphical Abstract The release pathways of bestatin inside active site of aminopeptidase N were simulated using RAMD simulation

     

Polymerized liposome as ligand carrier for affinity precipitation of proteins

A polymerized liposome (PLS) was prepared using a synthesized phospholipid with a diacetylene moiety in the hydrophobic chain and an amino group in the hydrophilic head. The PLS was used as a novel ligand carrier for affinity precipitation of proteins because it showed a reversibly precipitable property on salt addition and removal. Soybean trypsin inhibitor (STI) was easily immobilized on the PLS by a one-step carbodiimide reaction. The PLS showed no nonspecific adsoprtion of proteins. It had a large ligand coupling capacity, and then a large adsorption capacity for trypsin after STI immobilization. The PLS with immpbilized STI was recycled three times for the purification of trypsin from a crude pancreatic extract. Although the degree of purification was compromised by the impurity of the STI employed, in each run the purification factor reached about 6 and more than 80% of trypsin activity was recovered. The results indicated that the PLS was a potential ligand carrier for affinity precipitation of proteins. (c) 1995 John Wiley & Sons, Inc.     

Analysis of ligand binding to proteins using molecular dynamics simulations

This work aims to explore theoretically the molecular mechanisms of ligand binding to proteins through the use of molecular dynamics simulations. The binding of sodium dodecyl sulfate (SDS) to cobra cardio toxin A3 (CTX A3) and thiourea (TOU) to lysozyme have been chosen as the two model systems. Data acquisitions were made by Gromacs software. To begin with, the collisions of ligand molecules with every residue of CTX A3 and lysozyme were evaluated. With this information in hand, the average numbers of collisions with each residue was defined and then assessed. Next, a measure of the affinity of a residue, P_i, referred to as conformational factor, toward a ligand molecule was established. Based on the results provided, all site-making residues for CTX A3 and lysozyme were identified. The results are in good agreement with the experimental data. Finally, based on this method, all site-making residues of bovine carbonic anhydrase (BCA) toward the SDS ligand were predicted.     

Calorimetric studies of oxyhemoglobin ligand dissociation. III. Evidence for a hemoglobin-catalase-superoxide dismutase integrated system in the oxygen depletion by dithionite

Calorimetric studies of the effect of superoxide dismutase and/or catalase on the reduction of dioxygen into water by dithionite in oxyhemoglobin have been carried out and the results compared with those in red cell hemolysates. In the absence of the enzymes the stoichiometry (moles dithionite/mole dioxygen) is less than the value of 2:1 which was found previously in red cell hemolysates [Forlani et al., J. Inorg. Biochem. 20, 147-155 (1984)]. In the presence of either superoxide dismutase or catalase alone the stoichiometry increases but is still less than 2:1. In the presence of both enzymes the stoichiometry and the shape of the thermogram is that previously observed for hemolysates, suggesting the presence of a hemoglobin-catalase-superoxide dismutase integrated system. The absence of a calorimetric signal for hydrogen peroxide in the reduction of oxyhemoglobin in the presence of superoxide dismutase suggests a wider biological role of superoxide dismutase than previously thought.     

Metabolically 35S-labeled recombinant calmodulin as a ligand for the detection of calmodulin-binding proteins

We have developed a simplified procedure for the production of metabolically labeled calmodulin. We used bacterial clones (Escherichia coli) that were found to express VU-1 calmodulin, a calmodulin that is fully active with a variety of calmodulin-regulated enzymes. VU-1 calmodulin was labeled with sulfur-35 in bacteria maintained in a sulfur-free medium. Calmodulin was then purified by chromatography on phenyl-Sepharose. Under these conditions, the specific activity of the proteins was 150 to 400 cpm/fmol of calmodulin. To demonstrate the utility of this labeled VU-1 calmodulin, we examined the calmodulin-binding proteins in aortic myocyte preparation from Day 0 and Day 15 cultures by using both the gel and the nitrocellulose overlay protocols. The results showed that calmodulin-binding proteins are easily detected by the two procedures and that the profile of these target proteins changed in myocyte with time in culture. While most of these calmodulin-binding proteins have not been identified, the relative mobility on SDS-PAGE gels suggests that myosin light chain kinase (Mr approximately 137,000) was detected by these methods. We demonstrated here that the nitrocellulose overlay was faster than the gel overlay and that this technique can be useful for the study of calmodulin-binding proteins.     

Copper dissociation as a mechanism of fungal laccase denaturation by humic acid

Effects of humic acid on removal of hydroxy polychlorobiphenyls (PCBs) with laccase from Trametes versicolor were studied. In the absence of humic acid, hydroxy PCBs were rapidly degraded by laccase. However, the rate constants decreased with increasing humic acid concentration, the reactions being completely inhibited at 150 mg l^-1 of humic acid. Peroxidase from Arthromyces ramosus was not inhibited by the same treatment. The activity of humic acid-deactivated laccase was completely restored by copper ions (500 M of Cu²⁺ in 150 mg l^-1 of humic acid), but not by other metal ions (Zn²⁺, Fe²⁺ and Hg²⁺). Humic acid-deactivated laccase purified by gel permeation chromatography (GPC) showed no activity against 2,2-azino-bis(3-ethylbenzthiazoline sulfonic acid) diammonium salt and 3,5-dichloro-4-hydroxybiphenyl, but its activity was restored by copper ion treatment. Humic acid-deactivated laccase showed similar properties, such as GPC retention time and copper ion requirements for activity, to those of laccase deactivated by nitrilotriacetic acid. The extent of humic acid inhibition, expressed as activity non-recoverable by copper ion treatment, increased over time more rapidly than that of the humic acid-free control. These results suggest that short-term inactivation of laccase, i.e., less than 1 day, is attributable to a depletion of copper ion.     

Covalent modification of proteins as a threshold mechanism in development

Thresholds are a central but somewhat neglected aspect of cellular processes in development. An analysis has been made of the conditions in which different thresholds can be generated in the covalent modification of a number of target proteins when the concentration of an effector is continuously increased. It is assumed that the effector, which could represent a morphogen, activates, for example, kinases that phosphorylate the proteins. Thresholds are found when the modifying enzymes are saturated by their protein substrates, i.e. in conditions of zero-order ultrasensitivity (Goldbeter, A. & Koshland, D. E. 1981. An amplified sensitivity arising from covalent modification in biological systems. Proc. natn. Acad. Sci. U.S.A. 78, 6840-6844). Sequential thresholds can be generated when the kinase/phosphatase pairs differ either in the ratio of maximum modification rates or in the affinity of the effector for each kinase.     

The role of dynamics in modulating ligand exchange in intracellular lipid binding proteins

Lipids are essential for many biological processes and crucial in the pathogenesis of several diseases. Intracellular lipid-binding proteins (iLBPs) provide mobile hydrophobic binding sites that allow hydrophobic or amphipathic lipid molecules to penetrate into and across aqueous layers. Thus iLBPs mediate the lipid transport within the cell and participate to a spectrum of tissue-specific pathways involved in lipid homeostasis. Structural studies have shown that iLBPs' binding sites are inaccessible from the bulk, implying that substrate binding should involve a conformational change able to produce a ligand entry portal. Many studies have been reported in the last two decades on iLBPs indicating that their dynamics play a pivotal role in regulating ligand binding and targeted release. The ensemble of reported data has not been reviewed until today. This review is thus intended to summarize and possibly generalize the results up to now described, providing a picture which could help to identify the missing notions necessary to improve our understanding of the role of dynamics in iLBPs' molecular recognition. Such notions would clarify the chemistry of lipid binding to iLBPs and set the basis for the development of new drugs.