Exploring isospectral spring-mass systems with firefly algorithm

This paper investigates in-line spring-mass systems (An), fixed at one end and free at the other, with n-degrees of freedom (d.f.). The objective is to find feasible in-line systems (B(n)) that are isospectral to a given system. The spring-mass systems, A(n) and B(n), are represented by Jacobi matrices. An error function is developed with the help of the Jacobi matrices A(n) and B(n). The problem of finding the isospectral systems is posed as an optimization problem with the aim of minimizing the error function. The approach for creating isospectral systems uses the fact that the trace of two isospectral Jacobi matrices A(n) and B(n) should be identical. A modification is made to the diagonal elements of the given Jacobi matrix (A(n)), to create the isospectral systems. The optimization problem is solved using the firefly algorithm augmented by a local search procedure. Numerical results are obtained and resulting isospectral systems are shown for 4 d.f. and 10 d.f. systems.

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order +35 > +1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.

The GAF Domain of the cGMP-Binding, cGMP-Specific Phosphodiesterase (PDE5) Is a Sensor and a Sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFaF163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET2 assays. BRET2 ratios of the wild type GAFa fusion protein, but not GAFaF163A, increased in the presence of cGMP but not cAMP. Higher basal BRET2 ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFaF163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET2 technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET2 sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.

Distinct Allostery Induced in the Cyclic GMP-binding, Cyclic GMP-specific Phosphodiesterase (PDE5) by Cyclic GMP, Sildenafil, and Metal Ions

The activity of many proteins orchestrating different biological processes is regulated by allostery, where ligand binding at one site alters the function of another site. Allosteric changes can be brought about by either a change in the dynamics of a protein, or alteration in its mean structure. We have investigated the mechanisms of allostery induced by chemically distinct ligands in the cGMP-binding, cGMP-specific phosphodiesterase, PDE5. PDE5 is the target for catalytic site inhibitors, such as sildenafil, that are used for the treatment of erectile dysfunction and pulmonary hypertension. PDE5 is a multidomain protein and contains two N-terminal cGMP-specific phosphodiesterase, bacterial adenylyl cyclase, FhLA transcriptional regulator (GAF) domains, and a C-terminal catalytic domain. Cyclic GMP binding to the GAFa domain and sildenafil binding to the catalytic domain result in conformational changes, which to date have been studied either with individual domains or with purified enzyme. Employing intramolecular bioluminescence resonance energy transfer, which can monitor conformational changes both in vitro and in intact cells, we show that binding of cGMP and sildenafil to PDE5 results in distinct conformations of the protein. Metal ions bound to the catalytic site also allosterically modulated cGMP- and sildenafil-induced conformational changes. The sildenafil-induced conformational change was temperature-sensitive, whereas cGMP-induced conformational change was independent of temperature. This indicates that different allosteric ligands can regulate the conformation of a multidomain protein by distinct mechanisms. Importantly, this novel PDE5 sensor has general physiological and clinical relevance because it allows the identification of regulators that can modulate PDE5 conformation in vivo.

Baculovirus mediated high-level expression of luciferase in silkworm cells and larvae

Bombyx mori nuclear polyhedrosis virus (BmNPV)-based baculovirus expression system exploits silkworm larvae as an economical alternative to large-scale cell cultures for production of biomolecules. To generate recombinant BmNPV at high efficiency, we have achieved high efficiency transfection of B. mori cells, BmN, through lipofection. Optimal conditions for lipofection were standardized by quantification of the transient expression level of firefly luciferase (luc) reporter gene under control of an immediate early gene promoter of BmNPV Lipofection was 50-fold and 100-fold more efficient than the calcium phosphate method for transfecting BmN and Sf9 cells, respectively. Lipofection enabled us to generate a recombinant BmNPV (vBmluc), harboring luc under control of the strong polyhedrin promoter On infection with vBmluc, luciferase was expressed at very high levels, 170 mu g/10(6) BmN cells or 13 mg/larva. Expression of luciferase in vBmluc-infected larvae was visualized by luminescence emission instantaneously following luciferin injection generating ''glowing silkworms''.

Cyclic AMP-induced Conformational Changes in Mycobacterial Protein Acetyltransferases

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.

Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2

GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem(GAFab domain). In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET) experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.