The Evolution of Guanylyl Cyclases as Multidomain Proteins: Conserved Features of Kinase-Cyclase Domain Fusions

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signa transduction.

L2-stability of nonstationary feedback systems: Frequency-domain criteria

A frequency-domain positivity condition is derived for linear time-varying operators in2and is used to develop2stability criteria for linear and nonlinear feedback systems. These criteria permit the use of a very general class of operators in2with nonstationary kernels, as multipliers. More specific results are obtained by using a first-order differential operator with a time-varying coefficient as multiplier. Finally, by employing periodic multipliers, improved stability criteria are derived for the nonlinear damped Mathieu equation with a forcing function.

Genetic studies of the PRP17 gene of Saccharomyces cerevisiae: a domain essential for function maps to a nonconserved region of the protein

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the beta transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the beta transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between Prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is the N terminal region of the protein.

Architecture of a polymorphic ASIC for interoperability across multi-mode H.264 decoders

Run-time interoperability between different applications based on H.264/AVC is an emerging need in networked infotainment, where media delivery must match the desired resolution and quality of the end terminals. In this paper, we describe the architecture and design of a polymorphic ASIC to support this. The H.264 decoding flow is partitioned into modules, such that the polymorphic ASIC meets the design goals of low-power, low-area, high flexibility, high throughput and fast interoperability between different profiles and levels of H.264. We demonstrate the idea with a multi-mode decoder that can decode baseline, main and high profile H.264 streams and can interoperate at run.time across these profiles. The decoder is capable of processing frame sizes of up to 1024 times 768 at 30 fps. The design synthesized with UMC 0.13 mum technology, occupies 250 k gates and runs at 100 MHz.

Reconfigurable Viterbi decoder on mesh connected multiprocessor architecture

In modern wireline and wireless communication systems, Viterbi decoder is one of the most compute intensive and essential elements. Each standard requires a different configuration of Viterbi decoder. Hence there is a need to design a flexible reconfigurable Viterbi decoder to support different configurations on a single platform. In this paper we present a reconfigurable Viterbi decoder which can be reconfigured for standards such as WCDMA, CDMA2000, IEEE 802.11, DAB, DVB, and GSM. Different parameters like code rate, constraint length, polynomials and truncation length can be configured to map any of the above mentioned standards. Our design provides higher throughput and scalable power consumption in various configuration of the reconfigurable Viterbi decoder. The power and throughput can also be optimized for different standards.

RETHROTTLE: Execution Throttling in the REDEFINE SoC Architecture

REDEFINE is a reconfigurable SoC architecture that provides a unique platform for high performance and low power computing by exploiting the synergistic interaction between coarse grain dynamic dataflow model of computation (to expose abundant parallelism in applications) and runtime composition of efficient compute structures (on the reconfigurable computation resources). We propose and study the throttling of execution in REDEFINE to maximize the architecture efficiency. A feature specific fast hybrid (mixed level) simulation framework for early in design phase study is developed and implemented to make the huge design space exploration practical. We do performance modeling in terms of selection of important performance criteria, ranking of the explored throttling schemes and investigate effectiveness of the design space exploration using statistical hypothesis testing. We find throttling schemes which give appreciable (24.8%) overall performance gain in the architecture and 37% resource usage gain in the throttling unit simultaneously.

High performance VLSI architecture design for H.264 CAVLC decoder

H.264 video standard achieves high quality video along with high data compression when compared to other existing video standards. H.264 uses context-based adaptive variable length coding (CAVLC) to code residual data in Baseline profile. In this paper we describe a novel architecture for CAVLC decoder including coeff-token decoder, level decoder total-zeros decoder and run-before decoder UMC library in 0.13 mu CMOS technology is used to synthesize the proposed design. The proposed design reduces chip area and improves critical path performance of CAVLC decoder in comparison with [1]. Macroblock level (including luma and chroma) pipeline processing for CAVLC is implemented with an average of 141 cycles (including pipeline buffering) per macroblock at 250MHz clock frequency. To compare our results with [1] clock frequency is constrained to 125MHz. The area required for the proposed architecture is 17586 gates, which is 22.1% improvement in comparison to [1]. We obtain a throughput of 1.73 * 10(6) macroblocks/second, which is 28% higher than that reported in [1]. The proposed design meets the processing requirement of 1080HD [5] video at 30frames/seconds.

Role of a PAS sensor domain in the Mycobacterium tuberculosis transcription regulator Rv1364c

The Mycobacterium tuberculosis transcriptional regulator Rv1364c regulates the activity of the stress response sigma factor sigma(F). This multi-domain protein has several components: a signaling PAS domain and an effector segment comprising of a phosphatase, a kinase and an anti-anti-sigma factor domain. Based on Small Angle X-ray Scattering (SAXS) data, Rv1364c was recently shown to be a homo-dimer and adopt an elongated conformation in solution. The PAS domain could not be modeled into the structural envelope due to poor sequence similarity with known PAS proteins. The crystal structure of the PAS domain described here provides a structural basis for the dimerization of Rv1364c. It thus appears likely that the PAS domain regulates the anti-sigma activity of Rv1364c by oligomerization. A structural comparison with other characterized PAS domains reveal several sequence and conformational features that could facilitate ligand binding - a feature which suggests that the function of Rv1364c could potentially be governed by specific cellular signals or metabolic cues. (C) 2010 Elsevier Inc. All rights reserved.

Crystal Structure of Staphylococcus aureus Metallopeptidase (Sapep) Reveals Large Domain Motions between the Manganese-bound and Apo-states

Proteases belonging to the M20 family are characterized by diverse substrate specificity and participate in several metabolic pathways. The Staphylococcus aureus metallopeptidase, Sapep, is a member of the aminoacylase-I/M20 protein family. This protein is a Mn2+-dependent dipeptidase. The crystal structure of this protein in the Mn2+-bound form and in the open, metal-free state suggests that large interdomain movements could potentially regulate the activity of this enzyme. We note that the extended inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys(155) and Cys(178), are not active site residues, the reduced form of this enzyme is substantially more active as a dipeptidase. These findings acquire further relevance given a recent observation that this enzyme is only active in methicillin-resistant S. aureus. The structural and biochemical features of this enzyme provide a template for the design of novel methicillin-resistant S. aureus-specific therapeutics.

Architecture of Physalis Mottle Tymovirus as Probed by Monoclonal Antibodies and Cross-Linking Studies

Physalis mottle tymovirus (previously named belladonna mottle virus, Iowa strain) RNA was cross-linked to its coat protein by exposure of the intact virus to ultraviolet light. The site of cross-linking of the coat protein with the RNA was identified as Lys-10 by sequencing the oligonucleotide-linked tryptic peptide obtained upon HPLC separation subsequent to enzymetic digestion of the cross-linked and dissociated virus. Three monoclonal antibodies PA3B2, PB5G9, and PF12C9, obtained using denatured coat protein as antigen, cross-reacted effectively with the intact virus indicating that the epitopes recognized by these monoclonals are on the surface of the virus. Using the peptides generated by digestion with CNBr, clostripain, V-8 protease, or trypsin and a recombinant protein lacking the N-terminal 21 residues expressed from a cDNA clone, it was shown that PA3B2 recognizes the sequence 22-36 on the coat protein while PB5G9 and PF12C9 recognize region 75-110. These results suggest that Lys-10 is one of the specific sites through which the RNA interacts in the intact virus. The polypeptide segment (region 22-36) following this buried portion as well as the epitope within the region 75-110 are exposed in the intact virus. These observations are consistent with the canonical β-barrel structure observed in certain other plant viruses.

Area Optimized H.264 Intra Prediction Architecture for 1080p HD Resolution

High performance video standards use prediction techniques to achieve high picture quality at low bit rates. The type of prediction decides the bit rates and the image quality. Intra Prediction achieves high video quality with significant reduction in bit rate. This paper present an area optimized architecture for Intra prediction, for H.264 decoding at HDTV resolution with a target of achieving 60 fps. The architecture was validated on Virtex-5 FPGA based platform. The architecture achieves a frame rate of 64 fps. The architecture is based on multi-level memory hierarchy to reduce latency and ensure optimum resources utilization. It removes redundancy by reusing same functional blocks across different modes. The proposed architecture uses only 13% of the total LUTs available on the Xilinx FPGA XC5VLX50T.

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.

Primary structure of sesbania mosaic virus coat protein: its implications to the assembly and architecture of the virus

Sesbania mosaic virus (SMV) is a plant virus that infects Sesbania grandiflora plants in Andhra Pradesh, India. The amino acid sequence of the coat protein of SMV was determined using purified peptides generated by cleavage with trypsin, chymotrypsin, V8 protease and clostripain. The 230 residues so far determined were compared to the corresponding residues of southern bean mosaic virus (SBMV), the type member of sobemoviruses. The overall identity between the sequences is 61.7%. The amino terminal 64 residues, which constitute an independent domain (R-domain) known to interact with RNA, are conserved to a lower extent (52.5%). Comparison of the positively charged residues in this domain suggests that the RNA-protein interactions are considerably weaker in SMV. The residues that constitute the major domain of the coat protein, the surface domain (S-domain, residues 65-260), are better conserved (66.5%). The positively charged residues of this domain that face the nucleic acid are well conserved. The longest conserved stretch of residues (131-142) corresponds to the loop involved in intersubunit interactions between subunits related by the quasi 3-fold symmetry. A unique cation binding site located on the quasi 3-fold axis contributes to the stability of SMV. These differences are reflected in the increased stability of the SMV coat protein and its ability to be reconstituted with RNA at pH 7.5. A major epitope was identified using monoclonal antibodies to SMV in the segment 201-223 which contains an exposed helix in the capsid structure. This region is highly conserved between SMV and SBMV (70%) suggesting that it could represent the site of an important function such as vector recognition.

Testis-specific histone (H1t) is not phosphorylated and has a weak interaction with chromatin

Soluble chromatin was prepared from rat testes after a brief micrococcal nuclease digestion. After adsorption onto hydroxylapatite at low ionic strength, the histone Hl subtypes were eluted with a shallow salt gradient of 0.3 M NaCl to 0.7 M NaCl. Histone Hlt was eluted at 0.4 MNaCl, while histones H1a and Hlc were eluted at 0.43 M NaCl and 0.45 M respectively. The extreme divergence of the amino acid sequence of the C-terminal half of histone Hlt, the major DNA binding domain of histone Hl, from that of the somatic consensus sequence may contribute to the weaker interaction of histone Hlt with the rat testis chromatin. Further, histone Hlt was not phosphorylated in vivo in contrast to histone Hla and Hlc, as is evident from the observation that histone Hlt lacks the SPKK motif recognized by the CDC-2kinase or the RR/KXS motif recognized by protein kinase A.

The complete primary structure of a unique mannose/glucose-specific lectin from field bean (Dolichos lab lab)

The complete amino acid sequence of two non identical subunits of the glucose/mannose-specific lectin from Dolichos lab lab (field bean) has been determined by sequential Edman analyses of the intact subunits and peptides derived by enzymatic and chemical cleavage. Peptides were purified by reverse phase high performance liquid chromatography and ion pair chromatography. The D. lab lab lectin is a glycoprotein having two polypeptide chains of 132 and 105 amino acid residues. The amino acid sequence of the D. Lab lab lectin is compared with the various lectins of the family Leguminosae. The D. lab lab lectin is the only species of the tribe Phaseoleae that contains two nonidentical subunits of almost equal size and that shows a specificity to glucose/ mannose. The lectin shows a greater homology to the glucose/mannose specific lectins, especially concanavalin A. The unique subunit architecture of the D. lab lab lectin indicates the presence of new post translational cleavage sites.