CLAP: A web-server for automatic classification of proteins with special reference to multi-domain proteins

Background: The function of a protein can be deciphered with higher accuracy from its structure than from its amino acid sequence. Due to the huge gap in the available protein sequence and structural space, tools that can generate functionally homogeneous clusters using only the sequence information, hold great importance. For this, traditional alignment-based tools work well in most cases and clustering is performed on the basis of sequence similarity. But, in the case of multi-domain proteins, the alignment quality might be poor due to varied lengths of the proteins, domain shuffling or circular permutations. Multi-domain proteins are ubiquitous in nature, hence alignment-free tools, which overcome the shortcomings of alignment-based protein comparison methods, are required. Further, existing tools classify proteins using only domain-level information and hence miss out on the information encoded in the tethered regions or accessory domains. Our method, on the other hand, takes into account the full-length sequence of a protein, consolidating the complete sequence information to understand a given protein better. Results: Our web-server, CLAP (Classification of Proteins), is one such alignment-free software for automatic classification of protein sequences. It utilizes a pattern-matching algorithm that assigns local matching scores (LMS) to residues that are a part of the matched patterns between two sequences being compared. CLAP works on full-length sequences and does not require prior domain definitions. Pilot studies undertaken previously on protein kinases and immunoglobulins have shown that CLAP yields clusters, which have high functional and domain architectural similarity. Moreover, parsing at a statistically determined cut-off resulted in clusters that corroborated with the sub-family level classification of that particular domain family. Conclusions: CLAP is a useful protein-clustering tool, independent of domain assignment, domain order, sequence length and domain diversity. Our method can be used for any set of protein sequences, yielding functionally relevant clusters with high domain architectural homogeneity. The CLAP web server is freely available for academic use at http://nslab.mbu.iisc.ernet.in/clap/.

Sequence and conformational preferences at termini of alpha-helices in membrane proteins: Role of the helix environment

-helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These -helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C-termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze -helices in a high-resolution dataset of integral -helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C-termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near-helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420-3436. (c) 2014 Wiley Periodicals, Inc.

Identifying functionally important cis-peptide containing segments in proteins and their utility in molecular function annotation

Cis-peptide embedded segments are rare in proteins but often highlight their important role in molecular function when they do occur. The high evolutionary conservation of these segments illustrates this observation almost universally, although no attempt has been made to systematically use this information for the purpose of function annotation. In the present study, we demonstrate how geometric clustering and level-specific Gene Ontology molecular-function terms (also known as annotations) can be used in a statistically significant manner to identify cis-embedded segments in a protein linked to its molecular function. The present study identifies novel cis-peptide fragments, which are subsequently used for fragment-based function annotation. Annotation recall benchmarks interpreted using the receiver-operator characteristic plot returned an area-under-curve >0.9, corroborating the utility of the annotation method. In addition, we identified cis-peptide fragments occurring in conjunction with functionally important trans-peptide fragments, providing additional insights into molecular function. We further illustrate the applicability of our method in function annotation where homology-based annotation transfer is not possible. The findings of the present study add to the repertoire of function annotation approaches and also facilitate engineering, design and allied studies around the cis-peptide neighborhood of proteins.

Streptococcus pneumoniae Genome Database (SPGDB): A database for strain specific comparative analysis of Streptococcus pneumoniae genes and proteins

Streptococcus pneumoniae causes pneumonia, septicemia and meningitis. S. pneumoniae is responsible for significant mortality both in children and in the elderly. In recent years, the whole genome sequencing of various S. pneumoniae strains have increased manifold and there is an urgent need to provide organism specific annotations to the scientific community. This prompted us to develop the Streptococcus pneumoniae Genome Database (SPGDB) to integrate and analyze the completely sequenced and available S. pneumoniae genome sequences. Further, links to several tools are provided to compare the pool of gene and protein sequences, and proteins structure across different strains of S. pneumoniae. SPGDB aids in the analysis of phenotypic variations as well as to perform extensive genomics and evolutionary studies with reference to S. pneumoniae. (C) 2014 Elsevier Inc. All rights reserved.

Development, in vitro and in vivo characterization of zoledronic acid functionalized hydroxyapatite nanoparticle based formulation for treatment of osteoporosis in animal model

We investigated the potential of using novel zoledronic acid (ZOL)-hydroxyapatite (HA) nanoparticle based drug formulation in a rat model of postmenopausal osteoporosis. By a classical adsorption method, nanoparticles of HA loaded with ZOL (HNLZ) drug formulation with a size range of 100-130 nm were prepared. 56 female Wistar rats were ovariectomized (OVX) or sham-operated at 3 months of age. Twelve weeks post surgery, rats were randomized into seven groups and treated with various doses of HNLZ (100, 50 and 25 mu g/kg, intravenous single dose), ZOL (100 mu g/kg, intravenous single dose) and HA nanoparticle (100 mu g/kg, intravenous single dose). Untreated OVX and sham OVX served as controls. After three months treatment period, we evaluated the mechanical properties of the lumbar vertebra and femoral mid-shaft. Femurs were also tested for trabecular microarchitecture. Sensitive biochemical markers of bone formation and bone resorption in serum were also determined. With respect to improvement in the mechanical strength of the lumbar spine and the femoral mid-shaft, the therapy with HNLZ drug formulation was more effective than ZOL therapy in OVX rats. Moreover, HNLZ drug therapy preserved the trabecular microarchitecture better than ZOL therapy in OVX rats. Furthermore, the HNLZ drug formulation corrected increase in serum levels of bone-specific alkaline phosphatase, procollagen type I N-terminal propeptide, osteocalcin, tartrate-resistant acid phosphatase 5b and C-telopeptide of type 1 collagen better than ZOL therapy in OVX rats. The results strongly suggest that HNLZ novel drug formulation appears to be more effective approach for treating severe osteoporosis in humans. (C) 2014 Elsevier B.V. All rights reserved.

Paralogous cAMP Receptor Proteins in Mycobacterium smegmatis Show Biochemical and Functional Divergence

The cyclic AMP receptor protein (CRP) family of transcription factors consists of global regulators of bacterial gene expression. Here, we identify two paralogous CRPs in the genome of Mycobacterium smegmatis that have 78% identical sequences and characterize them biochemically and functionally. The two proteins (MSMEG_0539 and MSMEG_6189) show differences in cAMP binding affinity, trypsin sensitivity, and binding to a CRP site that we have identified upstream of the msmeg_3781 gene. MSMEG_6189 binds to the CRP site readily in the absence of cAMP, while MSMEG_0539 binds in the presence of cAMP, albeit weakly. msmeg_6189 appears to be an essential gene, while the ?msmeg_0539 strain was readily obtained. Using promoter-reporter constructs, we show that msmeg_3781 is regulated by CRP binding, and its transcription is repressed by MSMEG_6189. Our results are the first to characterize two paralogous and functional CRPs in a single bacterial genome. This gene duplication event has subsequently led to the evolution of two proteins whose biochemical differences translate to differential gene regulation, thus catering to the specific needs of the organism.

Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance

In many organisms ``Universal Stress Proteins'' CUSPS) are induced in response to a variety of environmental stresses. Here we report the structures of two USPs, YnaF and YdaA from Salmonella typhimurium determined at 1.8 angstrom and 2.4 angstrom resolutions, respectively. YnaF consists of a single USP domain and forms a tetrameric organization stabilized by interactions mediated through chloride ions. YdaA is a larger protein consisting of two tandem USP domains. Two protomers of YdaA associate to form a structure similar to the YnaF tetramer. YdaA showed ATPase activity and an ATP binding motif G-2X-G-9X-G(S/T/N) was found in its C-terminal domain. The residues corresponding to this motif were not conserved in YnaF although YnaF could bind ATP. However, unlike YdaA, YnaF did not hydrolyse ATP in vitro. Disruption of interactions mediated through chloride ions by selected mutations converted YnaF into an ATPase. Residues that might be important for ATP hydrolysis could be identified by comparing the active sites of native and mutant structures. Only the C-terminal domain of YdaA appears to be involved in ATP hydrolysis. The structurally similar N-terminal domain was found to bind a zinc ion near the segment equivalent to the phosphate binding loop of the C-terminal domain. Mass spectrometric analysis showed that YdaA might bind a ligand of approximate molecular weight 800 daltons. Structural comparisons suggest that the ligand, probably related to an intermediate in lipid A biosynthesis, might bind at a site close to the zinc ion. Therefore, the N-terminal domain of YdaA binds zinc and might play a role in lipid metabolism. Thus, USPs appear to perform several distinct functions such as ATP hydrolysis, altering membrane properties and chloride sensing. (C) 2015 Elsevier Inc. All rights reserved.

The Evolution of Animal Welfare and the 3Rs in Brazil, China, and India

Increasingly, scientific collaborations and contracts cross country borders. The need for assurance that the quality of animal welfare and the caliber of animal research conducted are equivalent among research partners around the globe is of concern to the scientific and laboratory animal medicine communities, the general public, and other key stakeholders. Therefore, global harmonization of animal care and use standards and practices, with the welfare of the animals as a cornerstone, is essential. In the evolving global landscape of enhanced attention to animal welfare, a widely accepted path to achieving this goal is the successful integration of the 3Rs in animal care and use programs. Currently, awareness of the 3Rs, their implementation, and the resulting animal care and use standards and practices vary across countries. This variability has direct effects on the animals used in research and potentially the data generated and may also have secondary effects on the country's ability to be viewed as a global research partner. Here we review the status of implementation of the 3Rs worldwide and focus on 3 countries-Brazil, China and India-with increasing economic influence and an increasing footprint in the biomedical research enterprise.

Characterization of rice small heat shock proteins targeted to different cellular organelles

Small heat shock proteins (sHSPs) are a family of ATP-independent molecular chaperones which prevent cellular protein aggregation by binding to misfolded proteins. sHSPs form large oligomers that undergo drastic rearrangement/dissociation in order to execute their chaperone activity in protecting substrates from stress. Substrate-binding sites on sHSPs have been predominantly mapped on their intrinsically disordered N-terminal arms. This region is highly variable in sequence and length across species, and has been implicated in both oligomer formation and in mediating chaperone activity. Here, we present our results on the functional and structural characterization of five sHSPs in rice, each differing in their subcellular localisation, viz., cytoplasm, nucleus, chloroplast, mitochondria and peroxisome. We performed activity assays and dynamic light scattering studies to highlight differences in the chaperone activity and quaternary assembly of sHSPs targeted to various organelles. By cloning constructs that differ in the length and sequence of the tag in the N-terminal region, we have probed the sensitivity of sHSP oligomer assembly and chaperone activity to the length and amino acid composition of the N-terminus. In particular, we have shown that the incorporation of an N-terminal tag has significant consequences on sHSP quaternary structure.

Identification of local variations within secondary structures of proteins

Secondary-structure elements (SSEs) play an important role in the folding of proteins. Identification of SSEs in proteins is a common problem in structural biology. A new method, ASSP (Assignment of Secondary Structure in Proteins), using only the path traversed by the C atoms has been developed. The algorithm is based on the premise that the protein structure can be divided into continuous or uniform stretches, which can be defined in terms of helical parameters, and depending on their values the stretches can be classified into different SSEs, namely -helices, 3(10)-helices, -helices, extended -strands and polyproline II (PPII) and other left-handed helices. The methodology was validated using an unbiased clustering of these parameters for a protein data set consisting of 1008 protein chains, which suggested that there are seven well defined clusters associated with different SSEs. Apart from -helices and extended -strands, 3(10)-helices and -helices were also found to occur in substantial numbers. ASSP was able to discriminate non--helical segments from flanking -helices, which were often identified as part of -helices by other algorithms. ASSP can also lead to the identification of novel SSEs. It is believed that ASSP could provide a better understanding of the finer nuances of protein secondary structure and could make an important contribution to the better understanding of comparatively less frequently occurring structural motifs. At the same time, it can contribute to the identification of novel SSEs. A standalone version of the program for the Linux as well as the Windows operating systems is freely downloadable and a web-server version is also available at .

A cis-Regulatory Mutation in Troponin-I of Drosophila Reveals the Importance of Proper Stoichiometry of Structural Proteins During Muscle Assembly

Rapid and high wing-beat frequencies achieved during insect flight are powered by the indirect flight muscles, the largest group of muscles present in the thorax. Any anomaly during the assembly and/or structural impairment of the indirect flight muscles gives rise to a flightless phenotype. Multiple mutagenesis screens in Drosophila melanogaster for defective flight behavior have led to the isolation and characterization of mutations that have been instrumental in the identification of many proteins and residues that are important for muscle assembly, function, and disease. In this article, we present a molecular-genetic characterization of a flightless mutation, flightless-H (fliH), originally designated as heldup-a (hdp-a). We show that fliH is a cis-regulatory mutation of the wings up A (wupA) gene, which codes for the troponin-I protein, one of the troponin complex proteins, involved in regulation of muscle contraction. The mutation leads to reduced levels of troponin-I transcript and protein. In addition to this, there is also coordinated reduction in transcript and protein levels of other structural protein isoforms that are part of the troponin complex. The altered transcript and protein stoichiometry ultimately culminates in unregulated acto-myosin interactions and a hypercontraction muscle phenotype. Our results shed new insights into the importance of maintaining the stoichiometry of structural proteins during muscle assembly for proper function with implications for the identification of mutations and disease phenotypes in other species, including humans.

Suppression of protein aggregation by gold nanoparticles: a new way to store and transport proteins

Suppression of the aggregation of proteins has tremendous implications in biology and medicine. In the pharmaceuticals industry, aggregation of therapeutically important proteins and peptides while stored, reduces the efficacy and promptness of action leading to, in many instances, intoxication of the patient by the aggregate. Here we report the effect of gold nanoparticles (Au-NPs) in preventing the thermal and chemical aggregation of two unrelated proteins of different size, alcohol dehydrogenase (ADH, 84 kDa) and insulin (6 kDa), respectively, in physiological pH. Our principal observation is that there is a significant reduction (up to 95%) in the extent of aggregation of ADH and insulin in the presence of gold nanoparticles (Au-NPs). Aggregation of these proteins at micromolar concentration is prevented using nanomolar or less amounts of gold nanoparticles which is remarkable since chaperones which prevent such aggregation in vivo are required in micromolar quantity. The prevention of aggregation of these two different proteins under two different denaturing environments has established the role of Au-NPs as a protein aggregation prevention agent. The extent of prevention increases rapidly with the increase in the size of the gold nanoparticles. Protein molecules get physisorbed on the gold nanoparticle surface and thus become inaccessible by the denaturing agent in solution. This adsorption of proteins on AuNPs has been established by a variety of techniques and assays.

Viruses and viral proteins

For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes.

Design of symmetric TIM barrel proteins from first principles

Background: Computational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (alpha/beta)(8) TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts. Methods: Computational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics. Results: Extensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a T-m of 44 degrees C and a Gibbs free energy of unfolding (Delta G degrees) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a C-m of 1.6 M and a Delta G degrees of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics. Conclusions: Despite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.

Isotopic fractionation in proteins as a measure of hydrogen bond length

If a deuterated molecule containing strong intramolecular hydrogen bonds is placed in a hydrogenated solvent, it may preferentially exchange deuterium for hydrogen. This preference is due to the difference between the vibrational zero-point energy for hydrogen and deuterium. It is found that the associated fractionation factor (I) is correlated with the strength of the intramolecular hydrogen bonds. This correlation has been used to determine the length of the H-bonds (donor-acceptor separation) in a diverse range of enzymes and has been argued to support the existence of short low-barrier H-bonds. Starting with a potential energy surface based on a simple diabatic state model for H-bonds, we calculate (I) as a function of the proton donor-acceptor distance R. For numerical results, we use a parameterization of the model for symmetric 0-H. ``.0 bonds R. H. McKenzie, Chem. Phys. Lett. 535, 196 (2012)]. We consider the relative contributions of the 0-H stretch vibration, O-H bend vibrations (both in plane and out of plane), tunneling splitting effects at finite temperature, and the secondary geometric isotope effect. We compare our total (I) as a function of R with NMR experimental results for enzymes, and in particular with an earlier model parametrization (D(R), used previously to determine bond lengths. (C) 2015 AIP Publishing LLC.