THE KNICKKOPF DOMON DOMAIN IS ESSENTIAL FOR CUTICLE DIFFERENTIATION IN Drosophila melanogaster

The dopamine monoxygenase N-terminal (DOMON) domain is found in extracellular proteins across several eukaryotic and prokaryotic taxa. It has been proposed that this domain binds to heme or sugar moieties. Here, we have analyzed the role of four highly conserved amino acids in the DOMON domain of the Drosophila melanogaster Knickkopf protein that is inserted into the apical plasma membrane and assists extracellular chitin organization. In principal, we generated Knickkopf versions with exchanged residues tryptophan(299,) methionine(333), arginine(401), or histidine(437), and scored for the ability of the respective engineered protein to normalize the knickkopf mutant phenotype. Our results confirm the absolute necessity of tryptophan(299,) methionine(333), and histidine(437) for Knickkopf function and stability, the latter two being predicted to be critical for heme binding. In contrast, arginine(401) is required for full efficiency of Knickkopf activity. Taken together, our genetic data support the prediction of these residues to mediate the function of Knickkopf during cuticle differentiation in insects. Hence, the DOMON domain is apparently an essential factor contributing to the construction of polysaccharide-based extracellular matrices.

C. N. R. Rao and the Growth of Solid State and Materials Chemistry as a Central Domain of Research

In celebrating Professor C. N. R. Rao's 80th birthday, this article recalls his singular contributions to solid state and materials chemistry for about sixty years. In so doing, the article also traces the growth of the field as a central domain of research in chemical sciences from its early origins in Europe. Although Rao's major work lies in solid state and materials chemistry - a field which he started and nurtured in India while its importance was being recognized internationally - his contributions to other areas of chemistry (and physics), viz., molecular spectroscopy, phase transitions, fullerenes, graphene, nanomaterials and multiferroics are equally significant. Illustrative examples of his work devoted to rare earth and transition metal oxides, defects and nonstoichiometry, metal-insulator transitions, investigation of crystal and electronic structures of a variety of solids by means of electron microscopies and photoelectron spectroscopy, superconducting cuprates, magnetoresistive manganites, multiferroic metal oxides of various structures and, last but not the least, development of new strategies for chemical synthesis of a wide variety of solids including nanomaterials and framework solids in different dimensionalities, are highlighted. The article also captures his exemplary role as a science teacher, science educationist and institution builder in post-Independence India.

Bi-Domain Protein Tyrosine Phosphatases Reveal an Evolutionary Adaptation to Optimize Signal Transduction

Significance: The bi-domain protein tyrosine phosphatases (PTPs) exemplify functional evolution in signaling proteins for optimal spatiotemporal signal transduction. Bi-domain PTPs are products of gene duplication. The catalytic activity, however, is often localized to one PTP domain. The inactive PTP domain adopts multiple functional roles. These include modulation of catalytic activity, substrate specificity, and stability of the bi-domain enzyme. In some cases, the inactive PTP domain is a receptor for redox stimuli. Since multiple bi-domain PTPs are concurrently active in related cellular pathways, a stringent regulatory mechanism and selective cross-talk is essential to ensure fidelity in signal transduction. Recent Advances: The inactive PTP domain is an activator for the catalytic PTP domain in some cases, whereas it reduces catalytic activity in other bi-domain PTPs. The relative orientation of the two domains provides a conformational rationale for this regulatory mechanism. Recent structural and biochemical data reveal that these PTP domains participate in substrate recruitment. The inactive PTP domain has also been demonstrated to undergo substantial conformational rearrangement and oligomerization under oxidative stress. Critical Issues and Future Directions: The role of the inactive PTP domain in coupling environmental stimuli with catalytic activity needs to be further examined. Another aspect that merits attention is the role of this domain in substrate recruitment. These aspects have been poorly characterized in vivo. These lacunae currently restrict our understanding of neo-functionalization of the inactive PTP domain in the bi-domain enzyme. It appears likely that more data from these research themes could form the basis for understanding the fidelity in intracellular signal transduction.

Accurate prediction of interfacial residues in two-domain proteins using evolutionary information: Implications for three-dimensional modeling

With the preponderance of multidomain proteins in eukaryotic genomes, it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces, and the knowledge of the interacting sites is essential to our understanding of the structure-function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multidomain), we predict the interface residues corresponding to domains from the two-domain proteins. We also use information from the three-dimensional structures of individual domains of two-domain proteins to train naive Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (approximate to 85%) and specific (approximate to 95%) to the domain-domain interfaces. This method is specific to multidomain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain-domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. We believe that these results can be of considerable interest toward rational protein and interaction design, apart from providing us with valuable information on the nature of interactions. Proteins 2014; 82:1219-1234. (c) 2013 Wiley Periodicals, Inc.

The HORMA domain: an evolutionarily conserved domain discovered in chromatin-associated proteins, has unanticipated diverse functions

The HORMA domain (for Hop1p, Rev7p and MAD2) was discovered in three chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae. This domain has also been found in proteins with similar functions in organisms including plants, animals and nematodes. The HORMA domain containing proteins are thought to function as adaptors for meiotic checkpoint protein signaling and in the regulation of meiotic recombination. Surprisingly, new work has disclosed completely unanticipated and diverse functions for the HORMA domain containing proteins. A. M. Villeneuve and colleagues (Schvarzstein et al., 2013) show that meiosis-specific HORMA domain containing proteins plays a vital role in preventing centriole disengagement during Caenorhabditis elegans spermatocyte meiosis. Another recent study reveals that S. cerevisiae Atg13 HORMA domain acts as a phosphorylation-dependent conformational switch in the cellular autophagic process. (C) 2014 Elsevier B.V. All rights reserved.

Frequency Domain Based Solution for Certain Class of Wave Equations: An exhaustive study of Numerical Solutions

The paper discusses the frequency domain based solution for a certain class of wave equations such as: a second order partial differential equation in one variable with constant and varying coefficients (Cantilever beam) and a coupled second order partial differential equation in two variables with constant and varying coefficients (Timoshenko beam). The exact solution of the Cantilever beam with uniform and varying cross-section and the Timoshenko beam with uniform cross-section is available. However, the exact solution for Timoshenko beam with varying cross-section is not available. Laplace spectral methods are used to solve these problems exactly in frequency domain. The numerical solution in frequency domain is done by discretisation in space by approximating the unknown function using spectral functions like Chebyshev polynomials, Legendre polynomials and also Normal polynomials. Different numerical methods such as Galerkin Method, Petrov- Galerkin method, Method of moments and Collocation method or the Pseudo-spectral method in frequency domain are studied and compared with the available exact solution. An approximate solution is also obtained for the Timoshenko beam with varying cross-section using Laplace Spectral Element Method (LSEM). The group speeds are computed exactly for the Cantilever beam and Timoshenko beam with uniform cross-section and is compared with the group speeds obtained numerically. The shear mode and the bending modes of the Timoshenko beam with uniform cross-section are separated numerically by applying a modulated pulse as the shear force and the corresponding group speeds for varying taper parameter in are obtained numerically by varying the frequency of the input pulse. An approximate expression for calculating group speeds corresponding to the shear mode and the bending mode, and also the cut-off frequency is obtained. Finally, we show that the cut-off frequency disappears for large in, for epsilon > 0 and increases for large in, for epsilon < 0.

Groundnut Bud Necrosis Virus Encoded NSm Associates with Membranes via Its C-Terminal Domain

Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell.

Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16

We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsinf-old and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977. (C) 2013 The Authors. Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies. All rights reserved.

Role of the loop L-4,L-5 in allosteric regulation in mtHsp70s: in vivo significance of domain communication and its implications in protein translocation

Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L-4,L-5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L-4,L-5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L-4,L-5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated within the SBD suppress domain communication defects by conformationally altering the allosteric interface, thereby restoring import and growth phenotypes. Strikingly, the suppressor mutations highlight that restoration of communication from NBD to SBD alone is the minimum essential requirement for effective in vivo function when primed at higher basal ATPase activity, mimicking the J-protein-bound state. Together these findings provide the first mechanistic insights into critical regions within the SBD of mtHsp70s regulating interdomain communication, thus highlighting its importance in protein translocation and mitochondrial biogenesis.

Fienup Algorithm With Sparsity Constraints: Application to Frequency-Domain Optical-Coherence Tomography

We address the problem of reconstructing a sparse signal from its DFT magnitude. We refer to this problem as the sparse phase retrieval (SPR) problem, which finds applications in tomography, digital holography, electron microscopy, etc. We develop a Fienup-type iterative algorithm, referred to as the Max-K algorithm, to enforce sparsity and successively refine the estimate of phase. We show that the Max-K algorithm possesses Cauchy convergence properties under certain conditions, that is, the MSE of reconstruction does not increase with iterations. We also formulate the problem of SPR as a feasibility problem, where the goal is to find a signal that is sparse in a known basis and whose Fourier transform magnitude is consistent with the measurement. Subsequently, we interpret the Max-K algorithm as alternating projections onto the object-domain and measurement-domain constraint sets and generalize it to a parameterized relaxation, known as the relaxed averaged alternating reflections (RAAR) algorithm. On the application front, we work with measurements acquired using a frequency-domain optical-coherence tomography (FDOCT) experimental setup. Experimental results on measured data show that the proposed algorithms exhibit good reconstruction performance compared with the direct inversion technique, homomorphic technique, and the classical Fienup algorithm without sparsity constraint; specifically, the autocorrelation artifacts and background noise are suppressed to a significant extent. We also demonstrate that the RAAR algorithm offers a broader framework for FDOCT reconstruction, of which the direct inversion technique and the proposed Max-K algorithm become special instances corresponding to specific values of the relaxation parameter.

Frequency-domain stimulated and spontaneous light emission signals at molecular junctions

Using a diagrammatic superoperator formalism we calculate optical signals at molecular junctions where a single molecule is coupled to two metal leads which are held at different chemical potentials. The molecule starts in a nonequilibrium steady state whereby it continuously exchanges electrons with the leads with a constant electron flux. Expressions for frequency domain optical signals measured in response to continuous laser fields are derived by expanding the molecular correlation functions in terms of its many-body states. The nonunitary evolution of molecular states is described by the quantum master equation. (C) 2014 AIP Publishing LLC.

The Non-catalytic ``Cap Domain'' of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Despite highly conserved core catalytic domains, members of the metallophosphoesterase (MPE) superfamily perform diverse and crucial functions ranging from nucleotide and nucleic acid metabolism to phospholipid hydrolysis. Unique structural elements outside of the catalytic core called ``cap domains'' are thought to provide specialization to these enzymes; however, no directed study has been performed to substantiate this. The cap domain of Rv0805, an MPE from Mycobacterium tuberculosis, is located C-terminal to its catalytic domain and is dispensable for the catalytic activity of this enzyme in vitro. We show here that this C-terminal extension (CTE) mediates in vivo localization of the protein to the cell membrane and cell wall as well as modulates expression levels of Rv0805 in mycobacteria. We also demonstrate that Rv0805 interacts with the cell wall of mycobacteria, possibly with the mycolyl-arabinogalactan-peptidoglycan complex, by virtue of its C terminus, a hitherto unknown property of this MPE. Using a panel of mutant proteins, we identify interactions between active site residues of Rv0805 and the CTE that determine its association with the cell wall. Finally, we show that Rv0805 and a truncated mutant devoid of the CTE produce different phenotypic effects when expressed in mycobacteria. Our study thus provides a detailed dissection of the functions of the cap domain of an MPE and suggests that the repertoire of cellular functions of MPEs cannot be understood without exploring the modulatory effects of these subdomains.

Super-Resolution Reconstruction in Frequency-Domain Optical-Coherence Tomography Using the Finite-Rate-of-Innovation Principle

The standard approach to signal reconstruction in frequency-domain optical-coherence tomography (FDOCT) is to apply the inverse Fourier transform to the measurements. This technique offers limited resolution (due to Heisenberg's uncertainty principle). We propose a new super-resolution reconstruction method based on a parametric representation. We consider multilayer specimens, wherein each layer has a constant refractive index and show that the backscattered signal from such a specimen fits accurately in to the framework of finite-rate-of-innovation (FRI) signal model and is represented by a finite number of free parameters. We deploy the high-resolution Prony method and show that high-quality, super-resolved reconstruction is possible with fewer measurements (about one-fourth of the number required for the standard Fourier technique). To further improve robustness to noise in practical scenarios, we take advantage of an iterated singular-value decomposition algorithm (Cadzow denoiser). We present results of Monte Carlo analyses, and assess statistical efficiency of the reconstruction techniques by comparing their performance against the Cramer-Rao bound. Reconstruction results on experimental data obtained from technical as well as biological specimens show a distinct improvement in resolution and signal-to-reconstruction noise offered by the proposed method in comparison with the standard approach.

Time-Domain Approach to Estimate Series Capacitance of an Isolated Phase Winding of a Transformer

Information available in frequency response data is equivalently available in the time domain as a response due to an impulse excitation. The idea to pursue this equivalence to estimate series capacitance is linked to the well-known fact that under impulse excitation, the line/neutral current in a transformer has three distinct components, of which, the initial capacitive component is the first to manifest, followed by the oscillatory and inductive components. Of these, the capacitive component is temporally well separated from the rest-a crucial feature permitting its direct access and analysis. Further, the winding initially behaves as a pure capacitive network, so the initial component must obviously originate from only the (series and shunt) capacitances. With this logic, it should therefore be possible to estimate series capacitance, just by measuring the initial capacitive component of line current and the total shunt capacitance. The principle of the method and details of its implementation on two actual isolated transformerwindings (uniformly wound) are presented. For implementation, a low-voltage recurrent surge generator, a current probe, and a digital oscilloscope are all that is needed. The method is simple and requires no programming and needs least user intervention, thus paving the way for its widespread use.

Structure, interactions and evolutionary implications of a domain-swapped lectin dimer from Mycobacterium smegmatis

Crystal structure determination of the lectin domain of MSMEG_3662 from Mycobacterium smegmatis and its complexes with mannose and methyl-alpha-mannose, the first effort of its kind on a mycobacterial lectin, reveals a structure very similar to beta-prism II fold lectins from plant sources, but with extensive unprecedented domain swapping in dimer formation. The two subunits in a dimer often show small differences in structure, but the two domains, not always related by 2-fold symmetry, have the same structure. Each domain carries three sugar-binding sites, similar to those in plant lectins, one on each Greek key motif. The occurrence of beta-prism II fold lectins in bacteria, with characteristics similar to those from plants, indicates that this family of lectins is of ancient origin and had evolved into a mature system before bacteria and plants diverged. In plants, the number of binding sites per domain varies between one and three, whereas the number is two in the recently reported lectin domains from Pseudomonas putida and Pseudomonas aeruginosa. An analysis of the sequences of the lectins and the lectin domains shows that the level of sequence similarity among the three Greek keys in each domain has a correlation with the number of binding sites in it. Furthermore, sequence conservation among the lectins from different species is the highest for that Greek key which carries a binding site in all of them. Thus, it would appear that carbohydrate binding influences the course of the evolution of the lectin.