bPE toolkit: toolkit for computational protein engineering

We present a computational toolkit consisting of five utility tools, for performing basic operations on a protein structure file in PDB format. The toolkit consists of five different programs which can be integrated as part of a pipeline for computational protein structure characterization or as a standalone analysis package. The programs include tools for chirality check for amino acids (ProChiral), contact map generation (CoMa), data redundancy (DaRe), hydrogen bond potential energy (HyPE) and electrostatic interaction energy (EsInE). All programs in the toolkit can be accessed and downloaded through the following link: http://www.iitg.ac.in/bpetoolkit/.     

Nanodiscs: A toolkit for membrane protein science

Membrane proteins are involved in numerous vital biological processes, including transport, signal transduction and the enzymes in a variety of metabolic pathways. Integral membrane proteins account for up to 30% of the human proteome and they make up more than half of all currently marketed therapeutic targets. Unfortunately, membrane proteins are inherently recalcitrant to study using the normal toolkit available to scientists, and one is most often left with the challenge of finding inhibitors, activators and specific antibodies using a denatured or detergent solubilized aggregate. The Nanodisc platform circumvents these challenges by providing a self‐assembled system that renders typically insoluble, yet biologically and pharmacologically significant, targets such as receptors, transporters, enzymes, and viral antigens soluble in aqueous media in a native‐like bilayer environment that maintain a target''s functional activity. By providing a bilayer surface of defined composition and structure, Nanodiscs have found great utility in the study of cellular signaling complexes that assemble on a membrane surface. Nanodiscs provide a nanometer scale vehicle for the in vivo delivery of amphipathic drugs, therapeutic lipids, tethered nucleic acids, imaging agents and active protein complexes. This means for generating nanoscale lipid bilayers has spawned the successful use of numerous other polymer and peptide amphipathic systems. This review, in celebration of the Anfinsen Award, summarizes some recent results and provides an inroad into the current and historical literature.     

A new family of global protein shape descriptors

A family of global geometric measures is constructed for protein structure classification. These measures originate from integral formulas of Vassiliev knot invariants and give rise to a unique classification scheme. Our measures can better discriminate between many known protein structures than the simple measures of the secondary structure content of these protein structures.     

The protein fluorescence and structural toolkit: Database and programs for the analysis of protein fluorescence and structural data

Protein fluorescence is a powerful tool for studying protein structure and dynamics if we have a means to interpret the spectral data in terms of protein structural properties. Our previous research successfully provided this support through the development of individual software modules implementing the algorithms for fluorescence and structural analyses. Now we have integrated the developed software modules, introduced a new program for the assignment of tryptophan residues to spectral-structural classes, and created a web-based toolkit PFAST: protein fluorescence and structural toolkit: http://pfast.phys.uri.edu/. PFAST contains three modules: (1) FCAT is a fluorescence-correlation analysis tool, which decomposes protein fluorescence spectra to reveal the spectral components of individual tryptophan residues or groups of tryptophan residues located close to each other, and assigns spectral components to one of five previously established spectral-structural classes. (2) SCAT is a structural-correlation analysis tool for the calculation of the structural parameters of the environment of tryptophan residues from the atomic structures of the proteins from the Protein Data Bank (PDB), and for the assignment of tryptophan residues to one of five spectral-structural classes. (3) The last module is a PFAST database that contains protein fluorescence and structural data obtained from results of the FCAT and SCAT analyses.     

A new tool for G protein analysis.

G proteins play a pivotal role in cellular signaling by acting as molecular switches that undergo conformational changes upon binding GTP. The primary sequence constituting the binding cleft among the >160 G proteins in the human genome is highly conserved, consistent with the fact that these proteins share similar guanine nucleotide-binding characteristics. Recent work has demonstrated the feasibility of designing new analogs of GTP that can specifically activate G proteins whose nucleotide-binding sites have been remodeled through mutagenesis. This strategy has the potential to provide new insights into how G proteins act as molecular switches that engage their downstream target/effector proteins to generate specific signaling outputs.     

A toolkit for computational molecular biology I: packing and unpacking of protein coordinate sets

Many investigations in the field of computational molecular biology require access to data sets which contain atomic coordinates of proteins. Programs are described which compress (by a factor of approximately 10) and recreate such data sets. These programs should be useful support for research into the computational analysis of protein structures, as they allow memory and storage devices to be used more efficiently.     

Mdt1/ASCIZ: A new DNA damage response protein family

DNA damage response pathways are crucial for genome stability and prevention of cancer, and are overall remarkably conserved from yeast to mammals. Two novel DNA damage response proteins, yeast Mdt1 (Modifier of DNA damage tolerance 1) and human ASCIZ (ATM/ATR-substrate Chk2-interacting Zn2+-finger protein), were recently identified based on their interactions with the N-terminal FHA domains of the conserved checkpoint kinases Rad53 and Chk2, respectively, and ASCIZ was subsequently re-isolated as an ATM-interacting protein (ATMIN). Mdt1 and ASCIZ share remarkable sequence similarity (36% highly conserved residues, 17% identity) and extended SQ/TQ cluster domains (SCDs) typical of DNA damage response proteins. However, despite their structural similarities and conserved interactions with the checkpoint machinery, the two proteins seem to respond to different DNA lesions: the strongest phenotypes of ASCIZ deficiency are increased sensitivity to DNA base damaging agents and altered immunoglobulin gene diversification following enzyme-induced base damage in B lymphocytes, whereas absence of Mdt1 leads to hypersensitivity to 3'-blocked DNA double-strand breaks and inefficient recombinational maintenance of telomeres. The Mdt1/ASCIZ family may function as structurally related scaffolds that facilitate efficient DNA repair, albeit with diverged lesion specificity.     

Human 76p: A new member of the gamma-tubulin-associated protein family

The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.     

A linkage analysis toolkit for studying allosteric networks in ion channels

A thermodynamic approach to studying allosterically regulated ion channels such as the large-conductance voltage- and Ca²⁺-dependent (BK) channel is presented, drawing from principles originally introduced to describe linkage phenomena in hemoglobin. In this paper, linkage between a principal channel component and secondary elements is derived from a four-state thermodynamic cycle. One set of parallel legs in the cycle describes the “work function,” or the free energy required to activate the principal component. The second are “lever operations” activating linked elements. The experimental embodiment of this linkage cycle is a plot of work function versus secondary force, whose asymptotes are a function of the parameters (displacements and interaction energies) of an allosteric network. Two essential work functions play a role in evaluating data from voltage-clamp experiments. The first is the conductance Hill energy W_H[g], which is a “local” work function for pore activation, and is defined as kT times the Hill transform of the conductance (G-V) curve. The second is the electrical capacitance energy W_C[q], representing “global” gating charge displacement, and is equal to the product of total gating charge per channel times the first moment (V_M) of normalized capacitance (slope of Q-V curve). Plots of W_H[g] and W_C[q] versus voltage and Ca²⁺ potential can be used to measure thermodynamic parameters in a model-independent fashion of the core gating constituents (pore, voltage-sensor, and Ca²⁺-binding domain) of BK channel. The method is easily generalized for use in studying other allosterically regulated ion channels. The feasibility of performing linkage analysis from patch-clamp data were explored by simulating gating and ionic currents of a 17-particle model BK channel in response to a slow voltage ramp, which yielded interaction energies deviating from their given values in the range of 1.3 to 7.2%.     

A phylogenetic analysis of the lipocalin protein family

The lipocalins are a family of extracellular proteins that bind and transport small hydrophobic molecules. They are found in eubacteria and a great variety of eukaryotic cells, in which they play diverse physiological roles. We report here the detection of two new eukaryotic lipocalins and a phylogenetic analysis of 113 lipocalin family members performed with maximum-likelihood and parsimony methods on their amino acid sequences. Lipocalins segregate into 13 monophyletic clades, some of which are grouped in well-supported superclades. An examination of the G + C content of the bacterial lipocalin genes and the detection of four new conceptual lipocalins in other eubacterial species argue against a recent horizontal transfer as the origin of prokaryotic lipocalins. Therefore, we rooted our lipocalin tree using the clade containing the prokaryotic lipocalins. The topology of the rooted lipocalin tree is in general agreement with the currently accepted view of the organismal phylogeny of arthropods and chordates. The rooted tree allows us to assign polarity to character changes and suggests a plausible scenario for the evolution of important lipocalin properties. More recently evolved lipocalins tend to (1) show greater rates of amino acid substitutions, (2) have more flexible protein structures, (3) bind smaller hydrophobic ligands, and (4) increase the efficiency of their ligand-binding contacts. Finally, we found that the family of fatty-acid-binding proteins originated from the more derived lipocalins and therefore cannot be considered a sister group of the lipocalin family.     

DIVERGE: phylogeny-based analysis for functional-structural divergence of a protein family

SUMMARY: DetectIng Variability in Evolutionary Rates among GEnes (DIVERGE) is a software system to study functional divergence of a protein family by detecting site-specific change in evolutionary rate using a multiple alignment of amino acid sequences for a given phylogenetic tree. The program first conducts a statistical test for site-specific rate shifts along the tree, and predicting candidate amino acid residues responsible for functional divergence based on posterior analysis. These results can then be mapped on the 3D protein structure if available. AVAILABILITY: DIVERGE is available free of charge from http://xgu1.zool.iastate.edu/. Distribution packages for both Linux and Microsoft Windows operating systems are available, including manual and example files.     

A Perl toolkit for LIMS development

Background  

High throughput laboratory techniques generate huge quantities of scientific data. Laboratory Information Management Systems (LIMS) are a necessary requirement, dealing with sample tracking, data storage and data reporting. Commercial LIMS solutions are available, but these can be both costly and overly complex for the task. The development of bespoke LIMS solutions offers a number of advantages, including the flexibility to fulfil all a laboratory's requirements at a fraction of the price of a commercial system. The programming language Perl is a perfect development solution for LIMS applications because of Perl's powerful but simple to use database and web interaction, it is also well known for enabling rapid application development and deployment, and boasts a very active and helpful developer community. The development of an in house LIMS from scratch however can take considerable time and resources, so programming tools that enable the rapid development of LIMS applications are essential but there are currently no LIMS development tools for Perl.     

Density-cluster NMA: A new protein decomposition technique for coarse-grained normal mode analysis

Normal mode analysis has emerged as a useful technique for investigating protein motions on long time scales. This is largely due to the advent of coarse-graining techniques, particularly Hooke's Law-based potentials and the rotational-translational blocking (RTB) method for reducing the size of the force-constant matrix, the Hessian. Here we present a new method for domain decomposition for use in RTB that is based on hierarchical clustering of atomic density gradients, which we call Density-Cluster RTB (DCRTB). The method reduces the number of degrees of freedom by 85-90% compared with the standard blocking approaches. We compared the normal modes from DCRTB against standard RTB using 1-4 residues in sequence in a single block, with good agreement between the two methods. We also show that Density-Cluster RTB and standard RTB perform well in capturing the experimentally determined direction of conformational change. Significantly, we report superior correlation of DCRTB with B-factors compared with 1-4 residue per block RTB. Finally, we show significant reduction in computational cost for Density-Cluster RTB that is nearly 100-fold for many examples.     

A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells

Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.     

R-spondin：一个新的Wnt信号通路相关蛋白家族的研究进展

R-spondin（Rspo）是近年来新发现的蛋白家族,包括4个成员（Rspo1～4）。已报道Rspo蛋白家族所有成员均为分泌性蛋白,均有两个富含半胱氨酸的furin-like结构域、1个TSP1结构域和富含碱性氨基酸的C端区域。Rspos通过激活并协同Wnt/β-catenin信号通路参与对细胞增殖和分化的调控,影响骨骼、肌肉、血管等组织的发育以及肢体和性腺的形成,并在多种疾病的发生过程中起重要作用。该文结合最新研究进展,就Rspo家族蛋白的结构、主要功能及其对经典Wnt信号通路的调控机理做一综述。