CARBANA: Carbon analysis program forprotein sequences

There are lots of works gone into proteins to understand the nature of proteins. Hydrophobic interaction is the dominant force that drives the proteins to carry out the biochemical reactions in all living system. Carbon is the only element that contributes towards this hydrophobic interaction. Studies find that globular proteins prefer to have 31.45% of carbon for its stability. Taking this as standard, a carbon analysis program has been developed to study the carbon distribution profile of protein sequences. This carbon analysis program has been made available online. This can be accessed at www.rajasekaran.net.in/tools/carbana.html. This new program is hoped to help in identification and development of active sites, study of protein stability, evolutionary understating of proteins, gene identification, ligand binding site identification, and to solve the long-standing problem of protein-protein and protein-DNA interactions.     

A simple and novel interpretation of the three-dimensional structure of globular proteins based on quantum-mechanical computations on small model molecules. I

It is suggested that the three-dimensional structure of globular proteins is partly determined by a framework of strengthened hydrogen bonds that involves both ionic side chains and water molecules in addition to the polypeptide backbone. This conclusion follows from a combination of the results of ab initio molecular-orbital computations on small model molecules and high-accuracy x-ray data on the rubredoxin molecule. The computations yield the idea of hydrogen-bonded bridges that are built from tens of atoms, and the experimental information yields the idea that the bridges are assembled into clusters, each of which is built from hundreds of atoms. Some 10 such clusters then form a globular protein.     

Possibility of biocompatibility of fullerene C60 with oligopeptides using a comparative analysis of their spatial form

It was proposed to elucidate the mechanism of unique biological biocompatibility of carbon materials used for making endoprostheses for medicinal practice. For this purpose, a method of comparing the geometry of individual globular and fibrillar proteins and carbon structures (fullerenes) was advanced, and a comparative analysis of the spatial structure of fullerene C60 and the amino acid sequences of 286 proteins was made. Based on a high degree of similarity in the positions of atoms of the polypeptide chains of proteins and peptides and the corresponding atoms of fullerene and of other structural parameters revealed by the comparison of the spatial structures using mathematical simulation, the phenomenon of biological compatibility was interpreted as an "insertion" of fullerenes into the structure of protein molecules in place of structurally similar amino acid sequences, i.e., as a "prosthetics" at the molecular level. It is proposed that fullerenes can "simulate" structurally similar short peptides in biological processes. It was shown that noncarbon biogenic atoms play a large role in the formation of specific structure of protein molecules.     

Decomposition of protein experimental compressibility into intrinsic and hydration shell contributions

The experimental determination of protein compressibility reflects both the protein intrinsic compressibility and the difference between the compressibility of water in the protein hydration shell and bulk water. We use molecular dynamics simulations to explore the dependence of the isothermal compressibility of the hydration shell surrounding globular proteins on differential contributions from charged, polar, and apolar protein-water interfaces. The compressibility of water in the protein hydration shell is accounted for by a linear combination of contributions from charged, polar, and apolar solvent-accessible surfaces. The results provide a formula for the deconvolution of experimental data into intrinsic and hydration contributions when a protein of known structure is investigated. The physical basis for the model is the variation in water density shown by the surface-specific radial distribution functions of water molecules around globular proteins. The compressibility of water hydrating charged atoms is lower than bulk water compressibility, the compressibility of water hydrating apolar atoms is somewhat larger than bulk water compressibility, and the compressibility of water around polar atoms is about the same as the compressibility of bulk water. We also assess whether hydration water compressibility determined from small compound data can be used to estimate the compressibility of hydration water surrounding proteins. The results, based on an analysis from four dipeptide solutions, indicate that small compound data cannot be used directly to estimate the compressibility of hydration water surrounding proteins.     

Stability of globular proteins in H2O and D2O

In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the stability of globular protein structure is determined in a systematic way. The differential scanning calorimetry technique is applied to allow for a thermodynamic analysis of two types of globular proteins: hen's egg lysozyme (LSZ) with relatively strong internal cohesion ("hard" globular protein) and bovine serum albumin (BSA), which is known for its conformational adaptability ("soft" globular protein). Both proteins tend to be more stable in D2O compared to H2O. We explain the increase of protein stability in D2O by the observation that D2O is a poorer solvent for nonpolar amino acids than H2O, implying that the hydrophobic effect is larger in D2O. In case of BSA the transitions between different isomeric forms, at low pH values the Nm and F forms, and at higher pH values Nm and B, were observed by the presence of a supplementary peak in the DSC thermogram. It appears that the pH-range for which the Nm form is the preferred one is wider in D2O than in H2O.     

Dynamical dimer structure and liquid structure of fatty acids in their binary liquid mixture: decanoic/octadecanoic acid and decanoic/dodecanoic acid systems

Dimer structure and liquid structure of fatty acids in their binary mixtures such as decanoic acid (DA)/octadecanoic acid (SA) and DA/dodecanoic acid (LA) were studied through the measurements of self-diffusion coefficient (D), differential scanning calorimetry (DSC), density and viscosity. The obtained phase diagrams showed that DA and SA form a eutectic in the solid state but partly a solid solution in the SA-rich region; DA and LA form an incongruent-melting compound which forms a eutectic with DA. In the liquid mixture of DA and SA, the D of DA is larger than that of SA over the entire range of compositions and tends to approach the D of SA with increasing SA-mole fraction; the D of DA in the DA/LA system is also larger than that of LA especially in the LA-poor region and steeply approaches that of LA with increasing LA-mole fraction. These D values and phase diagrams were compared with those for the binary mixtures of n-alkanes (C14/C20, C19/C20 and C20/C24); it is concluded that the two kinds of fatty acids always form their individual homodimers in their liquid mixtures regardless of their compositions and temperatures.     

Hydrogen bonds involving sulfur atoms in proteins.

Intrachain hydrogen bonds are a hallmark of globular proteins. Traditionally, these involve oxygen and nitrogen atoms. The electronic structure of sulfur is compatible with hydrogen bond formation as well. We surveyed a set of 85 high-resolution protein structures in order to evaluate the prevalence and geometry of sulfur-containing hydrogen bonds. This information should be of interest to experimentalists and theoreticians interested in protein structure and protein engineering.     

Subcellular distribution and membrane association of human neutrophil substrates for ADP-ribosylation by pertussis toxin and cholera toxin

Neutrophil guanine nucleotide-binding proteins are important components of receptor-mediated cellular responses such as degranulation, chemotaxis, and superoxide production. Because the cytoplasmic granules of neutrophils serve as an intracellular store of receptors and NADPH oxidase components, we investigated the subcellular distribution of substrates for ADP-ribosylation by both pertussis and cholera toxins. Cholera toxin substrates of Mr 43 and 52 kDa were present only in the plasma membrane fraction. A 39-kDa pertussis toxin substrate was present in the plasma membrane, cytosol, and a specific granule-enriched fraction. There were no substrates for either toxin in the primary granules. Quantitative GTP-gamma-5 binding was localized predominantly to the plasma membrane fraction (47%), but significant portions were found in the specific granule-enriched fractions (13%) and cytosol (34%) as well. Two-dimensional gel electrophoresis and chymotryptic digests of the pertussis toxin substrate from these three subcellular fractions suggested that they are highly homologous. Triton X-114 phase partitioning was used to investigate the hydrophobicity of the toxin substrates. The pertussis toxin substrates in the plasma membrane and granule fractions behaved like integral membrane proteins, whereas the cytosolic substrate partitioned into both lipophilic and aqueous fractions. ADP-ribosylation converted the substrates to a somewhat less lipophilic form. These data suggest that the specific granules or an organelle of similar density serve as an intracellular store of a G protein with a 39-kDa alpha-subunit and that the cytosolic fraction of neutrophils contains free alpha-subunits of the same size.     

Heterogeneity of packing: structural approach.

Analysis of the heterogeneity of packing in proteins showed that different groups of the protein preferentially contribute to low- or high-density regions. Statistical distribution reveals the two preferable values for packing density in the form of two peaks. One peak occurs in the range of densities 0.55-0.65, the other occurs in the range 0.75-0.8. The high-density peak is originated primarily by high packing inside the hydrogen bonded backbone and to some extent by side chains. Polar/charged and apolar side chains both contribute to the low-density peak. The average packing density values of individual atomic groups significantly vary for backbone atoms as well as for side chain atoms. The carbonyl oxygen atoms of protein backbone and the end groups of side chains show lower packing density than the rest of the protein. The side-chain atomic groups of a secondary structure element when packed against the neighboring secondary structure element form stronger contacts with the side chains of this element than with its backbone. Analysis of the low-density regions around each buried peptide group was done for the set of proteins with different types of packing, including alpha-alpha, alpha-beta, and beta-beta packing. It was shown that cavities are regularly situated in the groove of secondary structure element packed against neighboring elements for all types of packing. Low density in the regions surrounding the peptide groups and the end groups of side chains can be explained by their positioning next to a cavity formed upon the association of secondary structure elements. The model proposed can be applied to the analysis of protein internal motions, mechanisms of cellular signal transduction, diffusion through protein matrix, and other events.     

Reconstructing the protein–water interface

Using molecular dynamics simulations of fully hydrated proteins and analysis of crystal structures contained in the Protein Data Bank, we develop a transferable set of perpendicular radial distribution functions for water molecules around globular proteins. These universal functions may be used to reconstruct the unique three-dimensional solvent density distribution around every individual protein with a modest error. We discuss potential applications of this solvent treatment in protein x-ray crystallographic refinements and in theoretical modeling. We also present a fast, grid-based algorithm for construction of the perpendicular solvent density distributions. © 1998 John Wiley & Sons, Inc. Biopoly 45: 469–478, 1998     

SuperStar: a knowledge-based approach for identifying interaction sites in proteins.

An empirical method for identifying interaction sites in proteins is described and validated. The method is based entirely on experimental information about non-bonded interactions occurring in small-molecule crystal structures. These data are used in the form of scatterplots that show the experimentally observed distribution of one functional group (the "contact group" or "probe") around another. A template molecule (e.g. a protein binding site) is broken down into structure fragments and the scatterplots, showing the distribution of a chosen probe around these structure fragments, are superimposed on the corresponding parts of the template. The scatterplots are then translated into a three-dimensional map that shows the propensity of the probe at different positions around the template molecule. The method is illustrated for l -arabinose-binding protein, complexed with l -arabinose and with d -fucose, and for dihydrofolate reductase complexed with methotrexate. The method is validated on 122 X-ray structures of protein-ligand complexes. For all the binding sites of these proteins, propensity maps are generated for four different probes: a charged NH+3nitrogen, a carbonyl oxygen, a hydroxyl oxygen and a methyl carbon atom. Next, the maps are compared with the experimentally observed positions of ligand atoms of these types. For 74% of these ligand atoms (84% of the solvent-inaccessible ones) the calculated propensity of the matching probe at the experimental positions is higher than expected by chance. For 68% of the atoms (82% of the solvent-inaccessible ones) the propensity of the matching probe is higher than that of the other three probes. These results indicate that the approach generally gives good predictions for protein-ligand interactions. The potential applications of the propensity maps range from an aid in manual docking and structure-based drug design to their use in pharmacophore development.     

Cotranslational, cosecretory protein folding and its renaturation from a denatured state

On the basis of the available experimental data on structure, biosynthesis and secretion of globular proteins it is concluded that an alpha-helix is a starting conformation at formation of the native structure of any globular protein (alpha-helical model for initiation of protein folding). The structural invariant (clusterization of hydrophobic side chains on the alpha-helix surface) in the amino acid sequences of globular proteins is found which is predicted by alpha-helical model for the initiation of protein folding. The model predicts the pyramidization of the atoms C and N of peptide groups during the formation of spatial structure of proteins and a number of other effects that can be put to the experimental test. In the work the mechanism for protein translocation across membrane lipid bilayer is also suggested.     

Identification of GTP-Binding Proteins by ADP-Ribosylation in the Presence of Cholera Toxin, Pertussis Toxin and Botulinum Toxin D in Plasma Membrane Isolated from Eggs and Embryos of Sea Urchin

In plasma membrane fraction isolated from eggs and embryos of sea urchin, ³²P-labeled proteins were found on the fluorographs of SDS-polyacrylamide gel electrophoresis, performed after an exposure of the fraction to [adenylate-³²P] nicotinamide adenine dinucleotide in the presence of cholera toxin, pertussis toxin or botulinum toxin D. The molecular weights of proteins, thus ADP-ribosylated in the presence of cholera toxin and pertussis toxin are 45 and 39 K, which correspond to Gs and Gi or Go, respectively. Protein with the molecular weight of 24 K, labeled in the presence of botulinum toxin D, corresponds to small molecular weight G-protein. The labeling intensity of 45 K protein, probably proportional to its amount, became high at the blastula stage. The labeling intensity of 39 K protein was hardly altered up to the blastula stage. The labeling intensity of 24 K protein increased after fertilization and further increase occurred at the blastula stage. At the gastrula stage, the labeling intensities of these proteins became somewhat lower than at the blastula stage. Transmembrane signaling system, in which these G-proteins are involved, is probably altered in its function during early development.     

3D domain swapping: as domains continue to swap

Three-dimensional (3D) domain swapping creates a bond between two or more protein molecules as they exchange their identical domains. Since the term '3D domain swapping' was first used to describe the dimeric structure of diphtheria toxin, the database of domain-swapped proteins has greatly expanded. Analyses of the now about 40 structurally characterized cases of domain-swapped proteins reveal that most swapped domains are at either the N or C terminus and that the swapped domains are diverse in their primary and secondary structures. In addition to tabulating domain-swapped proteins, we describe in detail several examples of 3D domain swapping which show the swapping of more than one domain in a protein, the structural evidence for 3D domain swapping in amyloid proteins, and the flexibility of hinge loops. We also discuss the physiological relevance of 3D domain swapping and a possible mechanism for 3D domain swapping. The present state of knowledge leads us to suggest that 3D domain swapping can occur under appropriate conditions in any protein with an unconstrained terminus. As domains continue to swap, this review attempts not only a summary of the known domain-swapped proteins, but also a framework for understanding future findings of 3D domain swapping.     

Structural composition of betaI- and betaII-proteins

Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of alpha-rich proteins are similar to those of model alpha-helices, but beta-rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model beta-sheets or unordered polypeptides. The existence of these two types of CD spectra for beta-rich proteins form the basis for their classification as betaI- and betaII-proteins. Although the conformation of beta-sheets is largely responsible for the CD spectra of betaI-proteins, the source of betaII-protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II-type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the beta-sheet and P2 structure contents in beta-rich proteins to understand the origin of betaII-protein CD. We find that betaII-proteins have a ratio of P2 to beta-sheet content greater than 0.4, whereas for betaI-proteins this ratio is less than 0.4. The beta-sheet content in betaI-proteins is generally higher than that in betaII-proteins. The origin of two classes of CD spectra for beta-rich proteins appears to lie in their relative beta-sheet and P2 structure contents.     

Automated amino acid side-chain NMR assignment of proteins using <Superscript>13</Superscript>C- and <Superscript>15</Superscript>N-resolved 3D [<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY

ASCAN is a new algorithm for automatic sequence-specific NMR assignment of amino acid side-chains in proteins, which uses as input the primary structure of the protein, chemical shift lists of (1)H(N), (15)N, (13)C(alpha), (13)C(beta) and possibly (1)H(alpha) from the previous polypeptide backbone assignment, and one or several 3D (13)C- or (15)N-resolved [(1)H,(1)H]-NOESY spectra. ASCAN has also been laid out for the use of TOCSY-type data sets as supplementary input. The program assigns new resonances based on comparison of the NMR signals expected from the chemical structure with the experimentally observed NOESY peak patterns. The core parts of the algorithm are a procedure for generating expected peak positions, which is based on variable combinations of assigned and unassigned resonances that arise for the different amino acid types during the assignment procedure, and a corresponding set of acceptance criteria for assignments based on the NMR experiments used. Expected patterns of NOESY cross peaks involving unassigned resonances are generated using the list of previously assigned resonances, and tentative chemical shift values for the unassigned signals taken from the BMRB statistics for globular proteins. Use of this approach with the 101-amino acid residue protein FimD(25-125) resulted in 84% of the hydrogen atoms and their covalently bound heavy atoms being assigned with a correctness rate of 90%. Use of these side-chain assignments as input for automated NOE assignment and structure calculation with the ATNOS/CANDID/DYANA program suite yielded structure bundles of comparable quality, in terms of precision and accuracy of the atomic coordinates, as those of a reference structure determined with interactive assignment procedures. A rationale for the high quality of the ASCAN-based structure determination results from an analysis of the distribution of the assigned side chains, which revealed near-complete assignments in the core of the protein, with most of the incompletely assigned residues located at or near the protein surface.     

The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing

The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed “ruler” protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a “ball-and-chain” architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler protein''s N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems.     

The 3D solution structure of the C-terminal region of Ku86 (Ku86CTR)

In eukaryotes the non-homologous end-joining repair of double strand breaks in DNA is executed by a series of proteins that bring about the synapsis, preparation and ligation of the broken DNA ends. The mechanism of this process appears to be initiated by the obligate heterodimer (Ku70/Ku86) protein complex Ku that has affinity for DNA ends. Ku then recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The three-dimensional structures of the major part of the Ku heterodimer, representing the DNA-binding core, both free and bound to DNA are known from X-ray crystallography. However, these structures lack a region of ca 190 residues from the C-terminal region (CTR) of the Ku86 subunit (also known as Lupus Ku autoantigen p86, Ku80, or XRCC5) that includes the extreme C-terminal tail that is reported to be sufficient for DNA-PKcs-binding. We have examined the structural characteristics of the Ku86CTR protein expressed in bacteria. By deletion mutagenesis and heteronuclear NMR spectroscopy we localised a globular domain consisting of residues 592-709. Constructs comprising additional residues either to the N-terminal side (residues 543-709), or the C-terminal side (residues 592-732), which includes the putative DNA-PKcs-binding motif, yielded NMR spectra consistent with these extra regions lacking ordered structure. The three-dimensional solution structure of the core globular domain of the C-terminal region of Ku86 (Ku86CTR(592-709)) has been determined using heteronuclear NMR spectroscopy and dynamical simulated annealing using structural restraints from nuclear Overhauser effect spectroscopy, and scalar and residual dipolar couplings. The polypeptide fold comprises six regions of alpha-helical secondary structure that has an overall superhelical topology remotely homologous to the MIF4G homology domain of the human nuclear cap binding protein 80 kDa subunit and the VHS domain of the Drosophila protein Hrs, though strict analysis of the structures suggests that these domains are not functionally related. Two prominent hydrophobic pockets in the gap between helices alpha2 and alpha4 suggest a potential ligand-binding characteristic for this globular domain.     

General architecture of the alpha-helical globule

A model is presented for the arrangement of alpha-helices in globular proteins. In the model, helices are placed on certain ribs of "quasi-spherical" polyhedra. The polyhedra are chosen so as to allow the close packing of helices around a hydrophobic core and to stress the collective interactions of the individual helices. The model predicts a small set of stable architectures for alpha-helices in globular proteins and describes the geometries of the helix packings. Some of the predicted helix arrangements have already been observed in known protein structures; others are new. An analysis of the three-dimensional structures of all proteins for which co-ordinates are available shows that the model closely approximates the arrangements and packing of helices actually observed. The average deviations of the real helix axes from those in the model polyhedra is +/- 20 degrees in orientation and +/- 2 A in position (1 A = 0.1 nm). We also show that for proteins that are not homologous, but whose helix arrangements are described by the same polyhedron, the root-mean-square difference in the position of the C alpha atoms in the helices is 1.6 to 3.0 A.     

Expression of functional scorpion neurotoxin Lqq-V in E.coli

We report the results on the expression in Escherichia coli of a functional neurotoxin LqqV from the scorpion Leiurus quinquestriatus quinquestriatus. The gene for LqqV was synthesized using recursive PCR and expressed as a poly-histidine-tagged fusion protein in thioredoxin mutant E. coli strain [AD494(DE3)pLysS], thus permitting disulfide-bond formation. When cultured at 37 degrees C, about 50% of the expressed protein is contained as a monomer in the soluble fraction of the E. coli extract. The fusion protein from the soluble fraction was purified and the His-tag was cleaved by thrombin, resulting in a yield of about 1.5 mg/liter. The globular structure of the purified protein was confirmed by NMR and CD spectroscopy. Patch-clamp measurements using native sodium channels in guinea pig ventricular myocytes reveal (1) a slowing of inactivation and (2) a decrease in peak current upon application of toxin, thus confirming the alpha-toxin activity of the purified recombinant protein.