Membrane binding motif of the P-type cardiotoxin

Carditoxins (CTXs) from cobra snake venoms, the basic 60-62 residue all-beta sheet polypeptides, are known to bind to and impair the function of cell membranes. To assess the membrane induced conformation and orientation of CTXs, the interaction of the P-type cardiotoxin II from Naja oxiana snake venom (CTII) with perdeuterated dodecylphosphocholine (DPC) was studied using ( 1 )H-NMR spectroscopy and diffusion measurements. Under conditions where the toxin formed a well-defined complex with DPC, the spatial structure of CTII with respect to the presence of tightly bound water molecules in loop II, was calculated using the torsion angle dynamics program DYANA. The structure was found to be similar, except for subtle changes in the tips of all three loops, to the previously described "major" form of CTII in aqueous solution illustrated by the "trans" configuration of the Val7-Pro8 peptide bond. No "minor" form with the "cis" configuration of the above bond was found in the micelle-bound state. The broadening of the CTII backbone proton signals by 5, 16-doxylstearate relaxation probes, together with modeling based on the spatial structure of CTII, indicated a periphery mode of binding of the toxin molecule to the micelle and revealed its micelle interacting domain. The latter includes a hydrophobic region of CTII within the extremities of loops I and III (residues 5-11, 46-50), the basement of loop II (residues 24-29,31-37) and the belt of polar residues encircling these loops (lysines 4,5,12,23,50, serines 11,46, histidine 31, arginine 36). It is suggested that this structural motif and the mode of binding can be realized during interaction of CTXs with lipid and biological membranes.     

Tryptophanase from Proteus vulgaris: the conformational rearrangement in the active site, induced by the mutation of Tyrosine 72 to phenylalanine, and its mechanistic consequences

Tyr72 is located at the active site of tryptophanase (Trpase) from Proteus vulgaris. For the wild-type Trpase Tyr72 might be considered as the general acid catalyst at the stage of elimination of the leaving groups. The replacement of Tyr72 by Phe leads to a decrease in activity for L-tryptophan by 50,000-fold and to a considerable rearrangement of the active site of Trpase. This rearrangement leads to an increase of room around the alpha-C atom of any bound amino acid, such that covalent binding of alpha-methyl-substituted amino acids becomes possible (which cannot be realized in wild-type Trpase). The changes in reactivities of S-alkyl-L-cysteines provide evidence for an increase of congestion in the proximity of their side groups in the mutant enzyme as compared to wild-type enzyme. The observed alteration of catalytic properties in a large degree originates from a conformational change in the active site. The Y72F Trpase retains significant activity for L-serine, which allowed us to conclude that in the mutant enzyme, some functional group is present which fulfills the role of the general acid catalyst in reactions associated with elimination of small leaving groups.     

The Molecular Clock of Neutral Evolution Can Be Accelerated or Slowed by Asymmetric Spatial Structure

Over time, a population acquires neutral genetic substitutions as a consequence of random drift. A famous result in population genetics asserts that the rate, K, at which these substitutions accumulate in the population coincides with the mutation rate, u, at which they arise in individuals: K = u. This identity enables genetic sequence data to be used as a “molecular clock” to estimate the timing of evolutionary events. While the molecular clock is known to be perturbed by selection, it is thought that K = u holds very generally for neutral evolution. Here we show that asymmetric spatial population structure can alter the molecular clock rate for neutral mutations, leading to either K<u or K>u. Our results apply to a general class of haploid, asexually reproducing, spatially structured populations. Deviations from K = u occur because mutations arise unequally at different sites and have different probabilities of fixation depending on where they arise. If birth rates are uniform across sites, then K ≤ u. In general, K can take any value between 0 and Nu. Our model can be applied to a variety of population structures. In one example, we investigate the accumulation of genetic mutations in the small intestine. In another application, we analyze over 900 Twitter networks to study the effect of network topology on the fixation of neutral innovations in social evolution.     

Statistical modeling of biomedical corpora: mining the Caenorhabditis Genetic Center Bibliography for genes related to life span

Background  

The statistical modeling of biomedical corpora could yield integrated, coarse-to-fine views of biological phenomena that complement discoveries made from analysis of molecular sequence and profiling data. Here, the potential of such modeling is demonstrated by examining the 5,225 free-text items in the Caenorhabditis Genetic Center (CGC) Bibliography using techniques from statistical information retrieval. Items in the CGC biomedical text corpus were modeled using the Latent Dirichlet Allocation (LDA) model. LDA is a hierarchical Bayesian model which represents a document as a random mixture over latent topics; each topic is characterized by a distribution over words.     

Transient increase of tryptophan fluorescence of enzyme caused by photoexcitation of ligand in luciferase-luciferin complex

An experiment was proposed and accomplished that was based on the hypothesis of the dissociation of the luciferase-luciferin complex in photoexcitation. A pump-probe experiment was performed with the use of picosecond laser pulses and was based on the effect of quenching of enzyme tryptophan fluorescence caused by luciferin binding. A photoinduced increase of the tryptophan fluorescence intensity was detected. Experimental results were interpreted on the basis of the assumptions on photoinduced dissociation of the luciferin-luciferase complex and Forster energy transfer from tryptophan to luciferin. Under the assumption on the photoinduced dissociation and stationary quenching of tryptophan fluorescence the rate of propagation of the conformational changes in the protein caused by the complex dissociation was estimated to be >20 m/s.     

Hydrogel-based protein microchips: manufacturing,properties, and applications

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.     

A less laborious approach to the high-throughput production of recombinant proteins in Escherichia coli using 2-liter plastic bottles

Contemporary approaches to biology often call for the high-throughput production of large amounts of numerous proteins for structural or functional studies. Even with the highly efficient protein expression systems developed in Escherichia coli, production of these proteins is laborious and time-consuming. We have simplified established protocols by the use of disposable culture vessels: common 2-liter polyethylene terephthalate beverage bottles. The bottles are inexpensive, fit conveniently in commonly available flask holders, and, because they are notched, provide sufficient aeration to support the growth of high-density cultures. The use of antibiotics and freshly prepared media alleviates the need for sterilization of media and significantly reduces the labor involved. Uninoculated controls exhibited no growth during the time required for protein expression in experimental cultures. The yield, solubility, activity, and pattern of crystallization of proteins expressed in bottles were comparable to those obtained under conventional culture conditions. After use, the bottles are discarded, reducing the risk of cross-contamination of subsequent cultures. The approach appears to be suitable for high-throughput production of proteins for structural or functional studies.     

Fluorescent Properties of Firefly Luciferases and Their Complexes with Luciferin

Fluorescence of luciferases from Luciola mingrelica (single tryptophanresidue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417,Trp-426) was studied. Analysis of quenching of tryptophan fluorescenceshowed that the tryptophan residue conserved in all luciferases is notaccessible for charged quenchers, which is explained by the presence ofpositively and negatively charged amino acid residues in the close vicinityto it. An effective energy transfer from tryptophan to luciferin wasobserved during quenching of tryptophan fluorescence of both luciferaseswith luciferin. From the data on the energy transfer, the distance betweenthe luciferin molecule and Trp-417 (419) in the luciferin–luciferasecomplex was calculated: 11–15 for P. pyralis and 12–17 for L. mingrelica luciferases. The role of the conserved Trp residuein the catalysis is discussed.