Pathways,pathway tubes,pathway docking,and propagators in electron transfer proteins

The simplest views of long-range electron transfer utilize flat one-dimensional barrier tunneling models, neglecting structural details of the protein medium. The pathway model of protein electron transfer reintroduces structure by distinguishing between covalent bonds, hydrogen bonds, and van der Waals contacts. These three kinds of interactions in a tunneling pathway each have distinctive decay factors associated with them. The distribution and arrangement of these bonded and nonbonded contacts in a folded protein varies tremendously between structures, adding a richness to the tunneling problem that is absent in simpler views. We review the pathway model and the predictions that it makes for protein electron transfer rates in small proteins, docked proteins, and the photosynthetic reactions center. We also review the formulation of the protein electron transfer problem as an effective two-level system. New multi-pathway approaches and improved electronic Hamiltonians are described briefly as well.     

Highly specific prediction of phosphorylation sites in proteins

SUMMARY: The prediction of significant short functional protein sequences has inherent problems. In predicting phosphorylation sites, problems came from the shortness of phosphorylation sites, the difficulties in maintaining many different predefined models of binding sites, and the difficulties of obtaining highly sensitive predictions and of obtaining predictions with a constant sensitivity and specificity. The algorithm presented in this paper overcomes these problems. The proposed algorithm PHOSITE is based on the case-based sequence analysis. This enables the prediction of phosphorylation sites with constant specificity and sensitivity. Furthermore, this method leads not only to the prediction of phosphorylation sites in general but also predicts the most probable type of kinase involved. AVAILABILITY: The tool PHOSITE implementing the presented method can be evaluated under the website http://www.phosite.com.     

Electrostatic model of S4 motion in voltage-gated ion channels

The S4 transmembrane domain of the family of voltage-gated ion channels is generally thought to be the voltage sensor, whose translocation by an applied electric field produces the gating current. Experiments on hSkMI Na(+) channels and both Shaker and EAG K(+) channels indicate which S4 residues cross the membrane-solution interface during activation gating. Using this structural information, we derive the steady-state properties of gating-charge transfer for wild-type and mutant Shaker K(+) channels. Assuming that the energetics of gating is dominated by electrostatic forces between S4 charges and countercharges on neighboring transmembrane domains, we calculate the total energy as a function of transmembrane displacement and twist of the S4 domain. The resulting electrostatic energy surface exhibits a series of deep energy minima, corresponding to the transition states of the gating process. The steady-state gating-charge distribution is then given by a Boltzmann distribution among the transition states. The resulting gating-charge distributions are compared to experimental results on wild-type and charge-neutralized mutants of the Shaker K(+) channel.     

Simulating psoriasis by altering transit amplifying cells

Computational models of tissue homeostasis will facilitate a deeper understanding of many diseases. They link molecular networks, cellular differentiation and the spatial and temporal organization of tissues. Here we show an approach which is able to computationally turn a healthy in silico epidermis into one with four central properties of psoriatic epidermis. We achieve this by altering a single simulation parameter in the cellular differentiation program of the simulated epidermal keratinocytes: the fractional time period during which transit amplifying cells proliferate (tau). Prolonging tau results in the four main pathological characteristics of psoriatic skin: (1) an absolute increase of the germinative compartment, (2) an absolute increase of the differentiated compartment, (3) a higher proportion of germinative cells and (4) a marked reduction in turnover time. The prolongation of tau is able to increase the proliferation capacity of the epidermal tissue without altering the cell cycle frequency.     

Seasonal periodicity of decapod larvae and population dynamics of selected taxa in New Hampshire (USA) coastal waters

Decapod larvae were collected during night-time hours at threestations off the New Hampshire coast during 1978–1980.Samples were analyzed for species composition and abundance.Life stages were identified for two species of carideans (Eualuspusiolus and Crangon septemspinosa) and two species of brachyurans(Cancer spp. and Carcinus maenas). Production and mortalityestimates were developed for Cancer spp., and C. maenas, andproduction estimates only were developed for C. septemspinosa.     

Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains

The epithelial Na⁺ channel (ENaC) mediates the rate-limiting step in transepithelial Na⁺ transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.     

Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae)

Polymerase chain reaction (PCR) products corresponding to 803 bp of the cytochrome oxidase subunits I and II region of mitochondrial DNA (mtDNA COI-II) were deduced to consist of multiple haplotypes in three Sitobion species. We investigated the molecular basis of these observations. PCR products were cloned, and six clones from one individual per species were sequenced. In each individual, one sequence was found commonly, but also two or three divergent sequences were seen. The divergent sequences were shown to be nonmitochondrial by sequencing from purified mtDNA and Southern blotting experiments. All seven nonmitochondrial clones sequenced to completion were unique. Nonmitochondrial sequences have a high proportion of unique sites, and very few characters are shared between nonmitochondrial clones to the exclusion of mtDNA. From these data, we infer that fragments of mtDNA have been transposed separately (probably into aphid chromosomes), at a frequency only known to be equalled in humans. The transposition phenomenon appears to occur infrequently or not at all in closely related genera and other aphids investigated. Patterns of nucleotide substitution in mtDNA inferred over a parsimony tree are very different from those in transposed sequences. Compared with mtDNA, nonmitochondrial sequences have less codon position bias, more even exchanges between A, G, C and T, and a higher proportion of nonsynonymous replacements. Although these data are consistent with the transposed sequences being under less constraint than mtDNA, changes in the nonmitochondrial sequences are not random: there remains significant position bias, and probable excesses of synonymous replacements and of conservative inferred amino acid replacements. We conclude that a proportion of the inferred change in the nonmitochondrial sequences occurred before transposition. We believe that Sitobion aphids (and other species exhibiting mtDNA transposition) may be important for studying the molecular evolution of mtDNA and pseudogenes. However, our data highlight the need to establish the true evolutionary relationships between sequences in comparative investigations.      

Constraint-based, homology model of the extracellular domain of the epithelial Na+ channel α subunit reveals a mechanism of channel activation by proteases

The epithelial Na(+) channel (ENaC) mediates Na(+) transport across high resistance epithelia. This channel is assembled from three homologous subunits with the majority of the protein's mass found in the extracellular domains. Acid-sensing ion channel 1 (ASIC1) is homologous to ENaC, but a key functional domain is highly divergent. Here we present molecular models of the extracellular region of α ENaC based on a large data set of mutations that attenuate inhibitory peptide binding in combination with comparative modeling based on the resolved structure of ASIC1. The models successfully rationalized the data from the peptide binding screen. We engineered new mutants that had not been tested based on the models and successfully predict sites where mutations affected peptide binding. Thus, we were able to confirm the overall general fold of our structural models. Further analysis suggested that the α subunit-derived inhibitory peptide affects channel gating by constraining motions within two major domains in the extracellular region, the thumb and finger domains.     

Continuum Approaches to Understanding Ion and Peptide Interactions with the Membrane

Experimental and computational studies have shown that cellular membranes deform to stabilize the inclusion of transmembrane (TM) proteins harboring charge. Recent analysis suggests that membrane bending helps to expose charged and polar residues to the aqueous environment and polar head groups. We previously used elasticity theory to identify membrane distortions that minimize the insertion of charged TM peptides into the membrane. Here, we extend our work by showing that it also provides a novel, computationally efficient method for exploring the energetics of ion and small peptide penetration into membranes. First, we show that the continuum method accurately reproduces energy profiles and membrane shapes generated from molecular simulations of bare ion permeation at a fraction of the computational cost. Next, we demonstrate that the dependence of the ion insertion energy on the membrane thickness arises primarily from the elastic properties of the membrane. Moreover, the continuum model readily provides a free energy decomposition into components not easily determined from molecular dynamics. Finally, we show that the energetics of membrane deformation strongly depend on membrane patch size both for ions and peptides. This dependence is particularly strong for peptides based on simulations of a known amphipathic, membrane binding peptide from the human pathogen Toxoplasma gondii. In total, we address shortcomings and advantages that arise from using a variety of computational methods in distinct biological contexts.