Phylogenetic trees constructed from hydrophobicity values of protein sequences

Information about conformational properties of a protein is contained in the hydrophobicity values of the amino acids in its primary sequence. We have investigated the possibility of extracting meaningful evolutionary information from the comparison of the hydrophobicity values of the corresponding amino acids in the sequences of homologous proteins. Distance matrices for six families of homologous proteins were made on the basis of the differences in hydrophobicity values of the amino acids. The phylogenetic trees constructed from such matrices were at least as good (as judged from their faithful reflection of evolutionary relationships), as trees constructed from the usual minimum mutation distance matrix.     

From the genome sequence to the proteome and back: evaluation of E. coli genome annotation with a 2-D gel-based proteomics approach

The ambition of systems biology to understand complex biological systems at the molecular level implies that we need to have a concrete and correct understanding of each molecular entity and its function. However, even for the best-studied organism, Escherichia coli, a large number of proteins have never been identified and characterised from wild-type cells, and/or await unravelling of their biological role. Instead, the ORF models for these proteins have been predicted by suitable algorithms and/or through comparison with known, homologous proteins from other organisms, approaches which may be prone to error. In the present study, we used a combination of 2-DE, MALDI-TOF-MS and PMF to identify 1151 different proteins in E. coli K12 JM109. Comparison of the experimental with the theoretical Mr and pI values (4000 experimental values each) allowed the identification of numerous proteins with incorrect or incomplete ORF annotations in the current E. coli genome databases. Several inconsistencies in genome annotation were verified experimentally, and up to 55 candidates await further investigation. Our findings demonstrate how an up-to-date 2-D gel-based proteomics approach can be used for improving the annotation of prokaryotic genomes. They also highlight the need for harmonization among the different E. coli genome databases.     

Recognition of protein folds via dipolar couplings

Alignment of proteins in dilute liquid crystalline medium gives rise to residual dipolar couplings which provide orientational information of vectors connecting the interacting nuclei. Considering that proteins are mainly composed of regular secondary structures in a finite number of different mutual orientations, main chain dipolar couplings appear sufficient to reveal structural resemblance. Similarity between dipolar couplings measured from a protein and corresponding values computed from a known structure imply homologous structures. For dissimilar structures the agreement between experimental and calculated dipolar couplings remains poor. In this way protein folds can be readily recognized prior to a comprehensive structure determination. This approach has been demonstrated by showing the similarity in fold between the hitherto unknown structure of calerythrin and sarcoplasmic calcium-binding proteins from Nereis diversicolor and Branchiostoma lanceolatum with known crystal structures.     

Inter-species sequence conservation of single-spanning transmembrane regions

The extent of inter-species sequence identity in single-spanning transmembrane regions of integral membrane proteins was evaluated. The sequences of the 32 human transmembrane regions were compared with the respective rodent homologues. The identity between homologous transmembrane regions ranged from 32 to 100%, compared with a mean value of 14% identity between unrelated transmembrane sections. On average the identity between homologous transmembrane regions is slightly higher than for the rest of the chain. These values suggest that, in general, there are structural and/or functional constraints on the transmembrane regions beyond the simple requirement to act as a passive, nonpolar, connecting region across the cell membrane. Although there is limited experimental evidence available, the three transmembrane regions (CD2 antigen, MHC class I and ICAM-1) with particularly low values of inter-species identity (less than 50%) are probably not involved in an interaction with another transmembrane section in the same cell.     

Thermal Adaptation of Conformational Dynamics in Ribonuclease H

The relationship between inherent internal conformational processes and enzymatic activity or thermodynamic stability of proteins has proven difficult to characterize. The study of homologous proteins with differing thermostabilities offers an especially useful approach for understanding the functional aspects of conformational dynamics. In particular, ribonuclease HI (RNase H), an 18 kD globular protein that hydrolyzes the RNA strand of RNA:DNA hybrid substrates, has been extensively studied by NMR spectroscopy to characterize the differences in dynamics between homologs from the mesophilic organism E. coli and the thermophilic organism T. thermophilus. Herein, molecular dynamics simulations are reported for five homologous RNase H proteins of varying thermostabilities and enzymatic activities from organisms of markedly different preferred growth temperatures. For the E. coli and T. thermophilus proteins, strong agreement is obtained between simulated and experimental values for NMR order parameters and for dynamically averaged chemical shifts, suggesting that these simulations can be a productive platform for predicting the effects of individual amino acid residues on dynamic behavior. Analyses of the simulations reveal that a single residue differentiates between two different and otherwise conserved dynamic processes in a region of the protein known to form part of the substrate-binding interface. Additional key residues within these two categories are identified through the temperature-dependence of these conformational processes.     

Influence of protein net charge on the nucleic acid helix-destabilizing activity of various pancreatic ribonucleases

Summary Helix-destabilization of double-stranded poly[d(A-T)] induced by various homologous pancreatic ribonucleases which differ in their net charges has been studied under different ionic strength conditions.The response of the destabilizing activity of the various proteins to ionic strength is represented by bell-shaped curves, whose maxima are shifted to higher ionic strength values the higher the number of positive charges of the RNAase involved in the nucleic acid-protein complex.This observation is discussed, and a model proposed, that could explain the experimental results presented.     

Hsp70-2 is Required for Tumor Cell Growth and Survival

Heat shock protein 70 (Hsp70) family consists of at least eight chaperone proteins that differ from each other by their pattern of expression and intracellular localization. Whereas ample experimental and clinico-pathological data has implicated the major stress-inducible Hsp70-1 as a protein required for cancer cell survival, the study of the other family members has been limited by the lack of experimental tools to differentiate between the highly homologous family members. This limitation has been recently overcome by the RNA interference technology that for the first time allows targeted knockdown of the individual Hsp70 family members. Data based on this technology has revealed that also Hsp70-2, a protein essential for spermatogenesis, is required for cancer cell growth and survival. Remarkably, the highly homologous Hsp70 proteins enhance cancer cell growth and survival by distinct molecular mechanisms.     

The glycine-rich loop of adenylate kinase forms a giant anion hole

The conformation of the glycine-rich loop of adenylate kinase is described in detail. It forms a giant anion hole for a sulfate ion, which presumably mimicks a nucleotide phosphoryl group. This loop had been called flexible, because at pH values of 6 or below it is displaced in the crystal. In the region of this loop the adenylate kinases are probably homologous to the p21 proteins. Is is known that a mutation in this loop at residue 12 of p21 causes cell transformation and therefore cancer. Other potentially homologous proteins are indicated.     

Proteins of the Bacillus stearothermophilus ribosome. The amino acid sequences of proteins S5 and L30

The amino acid sequences of ribosomal proteins S5 and L30 from Bacillus stearothermophilus have been determined. These proteins have recently been crystallized in our institute. Sequence data were obtained by manual sequencing of peptides derived from cyanogen bromide cleavage and digestion with trypsin and chymotrypsin or thermolysin. Proteins S5 and L30 contain 166 and 62 amino acid residues and have calculated Mr values of 17,628 and 7,053, respectively. Comparison of the sequences with those of the homologous proteins from Escherichia coli shows 55% identical residues for S5 and 53% for L30. For both proteins, the distribution of conserved and substituted regions is not uniform throughout the molecule. Secondary structure predictions were carried out for the B. stearothermophilus proteins. Comparison with the results for the homologous E. coli proteins indicated similar secondary structural order for the molecules from the two species.     

One- and two-dimensional NMR spectral analysis of the consequences of single amino acid replacements in proteins

The traditional approach of using homologous sequences to elucidate the role of specific amino acid residues in protein structure and function becomes more meaningful as the number of differences is minimized, with the limit being alteration of a single residue. For small proteins in solution, NMR spectroscopy offers a means of obtaining detailed information about each residue and its response to a given change in the protein sequence. Extraction of this information has been aided by recent progress in spectrometer technology (higher magnetic fields, more sensitive signal detection, more sophisticated computers) and experimental strategies (new NMR pulse sequences including multiple-quantum and two-dimensional NMR methods). The set of avian ovomucoid third domains, which consists of the third domain proper plus a short leader (connecting peptide) and has a maximum of 56 amino acid residues, offers an attractive system for developing experimental methods for investigating sequence-structure and structure-function relationships in proteins. Our NMR results provide examples of sequence effects on pKa' values, average conformation, and internal motion of amino acid side chains.     

Analysis of shuffled gene libraries

In vitro recombination of homologous genes (family shuffling) has been proposed as an effective search strategy for laboratory evolution of genes and proteins. Few data are available, however, on the composition of shuffled gene libraries, from which one could assess the efficiency of recombination and optimize protocols. Here, probe hybridization is used in a macroarray format to analyze chimeric DNA libraries created by DNA shuffling. Characterization of hundreds of shuffled genes encoding dioxygenases has elucidated important biases in the shuffling reaction. As expected, crossovers are favored in regions of high sequence identity. A sequence-based model of homologous recombination that captures this observed bias was formulated using the experimental results. The chimeric genes were found to show biases in the incorporation of sequences from certain parents, even before selection. Statistically different patterns of parental incorporation in genes expressing functional proteins can help to identify key sequence-function relationships.     

Phenol-treatment and a homologous pairing-assay.

Homologous pairing is a key step in homologous genetic recombination. In the early stage of trials for the identification of homologous pairing-promoting proteins from a fission yeast, Schizosaccharomyces pombe, we treated DNA products with phenol in the presence of a salt for the removal of tightly bound proteins from DNA before the assay, but we found that this treatment caused very efficient protein-independent double-strand formation from complementary single-stranded DNAs. Using an assay including the phenol treatment, we detected another species of apparent homologous pairing-promoting proteins in the nuclei, in addition to a homologous pairing-promoting protein consisting of three components which we reported previously. However, studies involving the use of an assay without the phenol-treatments revealed that the second one was not really a homologous pairing-protein. Thus, the protein-independent double-strand formation by phenol-treatment in the presence of a salt could cause the erroneous identification of homologous pairing-promoting proteins.     

Effect of soil salinity on the electro-chemical and chemical kinetics of some plant nutrients in submerged soils

Summary The electro-chemical and chemical kinetics of six California rice soils were significantly influenced by the presence of salts up to an EC of 9 mmhos/cm in saturation extract (EC_e). Subsamples of each soil salinity treatment were incubated for periods up to 10 weeks after flooding. Most of the changes in Eh and pH values took place in the first 3–4 weeks after submergence. Salinity decreased pH values, but slightly increased the redox-potential. Both ammonification and nitrate reduction were significantly decreased, by increasing soil salinity. Salinity up to 9 mmhos/cm did not affect levels of Bray and Kurtz extractable P, but increased the water extractable Ca, Mg, K and Mn. In DTPA extract, salinity in incubated soils had no effect on Zn in 4 soils, but it decreased Fe in acid and neutral soils. Possible explanations for the electro-chemical and chemical kinetic changes due to flooding and salinity are discussed.     

Random Field Model Reveals Structure of the Protein Recombinational Landscape

We are interested in how intragenic recombination contributes to the evolution of proteins and how this mechanism complements and enhances the diversity generated by random mutation. Experiments have revealed that proteins are highly tolerant to recombination with homologous sequences (mutation by recombination is conservative); more surprisingly, they have also shown that homologous sequence fragments make largely additive contributions to biophysical properties such as stability. Here, we develop a random field model to describe the statistical features of the subset of protein space accessible by recombination, which we refer to as the recombinational landscape. This model shows quantitative agreement with experimental results compiled from eight libraries of proteins that were generated by recombining gene fragments from homologous proteins. The model reveals a recombinational landscape that is highly enriched in functional sequences, with properties dominated by a large-scale additive structure. It also quantifies the relative contributions of parent sequence identity, crossover locations, and protein fold to the tolerance of proteins to recombination. Intragenic recombination explores a unique subset of sequence space that promotes rapid molecular diversification and functional adaptation.     

Experimental manipulation of water levels in two French riverine grassland soils

In this experimental study, we simulated the effects of different river flooding regimes on soil nutrient availability, decomposition and plant production in floodplain grasslands. This was done to investigate the influences of soil water contents on nutrient cycling. Water levels were manipulated in mesocosms with intact soil turfs from two French floodplain grasslands. Three water levels were established: a `wet' (water level at the soil surface), an `intermediate' (water level at –20 cm) and a `dry' treatment (water level at –120 cm). With increasing soil moisture, soil pH became more neutral, while redox-potential and oxygen concentration decreased. The `dry' treatment showed much lower values for process rates in soil and vegetation than the `intermediate' and `wet' treatments. Regressions showed that soil C-evolution and N-mineralization were positively related to soil moisture content. Not all mineralized N was available for plant uptake in the wet treatment, as a considerable part was denitrified here. Denitrification was especially high as soil water contents increased to levels above field capacity, where redox-potentials sharply dropped. Further, soil P availability was higher under wet conditions. In the `dry' treatment, soil water content was close to the wilting point and plant production was low. In the `intermediate' treatment, plant production was most likely limited by nitrogen. The `wet' treatment did not result in a further increase in plant production. Dam construction and river bed degradation can result in lower river levels and summer drought on floodplains. This experimental study suggests that summer drought on floodplain soils reduces decomposition of soil organic matter, nutrient availability, denitrification, plant production and nutrient uptake. This can affect the capacity of floodplains to remove or retain nutrients from river water in a negative way.     

Molecular basis of voltage gating of OmpF porin.

OmpF and PhoE from Escherichia coli and related homologous proteins from other Gram-negative bacteria allow the passive transport of small polar molecules across the bacterial outer membrane. In vitro, they exhibit voltage gating depending on the experimental conditions. We review current hypotheses on the underlying molecular mechanism of voltage gating of OmpF porin and show how computer simulations can be used to examine each of the proposed mechanisms.     

Modeling Sequence-Space Exploration and Emergence of Epistatic Signals in Protein Evolution

During their evolution, proteins explore sequence space via an interplay between random mutations and phenotypic selection. Here, we build upon recent progress in reconstructing data-driven fitness landscapes for families of homologous proteins, to propose stochastic models of experimental protein evolution. These models predict quantitatively important features of experimentally evolved sequence libraries, like fitness distributions and position-specific mutational spectra. They also allow us to efficiently simulate sequence libraries for a vast array of combinations of experimental parameters like sequence divergence, selection strength, and library size. We showcase the potential of the approach in reanalyzing two recent experiments to determine protein structure from signals of epistasis emerging in experimental sequence libraries. To be detectable, these signals require sufficiently large and sufficiently diverged libraries. Our modeling framework offers a quantitative explanation for different outcomes of recently published experiments. Furthermore, we can forecast the outcome of time- and resource-intensive evolution experiments, opening thereby a way to computationally optimize experimental protocols.     

A docking approach to the study of copper trafficking proteins; interaction between metallochaperones and soluble domains of copper ATPases

A structural model of the transient complex between the yeast copper chaperone Atx1 and the first soluble domain of the copper transporting ATPase Ccc2 was obtained with HADDOCK, combining NMR chemical shift mapping information with in silico docking. These two proteins are involved in copper trafficking in yeast cells. Calculations were performed starting with the copper ion either bound to Atx1 or to Ccc2 and using the experimental structures of the copper-loaded and apo forms of each protein. The copper binding motifs of the two proteins are found in close proximity. Copper tends to move from Atx1 to Ccc2, consistent with the physiological direction of transfer, with concomitant structural rearrangements, in agreement with experimental observations. The interaction is mainly of an electrostatic nature with hydrogen bonds stabilizing the complex. The structural data are relevant for a number of proteins homologous to Atx1 and Ccc2 and conserved from bacteria to humans.     

Heat capacity-independent determination of differential free energy of stability between structurally homologous proteins

Under the assumption of equivalent heat capacity values, the differential free energy of stability for a pair of proteins midway between their thermal unfolding transition temperatures is shown to be independent of DeltaC(p) up to its cubic term in DeltaT(m). For model calculations reflecting the nearly 30 degrees C difference in T(m) for the adenylate kinases from the arctic bacterium Bacillus globisporus and the thermophilic bacterium Geobacillus stearothermophilus, the resultant error in estimating DeltaDeltaG by the formula 0.5 [DeltaS(T(m1))(1)+DeltaS(T(m2)) (2)] DeltaT(m) is less than 1%. Combined with the analogous thermal unfolding data for the adenylate kinase from Escherichia coli, these three homologous proteins exhibit T(m) and DeltaS(T(m)) values consistent with differential entropy and enthalpy contributions of equal magnitude. When entropy-enthalpy compensation holds for the differential free energy of stability, the incremental changes in T(m) values are shown to be proportionate to the changes in free energy.