Solution structure of the 162 residue C-terminal domain of human elongation factor 1Bgamma

The multisubunit elongation factor 1 (eEF1) is required for the elongation step of eukaryotic protein synthesis. The eEF1 complex consists of four subunits: eEF1A, a G-protein that shuttles aminoacylated tRNAs to the ribosome; eEF1Balpha and eEF1Bbeta, two guanine nucleotide exchange factors, and eEF1Bgamma. Although its exact function remains unknown, this latter subunit is present in all eukaryotes. Recombinant human eEF1Bgamma has been purified and shown to consist of two independent domains. We have utilized high resolution NMR to determine the three-dimensional structure of the 19 kDa C-terminal fragment (domain 2). The structure consists of a five-stranded anti-parallel beta-sheet surrounded by alpha-helices and resembles a contact lens. Highly conserved residues are mainly located on the concave face, suggesting thereby that this side of the molecule might be involved in some biologically relevant interface(s). Although the isolated domain 2 appears to be mostly monomeric in solution, biochemical and structural data indicate a potential homodimer. The proposed dimer model can be further positioned within the quaternary arrangement of the whole eEF1 assembly.     

Structural,energetic, and functional analysis of a protein-protein interface at distinct stages of affinity maturation

Due to a paucity of studies that synthesize structural, energetic, and functional analyses of a series of protein complexes representing distinct stages in an affinity maturation pathway, the biophysical basis for the molecular evolution of protein-protein interactions is poorly understood. Here, we combine crystal structures and binding-free energies of a series of variant superantigen (SAG)-major histocompatibility complex (MHC) class II complexes exhibiting increasingly higher affinity to reveal that this affinity maturation pathway is controlled largely by two biophysical factors: shape complementarity and buried hydrophobic surface. These factors, however, do not contribute equivalently to the affinity maturation of the interface, as the former dominates the early steps of the maturation process while the latter is responsible for improved binding in later steps. Functional assays reveal how affinity maturation of the SAG-MHC interface corresponds to T cell activation by SAGs.     

Site-directed mutagenesis within the central constriction site of ScrY (sucroseporin): effect on ion transport and comparison of maltooligosaccharide binding to LamB of Escherichia coli

The 3-D structures of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) and sucrose-specific ScrY (sucroseporin) are known from X-ray crystallography. The central constriction of the channels formed by the external loop 3 is controlled by a number of different amino acids. The most prominent one of these, N192, D201 and F204, were replaced by site-directed mutagenesis into those of LamB, which, according to the 3-D model of both channels are localized at similar places. The ScrY single mutants ScrYN192R, ScrYD201Y and ScrYF204D and the ScrY triple mutant ScrY3113 (N192R + D201Y + F204D) were created together with the triple mutant ScrY3213, which lacks also amino acids 1 to 61 from the N-terminal end. The mutant proteins were purified to homogeneity and were reconstituted into lipid bilayer membranes. In these experiments, the single-channel conductance of the mutants in different salt solutions and the stability constants for binding of different maltooligosaccharides to the mutant channels was measured using titration experiments with carbohydrates. The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant ScrY-channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate-binding to the binding site inside the channels. The results suggest that both on- and off-rate constants were affected by the mutations. Most of them showed a substantial effect on carbohydrate binding kinetics. Nevertheless, single-channel conductance and carbohydrate binding of ScrY3113 mutant were still different from that of LamB, suggesting that not only the amino acids of the central constriction but also the general architecture of both channels have a substantial influence on channel properties.     

The effects of fasting duration on the metabolic response to feeding in Python molurus: an evaluation of the energetic costs associated with gastrointestinal growth and upregulation

The oxygen uptake of Python molurus increases enormously following feeding, and the elevated metabolism coincides with rapid growth of the gastrointestinal organs. There are opposing views regarding the energetic costs of the gastrointestinal hypertrophy, and this study concerns the metabolic response to feeding after fasting periods of different duration. Since mass and function of the gastrointestinal organs remain elevated for several days after feeding, the metabolic increment following a second meal given soon after the first can reveal whether the metabolic costs relate to the upregulation of gastrointestinal organs or merely the metabolic cost of processing a meal. Eight juvenile pythons were kept on a regular feeding regime for 6 mo after hatching. At the beginning of the metabolic measurements, they were fed mice (20% of body mass), and the metabolic response to similarly sized meals was determined following 3, 5, 7, 14, 21, 30, and 60 d of fasting. Our data show that the metabolic response following feeding was large, ranging from 21% to 35% of ingested energy (mean=27%), but the metabolic response seems independent of fasting duration. Hence, the extraordinarily large cost of digestion in P. molurus does not appear to correlate with increased function and growth of gastrointestinal organs but must be associated with other physiological processes.     

A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes

We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-regulated during this differentiation process (adipsin, adipocyte complement-related protein 30 kDa, complement C3, and fibronectin), we identified four secreted molecules that have not been shown previously to be expressed differentially during the process of adipogenesis. Pigment epithelium-derived factor, a soluble molecule with potent antiangiogenic properties, was found to be highly secreted by preadipocytes but not adipocytes. Conversely, we found hippocampal cholinergic neurostimulating peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be expressed highly by mature adipocytes. We also used liquid chromatography-based separation followed by automated tandem mass spectrometry to identify proteins secreted by mature adipocytes. Several additional secreted proteins including resistin, secreted acidic cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1, cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this method. To our knowledge, this is the first study to identify several novel secreted proteins by adipocytes by a proteomic approach using mass spectrometry.     

Scoring functions for computational algorithms applicable to the design of spiked oligonucleotides.

Protein engineering by inserting stretches of random DNA sequences into target genes in combination with adequate screening or selection methods is a versatile technique to elucidate and improve protein functions. Established compounds for generating semi-random DNA sequences are spiked oligonucleotides which are synthesised by interspersing wild type (wt) nucleotides of the target sequence with certain amounts of other nucleotides. Directed spiking strategies reduce the complexity of a library to a manageable format compared with completely random libraries. Computational algorithms render feasible the calculation of appropriate nucleotide mixtures to encode specified amino acid subpopulations. The crucial element in the ranking of spiked codons generated during an iterative algorithm is the scoring function. In this report three scoring functions are analysed: the sum-of-square-differences function s, a modified cubic function c, and a scoring function m derived from maximum likelihood considerations. The impact of these scoring functions on calculated amino acid distributions is demonstrated by an example of mutagenising a domain surrounding the active site serine of subtilisin-like proteases. At default weight settings of one for each amino acid, the new scoring function m is superior to functions s and c in finding matches to a given amino acid population.     

CHARACTERIZATION AND PHYLOGENETIC POSITION OF THE ENIGMATIC GOLDEN ALGA PHAEOTHAMNION CONFERVICOLA: ULTRASTRUCTURE, PIGMENT COMPOSITION AND PARTIAL SSU rDNA SEQUENCE

The morphology, ultrastructure, photosynthetic pigments, and nuclear-encoded small subunit ribosomal DNA (SSU rDNA) were examined for Phaeothamnion confervicola Lagerheim strain SAG119.79. The morphology of the vegetative filaments, as viewed under light microscopy, was indistinguishable from the isotype. Light microscopy, including epifluorescence microscopy, also revealed the presence of one to three chloroplasts in both vegetative cells and zoospores. Vegetative filaments occasionally transformed to a palmelloid stage in old cultures. An eyespot was not visible in zoospores when examined with light microscopy, but small droplets, similar to eyespot droplets, were apparent beneath the shorter flagellum when cells were viewed with electron microscopy. Zoospores had two flagella that were laterally inserted in the cell approximately one-third of the cell length from the apex. The longer flagellum was directed anteriorly and the shorter flagellum was directed posteriorly. Electron microscopy revealed the presence of tubular tripartite flagellar hairs on the longer flagellum, but no lateral filaments were found on the tripartite hairs. The general organization of the flagellar root system was similar to that of zoospores belonging to the Xanthophyceae and Phaeophyceae. However, the transitional region of the flagella contained a transitional helix with four to six gyres. Microtubular root R₁ consisted of six microtubules at its proximal end and one microtubule at its distal end. Roots R₂ and R₄ consisted of one microtubule each and root R₃ consisted of two microtubules. No rhizoplast was found. Thin-layer chromatography revealed the presence of fucoxanthin, diadinoxanthin, neoxanthin, and heteroxanthin as well as chlorophylls a, c₁ and c₂. High-performance liquid chromatography revealed the presence of fucoxanthin, diadinoxanthin, diatoxanthin, heteroxanthin, and β,β-carotene as well as chlorophylls a and c. The complete sequence of the SSU rDNA could not be obtained, but a partial sequence (1201 bases) was determined. Parsimony and neighbor-joining distance analyses of SSU rDNA from Phaeothamnion and 36 other chromophyte algae (with two Öomycete fungi as the outgroup) indicated that Phaeothamnion was a weakly supported (bootstrap = <50%, 52%) sister taxon to the Xanthophyceae representatives and that this combined clade was in turn a weakly supported (bootstrap = <50%, 67%) sister to the Phaeophyceae. Based upon ultrastructural observations, pigment analysis, and SSU rDNA phylogenetic analysis, Phaeothamnion is not a member of the Chrysophyceae and should be classified as incertae sedis with affinities to the Xanthophyceae and Phaeophyceae.     

Replication Terminator Protein-Based Replication Fork-Arrest Systems in Various Bacillus Species

The replication terminator protein (RTP) of Bacillus subtilis interacts with its cognate DNA terminators to cause replication fork arrest, thereby ensuring that the forks approaching one another at the conclusion of a round of replication meet within a restricted terminus region. A similar situation exists in Escherichia coli, but it appears that the fork-arrest systems in these two organisms have evolved independently of one another. In the present work, RTP homologs in four species closely related to B. subtilis (B. atrophaeus, B. amyloliquefaciens, B. mojavensis, and B. vallismortis) have been identified and characterized. An RTP homolog could not be identified in another closely related species, B. licheniformis. The nucleotide and amino acid changes from B. subtilis among the four homologs are consistent with the recently established phylogenetic tree for these species. The GC contents of the rtp genes raise the possibility that these organisms arose within this branch of the tree by horizontal transfer into a common ancestor after their divergence from B. licheniformis. Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the importance of the precise folded structure to the functioning of RTP. The absence of any significant change in the proposed DNA-binding region of RTP emphasizes the importance of its high affinity for the DNA terminator in its functioning. By coincidence, the single change (E30K) found in the B. mojavensis RTP corresponds exactly to that purposefully introduced by others into B. subtilis RTP to implicate a crucial role for E30 in the fork-arrest mechanism. The natural occurrence of this variant is difficult to reconcile with such an implication, and it was shown directly that RTP.E30K functions normally in fork arrest in B. subtilis in vivo. Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing the definition of a Bacillus terminator consensus and identification of two more terminators in the B. subtilis 168 genome sequence to bring the total to nine.