Domain–domain motions in proteins from time-modulated pseudocontact shifts

In recent years paramagnetic NMR derived structural constraints have become increasingly popular for the study of biomolecules. Some of these are based on the distance and angular dependences of pseudo contact shifts (PCSs). When modulated by internal motions PCSs also become sensitive reporters on molecular dynamics. We present here an investigation of the domain–domain motion in a two domain protein (PA0128) through time-modulation of PCSs. PA0128 is a protein of unknown function from Pseudomonas aeruginosa (PA) and contains a Zn²⁺ binding site in the N-terminal domain. When substituted with Co²⁺ in the binding site, several resonances from the C-terminal domain showed severe line broadening along the ¹⁵N dimension. Relaxation compensated CPMG experiments revealed that the dramatic increase in the ¹⁵N linewidth came from contributions of chemical exchange. Since several sites with perturbed relaxation are localized to a single β-strand region, and since extracted timescales of motion for the perturbed sites are identical, and since the magnitude of the chemical exchange contributions is consistent with PCSs, the observed rate enhancements are interpreted as the result of concerted domain motion on the timescale of a few milliseconds. Given the predictability of PCS differences and the easy interpretation of the experimental results, we suggest that these effects might be useful in the study of molecular processes occurring on the millisecond to microsecond timescale. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Center for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) is a “specialized” or “technology development” center supported by the Protein Structure Initiative (PSI). CESG’s mission is to develop improved methods for the high-throughput solution of structures from eukaryotic proteins, with a very strong weighting toward human proteins of biomedical relevance. During the first three years of PSI-2, CESG selected targets representing 601 proteins from Homo sapiens, 33 from mouse, 10 from rat, 139 from Galdieria sulphuraria, 35 from Arabidopsis thaliana, 96 from Cyanidioschyzon merolae, 80 from Plasmodium falciparum, 24 from yeast, and about 25 from other eukaryotes. Notably, 30% of all structures of human proteins solved by the PSI Centers were determined at CESG. Whereas eukaryotic proteins generally are considered to be much more challenging targets than prokaryotic proteins, the technology now in place at CESG yields success rates that are comparable to those of the large production centers that work primarily on prokaryotic proteins. We describe here the technological innovations that underlie CESG’s platforms for bioinformatics and laboratory information management, target selection, protein production, and structure determination by X-ray crystallography or NMR spectroscopy.

What Was the Set of Ubiquitin and Ubiquitin-Like Conjugating Enzymes in the Eukaryote Common Ancestor?

Ubiquitin (Ub)-conjugating enzymes (E2) are key enzymes in ubiquitination or Ub-like modifications of proteins. We searched for all proteins belonging to the E2 enzyme super-family in seven species (Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Arabidopsis thaliana) to identify families and to reconstruct each family’s phylogeny. Our phylogenetic analysis of 207 genes led us to define 17 E2 families, with 37 E2 genes, in the human genome. The subdivision of E2 into four classes did not correspond to the phylogenetic tree. The sequence signature HPN (histidine–proline–asparagine), followed by a tryptophan residue at 16 (up to 29) amino acids, was highly conserved. When present, the active cysteine was found 7 to 8 amino acids from the C-terminal end of HPN. The secondary structures were characterized by a canonical alpha/beta fold. Only family 10 deviated from the common organization because the proteins were devoid of enzymatic activity. Family 7 had an insertion between beta strands 1 and 2; families 3, 5 and 14 had an insertion between the active cysteine and the conserved tryptophan. The three-dimensional data of these proteins highlight a strong structural conservation of the core domain. Our analysis shows that the primitive eukaryote ancestor possessed a diversified set of E2 enzymes, thus emphasizing the importance of the Ub pathway. This comprehensive overview of E2 enzymes emphasizes the diversity and evolution of this superfamily and helps clarify the nomenclature and true orthologies. A better understanding of the functions of these enzymes is necessary to decipher several human diseases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative analysis of essential genes and nonessential genes in Escherichia coli K12

Genes can be classified as essential or nonessential based on their indispensability for a living organism. Previous researches have suggested that essential genes evolve more slowly than nonessential genes and the impact of gene dispensability on a gene’s evolutionary rate is not as strong as expected. However, findings have not been consistent and evidence is controversial regarding the relationship between the gene indispensability and the rate of gene evolution. Understanding how different classes of genes evolve is essential for a full understanding of evolutionary biology, and may have medical relevance in the design of new antibacterial agents. We therefore performed an investigation into the properties of essential and nonessential genes. Analysis of evolutionary conservation, protein length distribution and amino acid usage between essential and nonessential genes in Escherichia coli K12 demonstrated that essential genes are relatively preserved throughout the bacterial kingdom when compared to nonessential genes. Furthermore, results show that essential genes, compared to nonessential genes, have a significantly higher proportion of large (>534 amino acids) and small proteins (<139 amino acids) relative to medium-sized proteins. The pattern of amino acids usage shows a similar trend for essential and nonessential genes, although some notable exceptions are observed. These findings help to clarify our understanding of the evolutionary mechanisms of essential and nonessential genes, relevant to the study of mutagenesis and possibly allowing prediction of gene properties in other poorly understood organisms.     

An interrupted beta-propeller and protein disorder: structural bioinformatics insights into the N-terminus of alsin

Defects in the human ALS2 gene, which encodes the 1,657-amino-acid residue protein alsin, are linked to several related motor neuron diseases. We created a structural model for the N-terminal 690-residue region of alsin through comparative modelling based on regulator of chromosome condensation 1 (RCC1). We propose that this alsin region contains seven RCC1-like repeats in a seven-bladed beta-propeller structure. The propeller is formed by a double clasp arrangement containing two segments (residues 1–218 and residues 525–690). The 306-residue insert region, predicted to lie within blade 5 and to be largely disordered, is poorly conserved across species. Surface patches of evolutionary conservation probably indicate locations of binding sites. Both disease-causing missense mutations—Cys157Tyr and Gly540Glu—are buried in the propeller and likely to be structurally disruptive. This study aids design of experimental studies by highlighting the importance of construct length, will enhance interpretation of protein–protein interactions, and enable rational site-directed mutagenesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Coevolutionary dynamics of adaptive radiation for food-web development

To investigate how complex food-webs can develop through repeated evolutionary diversification, a predator–prey model was analyzed. In the model, each individual has two traits: trait x as a predator and trait y as a prey. These traits constitute a two-dimensional phenotype space, in which the whole group of individuals are represented as a phenotype distribution. Predator–prey interactions among the phenotypes are determined by their relative positions in the phenotype space. Each phenotypic cluster was treated as a species. Each species evolves in y to escape from predation, while it evolves in x to chase their prey. Analytical investigation provided two predictions. First, coupled evolutionary diversifications of y and x may occur when the x of predators have caught up with their prey’s y, which may be repeated. Second, complex food-webs may develop when species’ competitive strengths are kept similar within the communities. If the functional response is close to the ratio-dependent response, the competitive strengths of all species are similar when the relationship between predators and prey corresponds to the ideal free distribution (IFD). These predictions were confirmed by numerical simulations. Electronic supplementary material  The online version of this article doi:() contains supplementary material, which is available to authorized users.     

Localization of ligand binding site in proteins identified in silico

Knowledge-based models for protein folding assume that the early-stage structural form of a polypeptide is determined by the backbone conformation, followed by hydrophobic collapse. Side chain–side chain interactions, mostly of hydrophobic character, lead to the formation of the hydrophobic core, which seems to stabilize the structure of the protein in its natural environment. The fuzzy-oil-drop model is employed to represent the idealized hydrophobicity distribution in the protein molecule. Comparing it with the one empirically observed in the protein molecule reveals that they are not in agreement. It is shown in this study that the irregularity of hydrophobic distributions is aim-oriented. The character and strength of these irregularities in the organization of the hydrophobic core point to the specificity of a particular protein’s structure/function. When the location of these irregularities is determined versus the idealized fuzzy-oil-drop, function-related areas in the protein molecule can be identified. The presented model can also be used to identify ways in which protein–protein complexes can possibly be created. Active sites can be predicted for any protein structure according to the presented model with the free prediction server at . The implication based on the model presented in this work suggests the necessity of active presence of ligand during the protein folding process simulation. Figure Fuzzy-oil-drop model applied to identify the ligation site in lysozyme complexed with N-acetylglucosamine (PDB ID:1LMQ) in form of hydrophobicity deficiency (ΔH) profile and three-dimensional distribution of on protein surface     

Predicting protein crystallization propensity from protein sequence

The high-throughput structure determination pipelines developed by structural genomics programs offer a unique opportunity for data mining. One important question is how protein properties derived from a primary sequence correlate with the protein’s propensity to yield X-ray quality crystals (crystallizability) and 3D X-ray structures. A set of protein properties were computed for over 1,300 proteins that expressed well but were insoluble, and for ~720 unique proteins that resulted in X-ray structures. The correlation of the protein’s iso-electric point and grand average hydropathy (GRAVY) with crystallizability was analyzed for full length and domain constructs of protein targets. In a second step, several additional properties that can be calculated from the protein sequence were added and evaluated. Using statistical analyses we have identified a set of the attributes correlating with a protein’s propensity to crystallize and implemented a Support Vector Machine (SVM) classifier based on these. We have created applications to analyze and provide optimal boundary information for query sequences and to visualize the data. These tools are available via the web site .     

Analysis of Protein Three-Dimension Structure Using Amino Acids Depths

The issue of amino acid depth in proteins gives important insights to our understanding of protein’s three-dimensional structure. There has already been much research done in mathematical and statistical sciences regarding the general definitions, properties and algorithms describing the particle depth of spatially extended systems. We constructed a method of calculating the amino acids depths and applied it to a set of 527 protein structures. We propose the introduction of amino acid depth tendency factors for three-dimensional structures of proteins. The depth tendency factors relate not only to the hydrophobicity indices but also to the electrostatic charge. We found a relationship between the protein size and the number of residues using the distance between the deepest residue and surface residues. We made a prediction regarding the number of residues on the surface of a protein, the deepest amino acid, and the average depth, all of which are fitted well to a linear functional relationship with the length of the protein. Finally, we have predicted the depths of multiple peptides in protein’s three-dimension structure. Electronic supplementary material The online version of this article () contains supplementary material, which is available to authorized users.     

Domain <Emphasis Type="Italic">a</Emphasis>’ of protein disulfide isomerase plays key role in inhibiting α-synuclein fibril formation

α-Synuclein (αSyn) is the main component of Lewy bodies formed in midbrain dopaminergic neurons which is a pathological characteristic of Parkinson's disease. It has been recently showed to induce endoplasmic reticulum (ER) stress and impair ER functions. However, the mechanism of how ER responds to αSyn toxicity is poorly understood. In the present study, we found that protein disulfide isomerase (PDI), a stress protein abundant in ER, effectively inhibits αSyn fibril formation in vitro. In PDI molecule with a structure of abb’xa’c, domain a’ was found to be essential and sufficient for PDI to inhibit αSyn fibril formation. PDI was further found to be more avid for binding with intermediate species formed during αSyn fibril formation, and the binding was more intensive in the later lag phase. Our results provide new insight into the role of PDI in protecting ER from the deleterious effects of misfolded protein accumulation in many neurodegenerative diseases.     

Molecular Evolution and Positive Selection of the Symbiotic Gene NORK in Medicago truncatula

Understanding the selective constraints of partner specificity in mutually beneficial symbiosis is a significant, yet largely unexplored, prospect of evolutionary biology. These selective constraints can be explored through the study of nucleotide polymorphism at loci controlling specificity. The membrane-anchored receptor NORK (nodulation receptor kinase) of the legume Medicago truncatula controls early steps of root infection by two symbiotic microorganisms: nitrogen-fixing bacteria (rhizobia) and endomycorrhizal fungi (Glomales). We analyzed the diversity of the gene NORK by sequencing 4 kilobases in 28 inbred lines sampled from natural populations. We detected 33 polymorphic sites with only one nonsynonymous change. Analysis based on Tajima’s D and Fay and Wu’s H summary statistics revealed no departure from the neutral model. We analyzed divergence using sequences from the closely related species M. coerulea. The McDonald-Kreitman test indicated a significant excess of nonsynonymous changes contributing to this divergence. Furthermore, maximum-likelihood analysis of a molecular phylogeny of a few legume species indicated that a number of amino acid sites, likely located in the receptor domain of the protein, evolved under the regime of positive selection. Further research should focus on the rate and direction of molecular coevolution between microorganisms’ signaling molecules and legumes’ receptors. [Reviewing Editor: Dr. Deborah Charlesworth] Sequence data were deposited in the GenBank database under accession nos. AY676428 to AY676457 and AJ884582.     

Multi-isomorphous replacement phasing of the earthworm fibrinolytic enzyme component A from Eisenia fetida

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida, a protein functioning not only as a direct fibrinolytic enzyme, but also as a plasminogen activator, has been crystallized in P2₁2₁2₁ space group with 3 protein molecules per asymmetric unit. Four heavy atom derivatives were prepared using a mother liquor containing 1.4 mol · L^-1 Li₂SO₄ and 0.1 mol · L^-1 MOPS buffer (pH7.2) and used to solve the protein’s diffraction phase. The heavy atom binding sites in the derivative crystals were determined using difference Patterson and difference Fourier methods and were refined in combination to yield the initial protein’s structure phase at 0.25 nm resolution. The non-crystallographic symmetry relationship of the three independent protein molecules in the asymmetric unit was determined using the correlative heavy atom sites and used for the averaging of the initial electron density. As a result, the electron density was significantly improved, providing a solid foundation for subsequent structure determination.     

Identifying protein interactions with metal-modified DNA using microarray technology

Protein microarrays have been used extensively to identify protein–protein interactions; however, this technology has not been widely applied to protein–DNA interactions. In particular, this work demonstrates the utility of this technique for rapidly identifying interactions of proteins with metal-modified DNA. Protein macroarray experiments were carried out with high mobility group protein 1 (HMG-1) and cisplatin- and chromium-modified 50-mer oligonucleotides to demonstrate “proof of principle.” Commercially available protein microarrays containing many different classes of human proteins were then employed to search for additional interactions with cisplatin-modified DNA. The results of the microarray experiments confirmed some known interactions and, more importantly, identified many novel protein interactions, demonstrating the utility of this method as a rapid, high-throughput technique to discover proteins that interact with metal-modified DNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Application of the random coil index to studying protein flexibility

Protein flexibility lies at the heart of many protein–ligand binding events and enzymatic activities. However, the experimental measurement of protein motions is often difficult, tedious and error-prone. As a result, there is a considerable interest in developing simpler and faster ways of quantifying protein flexibility. Recently, we described a method, called Random Coil Index (RCI), which appears to be able to quantitatively estimate model-free order parameters and flexibility in protein structural ensembles using only backbone chemical shifts. Because of its potential utility, we have undertaken a more detailed investigation of the RCI method in an attempt to ascertain its underlying principles, its general utility, its sensitivity to chemical shift errors, its sensitivity to data completeness, its applicability to other proteins, and its general strengths and weaknesses. Overall, we find that the RCI method is very robust and that it represents a useful addition to traditional methods of studying protein flexibility. We have implemented many of the findings and refinements reported here into a web server that allows facile, automated predictions of model-free order parameters, MD RMSF and NMR RMSD values directly from backbone ¹H, ¹³C and ¹⁵N chemical shift assignments. The server is available at . Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Species interactions contribute to the success of a global plant invader

Biological invasions are ubiquitous ecological phenomena that often impact native ecosystems. Some introduced species have evolved traits that enhance their ability to compete and dominate in recipient communities. However, it is still unknown if introduced species can evolve traits that may enhance their species interactions to fuel invasion success. We tested whether Centaurea solstitialis (yellow starthistle) from introduced populations have greater performance than native counterparts, and whether they generate more beneficial plant-soil interactions. We used common garden and plant-soil feedback experiments with soils and seeds from native Eurasian and introduced Californian populations. We found that performance of Centaurea did not differ among source genotypes, implying that the success of this invasive species is not due to evolutionary changes. However, Centaurea grew significantly larger in soils from introduced regions than from native regions, indicating a reduction in natural enemy pressure from native populations. We conclude that species interactions, not evolution, may contribute to Centaurea’s invasion success in introduced populations.     

Amino Acid Metabolic Origin as an Evolutionary Influence on Protein Sequence in Yeast

The metabolic cycle of Saccharomyces cerevisiae consists of alternating oxidative (respiration) and reductive (glycolysis) energy-yielding reactions. The intracellular concentrations of amino acid precursors generated by these reactions oscillate accordingly, attaining maximal concentration during the middle of their respective yeast metabolic cycle phases. Typically, the amino acids themselves are most abundant at the end of their precursor’s phase. We show that this metabolic cycling has likely biased the amino acid composition of proteins across the S. cerevisiae genome. In particular, we observed that the metabolic source of amino acids is the single most important source of variation in the amino acid compositions of functionally related proteins and that this signal appears only in (facultative) organisms using both oxidative and reductive metabolism. Periodically expressed proteins are enriched for amino acids generated in the preceding phase of the metabolic cycle. Proteins expressed during the oxidative phase contain more glycolysis-derived amino acids, whereas proteins expressed during the reductive phase contain more respiration-derived amino acids. Rare amino acids (e.g., tryptophan) are greatly overrepresented or underrepresented, relative to the proteomic average, in periodically expressed proteins, whereas common amino acids vary by a few percent. Genome-wide, we infer that 20,000 to 60,000 residues have been modified by this previously unappreciated pressure. This trend is strongest in ancient proteins, suggesting that oscillating endogenous amino acid availability exerted genome-wide selective pressure on protein sequences across evolutionary time. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Benjamin L. de Bivort and Ethan O. Perlstein have contributed equally to this work.     

Problems in Obtaining Diffraction-quality Crystals of Hetero-oligomeric Integral Membrane Proteins

The major barrier responsible for the slow pace of structure determination of integral membrane proteins is the difficulty of crystallizing detergent-solubilized hydrophobic proteins, particularly hetero-oligomeric integral membrane proteins. For the latter class of multi-subunit proteins, we have encountered the following problems in addition to the ubiquitous problem of detergent compatibility: (i) instability caused by over-purification that results in delipidation; (ii) protease activity degrading exposed loops and termini of subunits of the complex that could not be inhibited; (iii) poor protein–protein contacts presumably arising from masking by the detergent micelle. Problem (i) could be ameliorated in crystallization of the cytochrome b₆f complex by augmenting the delipidated complex with synthetic lipid. Problem (ii) has not been solved. Problem (iii) has been solved in other systems by the use of monoclonal antibodies (or other protein ligands) to increase the probability of protein–protein contacts. In the case of the complex formed by the cobalamin and colicin receptor, BtuB, and the receptor binding domain of colicin E3, the latter served as a ligand for protein–protein contacts that facilitated crystallization.     

FKBP immunophilins and Alzheimer’s disease: A chaperoned affair

The FK506-binding protein (FKBP) family of immunophilins consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. Analysis of the functions of immunophilins has been the focus of studies in recent years and has led to the identification of various molecular pathways in which FKBPs play an active role. All FKBPs contain a domain with prolyl cis/trans isomerase (PPIase) activity. Binding of the immunosuppressant molecule FK506 to this domain inhibits their PPIase activity while mediating immune suppression through inhibition of calcineurin. The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, FKBPs have been implicated with both neuroprotection and neurodegeneration. This review will focus on recent studies involving FKBP immunophilins in Alzheimer’s-disease-related pathways.