RNA structure and dynamics: A base pairing perspective

RNA is now known to possess various structural, regulatory and enzymatic functions for survival of cellular organisms. Functional RNA structures are generally created by three-dimensional organization of small structural motifs, formed by base pairing between self-complementary sequences from different parts of the RNA chain. In addition to the canonical Watson–Crick or wobble base pairs, several non-canonical base pairs are found to be crucial to the structural organization of RNA molecules. They appear within different structural motifs and are found to stabilize the molecule through long-range intra-molecular interactions between basic structural motifs like double helices and loops. These base pairs also impart functional variation to the minor groove of A-form RNA helices, thus forming anchoring site for metabolites and ligands. Non-canonical base pairs are formed by edge-to-edge hydrogen bonding interactions between the bases. A large number of theoretical studies have been done to detect and analyze these non-canonical base pairs within crystal or NMR derived structures of different functional RNA. Theoretical studies of these isolated base pairs using ab initio quantum chemical methods as well as molecular dynamics simulations of larger fragments have also established that many of these non-canonical base pairs are as stable as the canonical Watson–Crick base pairs. This review focuses on the various structural aspects of non-canonical base pairs in the organization of RNA molecules and the possible applications of these base pairs in predicting RNA structures with more accuracy.     

Additivity in both thermodynamic stability and thermal transition temperature for rubredoxin chimeras via hybrid native partitioning

Given any operational definition of pairwise interaction, the set of residues that differ between two structurally homologous proteins can be uniquely partitioned into subsets of clusters for which no such interactions occur between clusters. Although hybrid protein sequences that preserve such clustering are consistent with tertiary structures composed of only parental native-like interactions, the stability of such predicted structures will depend upon the physical robustness of the assumed interaction potential. A simple distance cutoff criterion was applied to the most thermostable protein known to predict such a seven-residue cluster in the metal binding site region of Pyrococcus furiosus rubredoxin and a mesophile homolog. Both conformational stability and thermal transition temperature measurements demonstrate that 39% of the differential stability arises from these seven residues.     

De novo design of α,β-didehydrophenylalanine containing peptides: from models to applications

The de novo design of peptides and proteins has emerged as an approach for investigating protein structure and function. The success relies heavily on the ability to design relatively short peptides that can adopt stable secondary structures. To this end, substitution with α,β-dehydroamino acids, especially α,β-didehydrophenylalanine (ΔPhe or ΔF) has blossomed in manifold directions, providing a rich diversity of well-defined structural motifs. Introduction of α,β-didehydrophenylalanine induces β-bends in small and 3(10)-helices in longer peptide sequences. Most favorable conformation of ΔF residues are (φ,ψ) ～(60°, 30°), (-60°, -30°), (-60°, 150°), and (60°, -150°). These features have been exploited in designing helix-turn-helix, helical bundle arrangements, and glycine zipper type super secondary structural motifs. The unusual capability of α,β-didehydrophenylalanine ring to form a variety of multicentered interactions (N-H…O, C-H…O, C-H…π, and N-H…π) suggests its possible exploitation for future de novo design of supramolecular structures. This work has now been extended to the de novo design of peptides with antibiotic, antifibrillization activity, etc. More recently, self-assembling properties of small dehydropeptides have been explored. This review focuses primarily on the structural and functional behavior of α,β-didehydrophenylalanine containing peptides.     

Adsorption Behavior of Polydisperse Polymeric Systems

Adsorption behavior of polydisperse polymers at interfaces is studied by the Monte Carlo simulation method based on a lattice model. Effects of temperature and adsorption energies on the polymer density profile, cluster distributions and adsorption layer thickness are evaluated in two different polydisperse systems. It is found that adsorption properties are greatly different in these two systems. In normal distribution polydisperse systems, polymers are more sensitive to the excluded volume effects while in average distribution polymers are more inclined to adsorb on adsorbing interfaces. As higher temperature and lower attraction constrain the polymer adsorption, more clusters are found under these conditions. When temperature approaches the critical temperature of monodisperse systems, stable and large clusters exist in these two polydisperse systems. These results suggest that micro phase transition may exist in polydisperse systems. Polydisperse systems take little change of phase transition temperatures but altered the clusters morphology to some extent. The adsorption layer thickness changes are more sensitive to both temperature and polymer–interface interactions in average distribution systems when the whole polymer concentration increases slightly. This work also suggests significant differences between the polydisperse and the monodisperse systems in adsorption behavior. Therefore, quantitative system errors may exist when the monodisperse system models are used in simulation to evaluate polymer adsorption properties.     

The building blocks and motifs of RNA architecture

RNA motifs can be defined broadly as recurrent structural elements containing multiple intramolecular RNA-RNA interactions, as observed in atomic-resolution RNA structures. They constitute the modular building blocks of RNA architecture, which is organized hierarchically. Recent work has focused on analyzing RNA backbone conformations to identify, define and search for new instances of recurrent motifs in X-ray structures. One current view asserts that recurrent RNA strand segments with characteristic backbone configurations qualify as independent motifs. Other considerations indicate that, to characterize modular motifs, one must take into account the larger structural context of such strand segments. This follows the biologically relevant motivation, which is to identify RNA structural characteristics that are subject to sequence constraints and that thus relate RNA architectures to sequences.     

Comprehensive analysis of distinctive polyketide and nonribosomal peptide structural motifs encoded in microbial genomes

We developed a highly accurate method to predict polyketide (PK) and nonribosomal peptide (NRP) structures encoded in microbial genomes. PKs/NRPs are polymers of carbonyl/peptidyl chains synthesized by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We analyzed domain sequences corresponding to specific substrates and physical interactions between PKSs/NRPSs in order to predict which substrates (carbonyl/peptidyl units) are selected and assembled into highly ordered chemical structures. The predicted PKs/NRPs were represented as the sequences of carbonyl/peptidyl units to extract the structural motifs efficiently. We applied our method to 4529 PKSs/NRPSs and found 619 PKs/NRPs. We also collected 1449 PKs/NRPs whose chemical structures have been determined experimentally. The structural sequences were compared using the Smith-Waterman algorithm, and clustered into 271 clusters. From the compound clusters, we extracted 33 structural motifs that are significantly related with their bioactivities. We used the structural motifs to infer functions of 13 novel PKs/NRPs clusters produced by Pseudomonas spp. and Burkholderia spp. and found a putative virulence factor. The integrative analysis of genomic and chemical information given here will provide a strategy to predict the chemical structures, the biosynthetic pathways, and the biological activities of PKs/NRPs, which is useful for the rational design of novel PKs/NRPs.     

Geometric Similarities of Protein-Protein Interfaces at Atomic Resolution Are Only Observed within Homologous Families: An Exhaustive Structural Classification Study

To elucidate the structural basis of the diversity and universality in protein-protein interactions, an exhaustive all-against-all structural comparison of all known protein interfaces in the Protein Data Bank was performed at atomic resolution. After similar interfaces were clustered, approximately 20,000 structural motifs with at least two members were identified, out of which 3678 motifs consisted of at least 10 interfaces. Except for some trivial interfaces involving single α helices, almost all motifs were found to be confined within single protein families. Furthermore, the interaction partners of each motif were found to be very limited, and, accordingly, the interaction networks of the motifs tend to be small and are much more restricted than the binding sites for small ligand molecules. These findings suggest that, at the level of atomic structures, protein-protein interactions are precisely designed; hence, protein interfaces with multiple interacting partners should involve incompletely overlapping multiple interfaces and/or accommodate structural changes upon binding to their targets.     

Selective localization of neptunium-237 in nuclei of mammalian cells]

After injection in the rat of soluble neptunium salt, the distribution of this element was studied at the subcellular level by electron microscopy and electron probe microanalysis. Abnormal structures have been observed by electron microscopy in the nuclei of hepatocytes, and the same structures have also been observed in the nuclei of the proximal tubules cells of the kidney. These structures are formed of clusters of very small and dense particles, several nanometers in diameter. The clusters are localized in the central part of the nuclei and they are separate from nucleoli and heterochromatin. Electron probe X-ray analysis of this cluster have shown that they contain neptunium associated with phosphorus. In the cell containing neptunium inclusions, other non specific lesions are also observed (nuclear pycnosis, mitochondrial depletion).     

Architecture, electronic structure and stability of TM@Ge(n) (TM = Ti, Zr and Hf; n = 1-20) clusters: a density functional modeling

The present study reports the geometry, electronic structure and properties of neutral and anionic transition metal (TM = Ti, Zr and Hf)) doped germanium clusters containing 1 to 20 germanium atoms within the framework of linear combination of atomic orbitals density functional theory under spin polarized generalized gradient approximation. Different parameters, like, binding energy (BE), embedding energy (EE), energy gap between the highest occupied and lowest unoccupied molecular orbitals (HOMO-LUMO), ionization energy (IP), electron affinity (EA), chemical potential etc. of the energetically stable clusters (ground state cluster) in each size are calculated. From the variation of these parameters with the size of the clusters the most stable cluster within the range of calculation is identified. It is found that the clusters having 20 valence electrons turn out to be relatively more stable in both the neutral and the anionic series. The sharp drop in IP as the valence electron count increases from 20 to 21 in neutral cluster is in agreement with predictions of shell models. To study the vibrational nature of the clusters, IR and Raman spectrum of some selected TM@Ge_n (n = 15,16,17) clusters are also calculated and compared. In the end, relevance of calculated results to the design of Ge-based super-atoms is discussed.     

Deciphering structural elements of mucin glycoprotein recognition

Mucin glycoproteins present a complex structural landscape arising from the multiplicity of glycosylation patterns afforded by their numerous serine and threonine glycosylation sites, often in clusters, and with variations in respective glycans. To explore the structural complexities in such glycoconjugates, we used NMR to systematically analyze the conformational effects of glycosylation density within a cluster of sites. This allows correlation with molecular recognition through analysis of interactions between these and other glycopeptides, with antibodies, lectins, and sera, using a glycopeptide microarray. Selective antibody interactions with discrete conformational elements, reflecting aspects of the peptide and disposition of GalNAc residues, are observed. Our results help bridge the gap between conformational properties and molecular recognition of these molecules, with implications for their physiological roles. Features of the native mucin motifs impact their relative immunogenicity and are accurately encoded in the antibody binding site, with the conformational integrity being preserved in isolated glycopeptides, as reflected in the antibody binding profile to array components.     

Mechanism of protein folding

The high structural resolution of the main transition states for the formation of native structure for the six small proteins of which Phi-values for a large set of mutants have become available, barstar, barnase, chymotrypsin inhibitor 2, Arc repressor, the src SH3 domain, and a tetrameric p53 domain reveals that for the first 5 of these proteins: (1) Residues that belong to regular secondary structure have a significantly larger average fraction of native structural consolidation than residues in loops; (2) on the other hand, secondary and tertiary structures have built up to the same degree, or at least a high degree, but nonuniformly distributed over the molecule; (3) the most consolidated parts of each protein molecule in the transition state cluster together, and these clusters contain a significantly higher percentage of residues that belong to regular secondary structure than the rest of the molecule. These observations further reconcile the framework model with the nucleation-condensation mechanism for folding: The amazing speed of protein folding can be understood as caused by the catalytic effect of the formation of clusters of residues which have particularly high preferences for the early formation of regular secondary structure in the presence of significant amounts of tertiary structure interactions.     

Multi-stemmed trees of Nothofagus pumilio second-growth forest in Patagonia are formed by highly related individuals

Background and Aims Multi-stemmed trees (tree clusters) in Nothofagus pumilio, a dominant tree species in Patagonia, are very uncommon and are restricted to the edge of second-growth forests following human-provoked fires. No vegetative reproduction has been reported so far. The genetic structure of multi-stemmed trees of this species was investigated and it was hypothesized that genets within a cluster were more closely related than average in the population. Methods Fifteen clusters (composed of at least three purported stems) and 15 single trees were sampled at the edge of a second-growth forest and genotyped using two amplified fragment length polymorphism (AFLP) primer pairs. We obtained 119 polymorphic markers that allowed clonality to be determined, together with sibship structure and relatedness among samples. Key Results Clonality was detected in seven clusters but all clusters had at least two different genotypes. Full sibs were found exclusively within clusters and in all clusters. Within a cluster, stems that were not identified as full sibs were often half sibs. Relatedness values for the full sibs and half sibs were higher than the theoretical values of 0·5 and 0·25 but the relatedness between clusters was very low. Conclusions Tree clusters that are merged at the edge of the second-growth forest of N. pumilio are composed of stems of the same genotype and of other genotypes that are highly related (but not always). It is suggested that this peculiar genetic structure results from a combination of several causes, including selection for merging of related individuals.     

Acetylcholine receptor clustering and nuclear movement in muscle fibers in culture

We have studied the formation of acetylcholine receptor (AChR) clusters and the behavior of myonuclei in rat and chick skeletal muscle cells grown in cell culture. These cells were treated with a factor derived from Torpedo electric extracellular matrix, which causes a large increase in their number of AChR clusters. We found that these clusters were located preferentially in membrane regions above myonuclei. This cluster-nucleus colocalization is explained by our finding that most of the nuclei near clusters remain relatively stationary, while most of those away from clusters are able to translocate throughout the myotube. In some cases, clusters clearly formed first, then nuclei migrated underneath and became immobilized. If clustered AChRs later dispersed, their associated nuclei resumed moving. These results suggest that AChR clustering initiates an extensive cytoskeletal rearrangement that causes the subcluster localization of organelles, potentially providing a stable source of newly synthesized AChRs for insertion into the cluster.     

Structure and stability of sodium and potassium complexes of dT4G4 and dT4G4T.

The ends of eukaryotic chromosomes contain specialized structures that include DNA with multiple tandem repeats of simple sequences containing clusters of G on one strand, together with proteins which synthesize and bind to these sequences. The unit repeat in the protozoan Oxytricha with the cluster dT4G4 can form structures containing tetrads of guanine residues, referred to G4 DNA, in the presence of metal ions such as Na+ or K+. We show here that, in the presence of Na+, dT4G4 forms a tetramer with parallel strands by means of a UV cross-linking assay. In the presence of K+, two further interactions are observed: at low temperature, higher order complexes are formed, provided the 3' end of the strand is G; a single 3'T inhibits this association in dT4G4T. At high temperature, these complexes dissociate, leading to a tetramer with a different ordered structure that melts only at very high temperatures. These results suggest that the cohesive properties of DNA containing G clusters might depend on associative interactions driven by a free 3'G terminus in the presence of K+, as well as by connecting antiparallel G hairpins as has been postulated.     

Automatic definition of recurrent local structure motifs in proteins

An automatic procedure for defining recurrent folding motifs in proteins of known structure is described. These motifs are formed by short polypeptide fragments of equal size containing between four and seven residues. The method applies a classical clustering algorithm that operates on distances between selected backbone atoms. In one application, we use it to cluster all protein fragments into only four structural classes. This classification is rough considering the observed diversity of local structures, but comparable in homogeneity to the four classes of secondary structure (alpha-helix, beta-strand, turn and coil). Yet, it discriminates between extended and curved coil and distinguishes beta-bulges from beta-strands. In a second application, the clustering procedure is combined with assignment of backbone dihedral angles to allowed regions in the Ramachandran map. This produces an exhaustive repertoire of highly homogeneous families of structural motifs that contains all the beta-hairpins, beta alpha- and alpha beta-loops previously defined by manual procedures, and new structural families of which two examples, a beta alpha-loop and an alpha-helix beginning, are analyzed in detail. The described automatic procedures should be useful in categorizing structure information in proteins, thereby increasing our ability to analyze relations between structure and sequence.     

A new, structurally nonredundant, diverse data set of protein-protein interfaces and its implications

Here, we present a diverse, structurally nonredundant data set of two-chain protein-protein interfaces derived from the PDB. Using a sequence order-independent structural comparison algorithm and hierarchical clustering, 3799 interface clusters are obtained. These yield 103 clusters with at least five nonhomologous members. We divide the clusters into three types. In Type I clusters, the global structures of the chains from which the interfaces are derived are also similar. This cluster type is expected because, in general, related proteins associate in similar ways. In Type II, the interfaces are similar; however, remarkably, the overall structures and functions of the chains are different. The functional spectrum is broad, from enzymes/inhibitors to immunoglobulins and toxins. The fact that structurally different monomers associate in similar ways, suggests "good" binding architectures. This observation extends a paradigm in protein science: It has been well known that proteins with similar structures may have different functions. Here, we show that it extends to interfaces. In Type III clusters, only one side of the interface is similar across the cluster. This structurally nonredundant data set provides rich data for studies of protein-protein interactions and recognition, cellular networks and drug design. In particular, it may be useful in addressing the difficult question of what are the favorable ways for proteins to interact. (The data set is available at http://protein3d.ncifcrf.gov/~keskino/ and http://home.ku.edu.tr/~okeskin/INTERFACE/INTERFACES.html.)