Secondary Structure Patterns Research Articles

Folding of pyrimidine-enriched RNA fragments from the vicinity of the internal ribosomal entry site of hepatitis A virus.

Two RNA fragments from the region just upstream of the internal ribosome entry site of Hepatitis A virus (HAV) were studied, a 35mer (HAV-35), 5'U4C3U3C3U4C3U3C2UAU2C3U33(4), and a 23mer (HAV-23), 5(4)U4C3U3C3U4C3U33(4). Secondary structural predictions and nuclease digestion patterns obtained with genomic RNAs suggested that they link two stable Watson-Crick (WC) hairpins in the genomic RNA and do not form conventional WC secondary structure, but do fold to form a condensed, stacked 'domain'. To obtain more information, folding of HAV-23 and -35 RNA fragments was characterized using 1H nuclear magnetic resonance, in H2O as a function of pH and temperature, circular dichroism as a function of NaCl concentration, pH and temperature, and square-wave voltammetry as a function of pH. The results indicate that these oligo-nucleotides form intramolecular structures that contain transient U*U base pairs at pH 7 and moderate ionic strength (100 mM NaCl). This folded structure becomes destabilized and loses the U*U base pairs above and below neutral pH, especially at ionic strengths above 0.1. All of the cytidine protons exchange relatively rapidly with solvent protons (exchange lifetimes shorter than 1 ms), so the structure contains few if any C*CH+base pairs at neutral pH, but can apparently form them at pH values below 6. We present a series of possible models in which chain folding draws the strand termini closer together, possibly serving to pull the attached WC hairpin domains together and providing a functional advantage by nucleating reversible formation of a more viable RNA substrate.

Preparation and Structure ofβ-Peptides Consisting of Geminally Disubstitutedβ2,2- andβ3,3-Amino Acids: A Turn Motif forβ-Peptides

We report on the synthesis of new and previously described β-peptides (1 – 6), consisting of up to twelve β2,2- or β3,3-geminally disubstituted β-amino acids which do not fit into any of the secondary structural patterns of β-peptides, hitherto disclosed. The required 2,2- and 3,3-dimethyl derivatives of 3-aminopropanoic acid are readily obtained from 3-methylbut-2-enoic acid and ammonia (Scheme 1) and from Boc-protected methyl 3-aminopropanoate by enolate methylation (Scheme 2). Protected (Boc for solution-, Fmoc for solid-phase syntheses) 1-(aminomethyl)cycloalkanecarboxylic-acid derivatives (with cyclopropane, cyclobutane, cyclopentane, and cyclohexane rings) are obtained from 1-cyanocycloalkanecarboxylates and the corresponding dihaloalkanes (Scheme 3). Fully 13C- and 15N-labeled 3-amino-2,2-dimethylpropanoic-acid derivatives were prepared from the corresponding labeled precursors (see asterixed formula numbers and Scheme 4). Coupling of these amino acids was achieved by methods which we had previously employed for other β-peptide syntheses (intermediates 18 – 23). Crystal structures of Boc-protected geminally disubstituted amino acids (16a – d) and of the corresponding tripeptide (23a), as well as NMR and IR spectra of an isotopically labeled β-hexapeptide (2a*) are presented (Figs. 1 – 4) and discussed. The tripeptide structure contains a ten-membered H-bonded ring which is proposed to be a turn-forming motif for β-peptides (Fig. 2).

Use of graph theory for secondary structure recognition and sequential assignment in heteronuclear (13C, 15N) NMR spectra: application to HU protein from Bacillus stearothermophilus.

A computer-assisted procedure, based upon a branch of mathematics known as graph theory, has been developed to recognize secondary structure elements in proteins from their corresponding nuclear Overhauser effect spectroscopy (NOESY)-type spectra and to carry out their sequential assignment. In the method, NOE connectivity templates characteristic of regular secondary structures are identified in the spectra. Resonance assignment is then achieved by connecting these NOE patterns of secondary structure together, and thereby matching connected spin systems to specific parts of the primary sequence. The range of NOE-graph templates of secondary structure motifs, incorporating alpha-helices and beta-strand motifs, has been examined for reliability and extent of secondary structure identification in a data base composed of the high resolution structures of 20 proteins. The analysis identified several robust NOE-graph templates and supports the implementation of an ordered search strategy. The method, known as SERENDIPITY, has been applied to the analysis of nuclear Overhauser effect data from a three-dimensional time-shared nuclear Overhauser effect spectroscopy (13C, 15N) heteronuclear single quantum correlation spectrum of the (alpha + beta) type protein HU from Bacillus stearothermophilus. The arrangement of the elucidated elements of secondary structure is very similar to that of the x-ray and nmr structures of HU. In addition, our analysis revealed a pattern of interstrand nuclear Overhauser effect in the beta-arm region (residues 53-76) of HU, which suggest irregularities, not reported in the x-ray structure, in both strands of the beta-arm at Ala57 and Pro72, respectively. At these residues, both strands of the beta-arm appear to flip inside out before continuing as a regular antiparallel beta-sheet.

Discovering knowledge in DNA and protein data

This research investigates a method for discovering knowledge in structural data. We have implemented the SUBDUE substructure discovery system which discovers interesting and repetitive subgraphs in a labeled graph representation using the minimum description length principle. Experiments have shown SUBDUE's applicability in a variety of domains. We are currently applying SUBDUE to both DNA and protein data from the Brookhaven PDB, where SUBDUE was able to find patterns in secondary structure that are both characteristic and unique to categories of proteins, such as hemoglobin and myoglobin. Ultimately, we plan to use SUBDUE to find structural patterns in functional groups of proteins and the boundaries of genes in DNA.

Structural characterization of human glyoxalase II as probed by limited proteolysis.

Human glyoxalase II is partially proteolyzed by trypsin, under non denaturing conditions, only at the level of the C-terminal region. The proteolytic cleavage resulted in an inactivation of the enzyme without loss of the secondary structure. Sodium dodecyl sulphate polyacrylamide gel-electrophoresis and microsequence analysis showed that the glyoxalase II is proteolyzed at the level of Arg 184 and Lys 230 and undergoes a third cleavage in a region located at the beginning of the supposed C-terminal domain. The proteolysis occurs either in the presence or in the absence of specific inhibitors. Our limited proteolysis experiments and secondary structure prediction give evidence for the presence of two domains characterized by different pattern of secondary structure.

Sequence determination and genetic analysis of the leader region of various equine arteritis virus isolates.

The entire leader sequence of ten equine arteritis virus (EAV) isolates including the Bucyrus reference strain was determined and analyzed at the primary nucleotide and secondary structure levels. The leader sequence of eight EAV isolates was determined to be 206 nucleotides (nt) in length, whereas those of the 86AB-A1 and 86NY-A1 isolates were found to be 205 and 207 nt in length, respectively. The sequence identity of the leader sequences between the different isolates and the Bucyrus reference strain ranged from 94.2 to 98.5%. An AUG start codon found at position 14 in all EAV isolates could initiate an open reading frame (ORF) that could produce a polypeptide of 37 amino acids, except for the 86NY-A1 isolate where the intraleader polypeptide would contain 54 amino acids. Five patterns of computer-predicted RNA secondary structures were identified in the ten EAV leader regions analyzed. All EAV isolates showed three conserved stem-loops (designated A, B and C). An additional conserved stem-loop (D) was observed in six EAV isolates, including the Bucyrus reference strain. Based on the presence or absence of stem-loop D, all EAV isolates analyzed in this study could be tentatively classified into two genogroups (I and II). The significance of the intraleader ORF and the predicted secondary structures has yet to be determined.

Sequence-specific 1H assignment and secondary structure of the bacteriocin AS-48 cyclic peptide.

The bacteriocin AS-48 is a cationic peptide (7149 Da) having a broad antimicrobial spectrum, encoded by the 68 kb conjugative plasmid pMB2 from Enterococcus faecalis S-48. It is a unique peptide since it has a cyclic structure, which is achieved by the formation of a tail-head peptide bond after ribosomal synthesis (Gálvez et al., 1989; Martínez-Bueno et al., 1994; Samyn et al., 1994). Preliminary CD and calorimetric studies (data not shown) pointed towards a highly helical and very stable three dimensional structure. All the information gathered until now indicates that the target of AS-48 is the cytoplasmic membrane in which it opens channels or pores, leading to dissipation of the proton motive force and cell death, which in some cases is also followed by bacterial lysis (Gálvez et al., 1991). This peptide is a suitable tool for studying protein-membrane interactions, and it also offers promising perspectives for biotechnological applications. Knowledge of the 3D structure of AS-48 is a first step in the conduct of further structure-function studies. Here we report the complete 1H NMR assignment of its proton resonances together with the resulting secondary structure pattern as prerequisites for the determination of a high-resolution 3D solution structure.

Modeling the paramyxovirus hemagglutinin-neuraminidase protein.

The paramyxovirus hemagglutinin-neuraminidase (HN) protein exhibits neuraminidase activity and has an active site functionally similar to that in influenza neuraminidases. Earlier work identified conserved amino acids among HN sequences and proposed similarity between HN and influenza neuraminidase sequences. In this work we identify the three-dimensional fold and develop a more detailed model for the HN protein, in the process we examine a variety of protein structure prediction methods. We use the known structures of viral and bacterial neuraminidases as controls in testing the success of protein structure prediction and modeling methods, including knowledge-based threading, discrete three-dimensional environmental profiles, hidden Markov models, neural network secondary structure prediction, pattern matching, and hydropathy plots. The results from threading show that the HN protein sequence has a 6 beta-sheet propellor fold and enable us to assign the locations of the individual beta-strands. The three-dimensional environmental profile and hidden Markov model methods were not successful in this work. The model developed in this work helps to understand better the biological function of the HN protein and design inhibitors of the enzyme and serves as an assessment of some protein structure prediction methods, especially after the x-ray crystallographic solution of its structure.

Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and bayesian scoring functions

We explore the ability of a simple simulated annealing procedure to assemble native-like structures from fragments of unrelated protein structures with similar local sequences using Bayesian scoring functions. Environment and residue pair specific contributions to the scoring functions appear as the first two terms in a series expansion for the residue probability distributions in the protein database; the decoupling of the distance and environment dependencies of the distributions resolves the major problems with current database-derived scoring functions noted by Thomas and Dill. The simulated annealing procedure rapidly and frequently generates native-like structures for small helical proteins and better than random structures for small β sheet containing proteins. Most of the simulated structures have native-like solvent accessibility and secondary structure patterns, and thus ensembles of these structures provide a particularly challenging set of decoys for evaluating scoring functions. We investigate the effects of multiple sequence information and different types of conformational constraints on the overall performance of the method, and the ability of a variety of recently developed scoring functions to recognize the native-like conformations in the ensembles of simulated structures.

Gene synthesis, high-level expression and assignment of backbone 15N and 13C resonances of soybean leghemoglobin

A synthetic gene for apoleghemoglobin-a from soybean, optimized for expression in Escherichia coli has been designed and synthesized by a recursive polymerase chain reaction technique. The protein has been expressed with high efficiency and a purification protocol has been developed. The holoprotein is readily reconstituted by the addition of heme. 15N- and 15N, 13C-labeled samples were produced and backbone 15N and 13C assignments were determined by 2D and 3D NMR spectroscopy. Comparison of the chemical shifts of 13C α and 13CO with random coil shifts revealed a pattern of secondary structure which correlates well with the one previously derived from homonuclear NMR data and low-resolution X-ray crystallography.

Evaluation of the secondary structure of vaccinia-virus thymidine kinase by circular-dichroism spectroscopy of overlapping synthetic peptides.

A new approach is reported that includes multiple-peptide synthesis and CD spectroscopy of overlapping peptides to evaluate the secondary structure of the vaccinia-virus thymidine kinase (TK). We divided the sequence of the vaccinia-virus TK into 82 peptides of 15 residues that overlapped by 13 residues and covered the complete sequence of vaccinia-virus TK. All peptides were synthesized by solid-phase multiple-peptide synthesis by means of the Fmoc/tert-butyl strategy. Subsequently, the secondary structure of each peptide was studied by means of CD spectroscopy in a mixture of 30% trifluoroethanol and sodium phosphate, pH 7. Secondary-structure evaluation led to determination of a vaccinia-virus-TK secondary-structure pattern. Consecutive peptides with alpha-helical content mainly showed CD spectra with increasing and decreasing Cotton effects typical of alpha-helices. This phenomenon was used to localize the helices on the sequence. In contrast, only single CD spectra with clear beta-sheet conformation, or CD spectra of mixed secondary-structure content were observed for beta-sheets. Therefore, the exact localization of beta-sheet-containing residues was deduced by comparison with isofunctional sequence-dissimilar proteins. We identified seven alpha-helices and six beta-sheet-containing regions, which we used for a secondary-structure model of the vaccinia-virus TK protein.

Assessment of a protein fold recognition method that takes into account four physicochemical properties: side-chain packing, solvation, hydrogen-bonding, and local conformation.

A protein fold recognition method was tested by the blind prediction of the structures of a set of proteins. The method evaluates the compatibility of an amino acid sequence with a three-dimensional structure using the four evaluation functions: side-chain packing, solvation, hydrogen-bonding, and local conformation functions. The structures of 14 proteins containing 19 sequences were predicted. The predictions were compared with the experimental structures. The experimental results showed that 9 of the 19 target sequences have known folds or portions of known folds. Among them, the folds of Klebsiella aerogenes urease beta subunit (KAUB) and pyruvate phosphate dikinase domain 4 (PPDK4) were successfully recognized; our method predicted that KAUB and PPDK4 would adopt the folds of macromomycin (Ig-fold) and phosphoribosylanthranilate isomerase:indoleglycerol-phosphate synthase (TIM barrel), respectively, and the experimental structure revealed that they actually adopt the predicted folds. The predictions for the other targets were not successful, but they often gave secondary structural patterns similar to those of the experimental structures.

Graph-theoretical assignment of secondary structure in multidimensional protein NMR spectra: Application to the lac repressor headpiece

A novel procedure is presented for the automatic identification of secondary structures in proteins from their corresponding NOE data. The method uses a branch of mathematics known as graph theory to identify prescribed NOE connectivity patterns characteristic of the regular secondary structures. Resonance assignment is achieved by connecting these patterns of secondary structure together, thereby matching the connected spin systems to specific segments of the protein sequence. The method known as SERENDIPITY refers to a set of routines developed in a modular fashion, where each program has one or several well-defined tasks. NOE templates for several secondary structure motifs have been developed and the method has been successfully applied to data obtained from NOESY-type spectra. The present report describes the application of the SERENDIPITY protocol to a 3D NOESY-HMQC spectrum of the 15N-labelled lac repressor headpiece protein. The application demonstrates that, under favourable conditions, fully automated identification of secondary structures and semi-automated assignment are feasible.

Omega loops; nonregular secondary structures significant in protein function and stability

Omega (omega-) loops, a nonregular secondary structure found in globular proteins, are characterized by a polypeptide chain that follows a loop-shaped course in three-dimensional space. They do not contain repeating backbone dihedral angles or regular patterns of hydrogen bonding; however, many omega-loops contain a large number of hydrogen bonds, therefore it is not correct to think of omega-loops as structures lacking in hydrogen bonds. omega-Loops are found almost exclusively at the protein surface and exhibit amino acid preferences consistent with this observation. Since the first description of omega-loops in 1986, experiments have been conducted to probe the role of these structures in protein function, stability, and folding. It has become clear that omega-loops are often involved in protein function and molecular recognition. One motif, an omega-loop lid, that is flexible and mobile until substrate or inhibitor is bound and which probably plays a role in one or more steps of enzymatic catalysis, has been described in a variety of enzymes. Because they lack the periodic hydrogen bonding patterns of the regular secondary structures, some omega-loops are well suited for such functional roles in proteins. However, loops with a higher-than-average number of hydrogen bonds or hydrophobic contacts may play roles in protein stability or folding. Rather than determining further geometric definitions of loops, it may be instructional to group them according to their roles in protein structure, i.e., as categories of functional omega-loops, stability omega-loops, and folding omega-loops.

The predicted secondary structure of the G-type glutamine amidotransferase is compatible with TIM-barrel topology.

Glutamine amidotransferase (GAT) subunits or domains catalyze an important partial reaction in many complex biosynthetic reactions. The structure of one member of the F-type GATs is known, but the structure of the unrelated G-type is still unknown. Because many protein sequences are available for anthranilate synthase component II (product of the trpG gene), we have predicted its average secondary structure by a joint prediction method [Niermann and Kirschner (1991a) Protein Engng, 4, 359-370]. The predicted eight beta-strands and seven alpha-helices follow an 8-fold cyclic repetition of a beta-strand-loop-alpha-helix-loop module with helix alpha 7 missing. This pattern of secondary structure suggests that the G-type GAT domain has an 8-fold beta alpha-barrel topology, as found first in triose phosphate isomerase (TIM-barrel). This model is supported by the location of known catalytically essential residues in loops between beta-strands and alpha-helices. Evidence from published sequencing and mutational studies on selected members of the GAT superfamily (carbamoyl phosphate, imidazoleglycerol phosphate, GMP and CTP synthases) support both the secondary structure prediction and the TIM-barrel topology.

Modeling large RNAs and ribonucleoprotein particles using molecular mechanics techniques

There is a growing body of low-resolution structural data that can be utilized to devise structural models for large RNAs and ribonucleoproteins. These models are routinely built manually. We introduce an automated refinement protocol to utilize such data for building low-resolution three-dimensional models using the tools of molecular mechanics. In addition to specifying the positions of each nucleotide, the protocol provides quantitative estimates of the uncertainties in those positions, i.e., the resolution of the model. In typical applications, the resolution of the models is about 10-20 A. Our method uses reduced representations and allows us to refine three-dimensional structures of systems as big as the 16S and 23S ribosomal RNAs, which are about one to two orders of magnitude larger than nucleic acids that can be examined by traditional all-atom modeling methods. Nonatomic resolution structural data--secondary structure, chemical cross-links, chemical and enzymatic footprinting patterns, protein positions, solvent accessibility, and so on--are combined with known motifs in RNA structure to predict low-resolution models of large RNAs. These structural constraints are imposed on the RNA chain using molecular mechanics-type potential functions with parameters based on the quality of experimental data. Surface potential functions are used to incorporate shape and positional data from electron microscopy image reconstruction experiments into our models. The structures are optimized using techniques of energy refinement to get RNA folding patterns. In addition to providing a consensus model, the method finds the range of models consistent with the data, which allows quantitative evaluation of the resolution of the model. The method also identifies conflicts in the experimental data. Although our protocol is aimed at much larger RNAs, we illustrate these techniques using the tRNA structure as an example and test-bed.

A new approach to artificial and modified proteins: theory-based design, synthesis in a cell-free system and fast testing of structural properties by radiolabels.

A novel approach to the creation of artificial and modified proteins has been elaborated. The approach includes a sequence design based on the molecular theory of protein secondary structure and folding patterns, gene expression in a cell-free system and testing of structural properties of the synthesized polypeptides at a nanogram level using radiolabelled chains. The approach has been applied to a new synthetic protein albebetin which has been designed to form a 3-D fold which does not contradict any structural rule but has been never observed up to now in natural proteins. Using size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis of a radiolabelled chain, it has been shown that the artificial protein is nearly as compact as natural proteins, cooperatively unfolds at high urea concentrations and has some structural features of a definite structure consistent with the designed one. As albebetin has been designed as consisting of two structural repeats, a 'half-albebetin' (one of these repeats) has also been synthesized and studied. It was shown that 'half-albebetin' is also compact.

Cardiotoxin III from the Taiwan Cobra ( Naja naja atra) : Determination of Structure in Solution and Comparison with Short Neurotoxins

The structure in solution of cardiotoxin III, a membrane toxin purified from the venom of the Taiwan cobra, Naja naja atra, is reported. Sequence-specific assignment of 1H-NMR lines was completed and the NMR data show the presence of a triple and a double-stranded antiparallel β -sheet. Many NOE cross peaks identified in NOESY spectra were applied as distance constraints based on a hybrid distance geometry/dynamical simulated annealing technique; 20 structures were found within a single family. The average value of atomic RMS differences between the 20 structures and their geometric mean is 0·087 nm for the backbone atoms and 0·152nm for all heavy atoms; they are 0·055nm and 0·12nm, respectively for the segments of secondary structure. In these selected structures the backbone of the polypeptide chain folds such that five strands emerge from a globular head. Three major loops link these strands to form a double and a triple-stranded antiparallel β-sheet. Comparison of the structures of the toxin in solution with the X-ray crystal structure of its homologous protein, cardiotoxin V II 4 from Naja mossambica mossambica, showed good agreement between the structures except at segments of the turns. As the functions of short neurotoxins and cardiotoxins are distinct, despite their similar secondary structural patterns and tertiary folding, a comparative analysis has been carried out between cardiotoxin III and short neurotoxins of known structures. We discuss their structural features in order to clarify relationships between their structure and function.

Secondary Structure and Protein Folding of Recombinant Chloroplastic Thioredoxin Ch2 from the Green Alga Chlamydomonas reinhardtii as Determined by 1H NMR1

The recombinant form of the chloroplastic thioredoxin Ch2 from the green alga Chlamydomonas reinhardtii [Jacquot et al. (1992) Nucleic Acids Res. 20, 617] that preferentially activates the NADP dependent malate dehydrogenase [EC 1.1.1.82] (m-type thioredoxin) through a light promoted reductive system, has been subjected to an extensive two-dimensional 1H NMR analysis. A complete 1H NMR assignment of the resonance lines in both the oxidized and the reduced states at pH 5.8 has been obtained allowing the recognition of the secondary structure patterns and the global protein folding. The single polypeptide chain, made of 106 residues plus one additional Met located at the N-terminal position (11.6 kDa) due to the protein expression system, folds into a pattern characteristic of the open twisted alpha/beta structures already found for Escherichia coli and human thioredoxins for which the protein shares 46 and 20% of sequence identity, respectively. The open alpha/beta structure is made of 5 beta-sheets associated in a parallel (beta 1 to beta 3) and anti parallel manner (beta 3 to beta 5) and surrounded by 4 helices. This represents the first structural exploratory study of the ubiquitous oxido-reductase thioredoxins in a photosynthetic living system.

Two-dimensional 1H Nuclear Magnetic Resonance Studies of the Half-saturated (Ca 2+) 1 State of Calbindin D 9k: Further Implications for the Molecular Basis of Cooperative Ca 2+ binding

Calbindin D 9k exhibits cooperative binding of two calcium ions, hence study of the half-saturated states of the protein is critical to understanding the binding process. However, the half-saturated states are not significantly populated under equilibrium conditions. To circumvent this problem, an absolutely conserved glutamic acid residue in the C-terminal binding site (site II) has been mutated to glutamine (E65Q), causing a substantial reduction in calcium affinity and permitting detailed two-dimensional 1H NMR analysis of calbindin D 9k with a calcium ion bound only in the N-terminal EF-hand. Complete 1H resonance assignments have been obtained for (Ca 2+) 1 E65Q, as well as near complete assignments for the apo and (Ca 2+) 2 states. A value of 1·1 (±0·2) × 10 3 M -1 has been determined for the calcium binding constant in site II, from an analysis of the chemical shift changes in response to titration with calcium. The elements of secondary structure and global folding patterns were identified from nuclear Overhauser effects, backbone spin-spin coupling constants and the exchange rates of backbone amide protons. Although the mutation has only very small effects on the secondary structure and global fold of the protein, it so drastically lowers affinity for Ca 2+ in the C-terminal site that (Ca 2+) 2 E65Q does not correspond to a standard (Ca 2+) 2 state. From the analysis of the half-saturated state, it is apparent that some reorganization of the structure and changes in the internal dynamics of calbindin D 9k does occur for each step of the apo → (Ca 2+) 1(I) → (Ca 2+) 2 binding pathway. When the first ion is bound to the N-terminal EF-hand, that half of the molecule adopts a conformation and dynamic state similar to the fully calcium-loaded protein state, whereas only minor change occur in the C-terminal EF-hand. It is only upon binding of the second calcium ion that the C-terminal EF-hand switches over to the fully calcium-loaded state. Together with the results from four earlier study of the apo → (Ca 2+) 1(II) → (Ca 2+) 2 binding pathway, these findings indicate that changes in protein conformation and dynamics associated with Ca 2+ binding contribute to the observed cooperatively, and that the molecular details of the cooperative binding events are different for the two binding pathways.