Simple Structural Motifs Research Articles

Mapping the Energy Landscape of Repeat Proteins using NMR-detected Hydrogen Exchange

Repeat proteins contain tandem arrays of a simple structural motif. In contrast to globular proteins, repeat proteins are stabilized only by interactions between residues that are relatively close together in the sequence, with no ”long-range” interactions. Our work focuses on the tetratricopeptide repeat (TPR), a 34 amino acid helix-turn-helix motif found in tandem arrays in many natural proteins. Earlier, we reported the design and characterization of a series of consensus TPR (CTPR) proteins, which are built as arrays of multiple tandem copies of a 34 amino acid consensus sequence. Here, we present the results of extensive hydrogen exchange (HX) studies of the folding–unfolding behavior of two CTPR proteins (CTPR2 and CTPR3). We used HX to detect and characterize partially folded species that are populated at low frequency in the nominally folded state. We show that for both proteins the equilibrium folding–unfolding transition is non-two-state, but sequential, with the outermost helices showing a significantly higher probability than inner helices of being unfolded. We show that the experimentally observed unfolding behavior is consistent with the predictions of a simple Ising model, in which individual helices are treated as ”spin-equivalents”. The results that we present have general implications for our understanding of the thermodynamic properties of repeat proteins.

Synthetic Approaches to Sterically Hindered N‐Arylimidazoles through Copper‐Catalyzed Coupling Reactions

Optimization studies allowed the efficient synthesis of a simple structural motif based on meta-bis(1-imidazolyl)benzenes 1 through copper-catalyzed coupling of 1,3-diiodobenzene and imidazole under mild reaction conditions. This protocol was then used to prepare a representative sterically hindered N-arylimidazole 2a, the most common structural motif among N-heterocyclic carbenes (NHC). Having optimized the main variables governing CuI-catalyzed imidazole N-arylation, the first Ullmann-type synthesis of N-mesitylimidazole (2a) is reported. Moreover, the coupling between boronic acids as the aryl donor partners and either imidazole or benzimidazole was examined; in all cases the reactions proceeded in very low yield. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Network motifs: structure does not determine function

BackgroundA number of publications have recently examined the occurrence and properties of the feed-forward motif in a variety of networks, including those that are of interest in genome biology, such as gene networks. The present work looks in some detail at the dynamics of the bi-fan motif, using systems of ordinary differential equations to model the populations of transcription factors, mRNA and protein, with the aim of extending our understanding of what appear to be important building blocks of gene network structure.ResultsWe develop an ordinary differential equation model of the bi-fan motif and analyse variants of the motif corresponding to its behaviour under various conditions. In particular, we examine the effects of different steady and pulsed inputs to five variants of the bifan motif, based on evidence in the literature of bifan motifs found in Saccharomyces cerevisiae (commonly known as baker's yeast). Using this model, we characterize the dynamical behaviour of the bi-fan motif for a wide range of biologically plausible parameters and configurations. We find that there is no characteristic behaviour for the motif, and with the correct choice of parameters and of internal structure, very different, indeed even opposite behaviours may be obtained.ConclusionEven with this relatively simple model, the bi-fan motif can exhibit a wide range of dynamical responses. This suggests that it is difficult to gain significant insights into biological function simply by considering the connection architecture of a gene network, or its decomposition into simple structural motifs. It is necessary to supplement such structural information by kinetic parameters, or dynamic time series experimental data, both of which are currently difficult to obtain.

Synthetic Approaches to Sterically Hindered N‐Arylimidazoles Through Copper‐Catalyzed Coupling Reactions.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthetic Approaches to Sterically Hindered N‐Arylimidazoles through Copper‐Catalyzed Coupling Reactions

AbstractOptimization studies allowed the efficient synthesis of a simple structural motif based on meta‐bis(1‐imidazolyl)benzenes 1 through copper‐catalyzed coupling of 1,3‐diiodobenzene and imidazole under mild reaction conditions. This protocol was then used to prepare a representative sterically hindered N‐arylimidazole 2a, the most common structural motif among N‐heterocyclic carbenes (NHC). Having optimized the main variables governing CuI‐catalyzed imidazole N‐arylation, the first Ullmann‐type synthesis of N‐mesitylimidazole (2a) is reported. Moreover, the coupling between boronic acids as the aryl donor partners and either imidazole or benzimidazole was examined; in all cases the reactions proceeded in very low yield. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

A Tweezer‐Like Peptide‐Binding Receptor

AbstractFor Abstract see ChemInform Abstract in Full Text.

A Tweezer-like Peptide-binding Receptor

In recent years, significant progress has been made in the construction of synthetic molecules that selectively bind peptide substrates and other small molecules. Some such molecules have the complex, cage-like structures, but a different and simple structural motif such as molecular tweezer with two substrate binding arms has emerged as having selective peptide-binding properties. Among those are two-armed receptors based on cyclic oligomers of 1,2diamine and isophthalic acid. Although such receptors showed remarkable binding properties, to have further possible applications its binding properties had to be improved. Here, a novel two-armed tweezer-like peptidebinding receptor is described. Receptor (1) consist of two different parts: A linker to which the tweezer arms are attached; and two substrate binding arms based on cyclic tetramer of two isophthalic acids and two 1,2-diaminocyclohexane. Particularly, linker in receptor (1) has transition metal ion (Ru). In this receptor-like molecule, metal ion acts to make potential substrate-binding sites to be preorganized for the effective complexation with suitable substrates. Furthermore, metal ion such as Ru can act as the sensitive probes for binding with substrates and provide the additional interactions between linkers and peptide substrates. Synthesis of 1 began with the preparation of monocyclic intermediates of 1,2-diaminocyclohexanes and isophthalic acid derivatives by the known procedures for the related molecules. Ester bond formation reaction between bis(pentafluorophenyl)ester (2) and 4,4'-hydroxymethyl-2,2'dipyridine (3), and the subsequent reaction with Ru(bpy)2Cl2 in refluxing EtOH provided 1 with 48.2% yield. Recently, combinatorial chemistry has become a major tool in the elucidation of the binding properties of receptors. By using such methods it has become possible to find the binding properties of receptors efficiently, and furthermore to detect subtle differences in receptor-substrate binding that could not have been studied by conventional experiments. Receptor 1 has the distinct red color due to transition metal ion (Ru), and thus ideal for solid phase color binding assay using encoded combinatorial library of peptide substrates. Receptor 1 was screened against a tripeptide library on Scheme 1. Two-armed Tweezerlike Receptor (1).

Synthesis and Characterization of an Extremely Versatile Structural Motif Called the “Plum-Pudding” Gel

The “plum-pudding” gel is a composite gel structure composed of responsive microgel particles randomly dispersed in a bulk gel medium. Microgel particles can be responsive to a variety of stimuli (temperature, pH, light, etc.), and the bulk gel can be any cross-linkable material. Thus an infinite variety of materials of different mechanical and responsive properties can be envisioned based on this simple structural motif. The novelty of the plum-pudding gel is that it separates the concepts of functionality and mechanical strength. The “plum-pudding” nature of the gel was confirmed by laser scanning confocal microscopy: the fluorescently labeled PNIPAM microgel particles resembled plums in a pudding. A model for the structure was proposed, where the growing network chains grew through the pores of the swollen microgel particles and were unaffected by their presence, resulting in an unchanged network structure at the concentrations of microgel particles studied (up to 20 mol % of the monomer content). Two examples of the diversity of the plum-pudding gel as a structural motif are illustrated. PNIPAM microgel particles are used in the first example to enhance the positive thermoresponsiveness of responsive bulk materials (NIPA-co-BAM-co-DAM gels) and in the second example to induce positive thermoresponsiveness into nonresponsive bulk gels (DAM gels).

Structural dependence of thermodynamic stability of unbranched catacondensed benzenoid hydrocarbons

Analysis of HF/6-31G** and B3LYP/6-311G** relative energies of 25 unbranched catacondensed benzenoid hydrocarbons with six rings reveals a well-pronounced dependence of their thermodynamic stabilities on the presence of simple structural motifs. In particular, the energy contribution due to steric overcrowding is proportional to the number of pairs of adjacent angular annelations of the same helicity, whereas the contribution due to conjugation effects is related to the number and mutual position of linear annelations. Although the arrangement of linear annelations also uniquely determines the number of Kekulé structures, the latter does not correlate directly with the non-steric energy component.

Adaptive crystal structures: CuAu and NiPt.

We discover that Au-rich Cu1-xAux and Pt-rich Ni1-xPtx contain a composition range in which there is a quasicontinuum of stable, ordered "adaptive structures" made of (001) repeat units of simple structural motifs. This is found by searching approximately 3x10(6) different fcc configurations whose energies are parametrized via a "cluster expansion" of first-principles-calculated total energies of just a few structures. This structural adaptivity is explained in terms of an anisotropic, long-range strain energy.

Dynamical View of the Positions of Key Side Chains in Protein-Protein Recognition

When a complex is constructed from the separately determined rigid structures of a receptor and its ligand, some key side chains are usually in wrong positions. These distortions of the interface yield an apparent loss in affinity and would unfavorably affect the kinetics of association. It is generally assumed that the interacting proteins should drive the appropriate conformational changes, leading to their complementarity, but this hypothesis does not explain their fast association rates. However, nanosecond explicit solvent molecular dynamics simulations of misfolded surface side chains from the independently solved structures of barstar, bovine pancreatic trypsin inhibitor, and lysozyme show that even before any receptor-ligand interaction, key side chains frequently visit the rotamer conformations seen in the complex. We show that these simple structural motifs can reconcile most of the binding affinity required for a rapid and highly specific association process. Side chains amenable to induced fit are also identified. These results corroborate that solvent-side chain interactions play a critical role in the recognition process. Our findings are also supported by crystallographic data.

Expanding the Potential of DNA for Binding and Catalysis: Highly Functionalized dUTP Derivatives That Are Substrates for Thermostable DNA Polymerases.

The discovery of a simple structural motif allows for the enzymatic synthesis by polymerase chain reactions (PCR) of modified DNA (see reaction scheme) bearing side chains similar or even identical to those of several amino acids. Libraries of DNA functionalized with both cationic and anionic groups may now be readily prepared. R=I, HgCl; X=functional group.

Expanding the Potential of DNA for Binding and Catalysis: Highly Functionalized dUTP Derivatives That Are Substrates for Thermostable DNA Polymerases

The discovery of a simple structural motif allows for the enzymatic synthesis by polymerase chain reactions (PCR) of modified DNA (see reaction scheme) bearing side chains similar or even identical to those of several amino acids. Libraries of DNA functionalized with both cationic and anionic groups may now be readily prepared. R=I, HgCl; X=functional group.

Equilibrium unfolding (folding) pathway of a model H-type pseudoknotted RNA: the role of magnesium ions in stability.

In T2 and related T-even bacteriophages, the upstream autoregulatory mRNA leader sequence of gene 32 folds into a simple tertiary structural motif, a hairpin (H)-type pseudoknot. This pseudoknot is derived from 32 contiguous nucleotides which form two coaxially stacked helical stems that adopt a pseudocontinuous A-form helical structure. These stems are connected by two nonequivalent single-stranded loops. The equilibrium unfolding pathway of a 36-nucleotide RNA fragment corresponding to the wild-type and sequence variants of the T2 gene 32 mRNA pseudoknot has been probed as a function of [Mg2+] by analysis of dual optical wavelength, equilibrium thermal melting profiles. A van't Hoff model based on multiple sequential, two-state unfolding transitions has been applied to the resultant data. Compensatory base pair substitutions incorporated into the helical stems have been used to assign optical melting transitions to molecular unfolding events. The optical melting profile of the wild-type RNA is minimally described by three sequential unfolding transitions. The helix-helix junction region melts first in a low-enthalpy transition, followed by the unfolding of the remainder of helical stem 2, and then, all of stem 1. The total enthalpy of unfolding (folding) at [Mg2+] >/= 1 mM is accounted for by the secondary structure alone, suggesting that, if any non-Watson-Crick or tertiary structure exists in this conformation, it makes little or no enthalpic contribution to the stability of the molecule. Consistent with this, the [Mg2+] dependence of individual unfolding transitions within the pseudoknot is well-described by differential extents of delocalized binding of Mg2+ to folded and unfolded regions of the molecule. At a fixed [Mg2+], the helix junction region and stem 2 sequester more Mg2+ ions than the stem 1 hairpin; a larger fraction of these ions are then released upon unfolding. Two base or base pair substitution mutant RNAs which destabilize the helical junction and/or the base of stem 2 appear to sequester fewer ions, with a correspondingly smaller number of these ions released upon unfolding.

Non-nearest neighbor effects on the thermodynamics of unfolding of a model mRNA pseudoknot

The upstream autoregulatory mRNA leader sequence of gene 32 of 17 T-even and related bacteriophages folds into a simple tertiary structural motif, a hairpin-type RNA pseudoknot. In phage T4, the pseudoknot is contained within 28 contiguous nucleotides which adopt a pseudocontinuous helical structure derived from two coaxially stacked helical stems of four (stem 1) and seven (stem 2) base-pairs connected by two inequivalent single-stranded loops of five and one nucleotide(s). These two loops cross the minor and major grooves of stems 1 and 2, respectively. In this study, the equilibrium unfolding pathway of a 35-nucleotide RNA fragment corresponding to the wild-type and sequence variants of the T4 gene 32 mRNA has been determined through analysis of dual-wavelength, equilibrium thermal melting profiles via application of a van’t Hoff model based on multiple sequential, two-state transitions. The melting profile of the wild-type RNA is well-described by two sequential melting transitions over a wide range of magnesium concentration. Compensatory base-pair substitutions incorporated into helical stems 1 and 2 were used to assign the first low enthalpy, moderate t m melting transition to the denaturation of the short three to four base-pair stem 1, followed by unfolding of the larger seven base-pair stem 2. We find that loop 1 substitution mutants (A10 to G10, C10, U10 or GA10) strikingly uncouple the melting of stems 1 and 2, with the U10 substitution and the GA10 loop expansion more destabilizing than the G10 and C10 substitutions. A significant increase in the extent of cleavage by RNase T 1 following the conserved G26 (the 3′ nucleotide in loop 2) in the U10, G10, and GA10 mutants suggests that an altered helix-helix junction region in this mutant may be responsible, at least in part, for this uncoupling. In addition to a modest destabilization of stem 2, the major effect of deletion or nucleotide substitution in the 3′ single-stranded tail is a destabilization of stem 1, a non-nearest neighbor tertiary structural effect, which may well be transmitted through an altered loop 1-core helix interaction. In contrast, truncation of the 5′ tail has no effect on the stability of the molecule.

A distinct 14 residue site triggers coiled-coil formation in cortexillin I.

We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311-Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four- rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled-coil 'trigger' site Arg311-Asp324 yielded approximately 30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311-Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I.

Cystine knots

Recent reports of the crystal structure of the glycoprotein hormone human chorionic gonadotrophin shown taht cystine knots are proving to be versatile structural motifs that enable the construction of variety of proteins with different functional properties. Because of their shape, there appears to be an intrisic requirement for the cystine-knot growth factors to form dimers. This extra level of organization increases the variety of structures built around this simple structural motif

RNA aptamers that bind flavin and nicotinamide redox cofactors.

RNA molecules that specifically bind riboflavin (Rb) and beta-nicotinamide mononucleotide (NMN) have been isolated by in vitro selection. A simple structural motif containing intramolecular G-quartets was found to bind tightly to oxidized riboflavin (Kd = 1-5 micromolar). DNA versions of the consensus sequence also bind, but with weaker affinity. DMS protection experiments show that the quartet structure of these aptamers is stabilized by interaction with the flavin. As a measure of their redox specificity, the aptamers do not show significant differential binding between oxidized and reduced forms of a 5-deazariboflavin derivative that is a close structural analog of riboflavin. In contrast to the lack of redox specificity of the riboflavin aptamers, RNAs selected for binding to the nicotinamide portion of NAD discriminate between NAD and NADH in solution by over an order of magnitude. A mutagenized pool based on one of the NMN aptamer sequences was used to reselect for NMN binding. Comparison of the reselected sequences led to the identification of the binding region of the aptamer. A complex secondary structure containing two interacting stem-loops is proposed for the minimal NMN-binding RNA. The same mutagenized pool was used to select for increased discrimination between NMN and NMNH. From these reselected sequences, a mutation within the binding region was identified that increases specificity for NMN. These experiments show that RNA can bind these cofactors with low micromolar affinity and, in the case of nicotinamide cofactors, can discriminate between the two redox states. These cofactor binding motifs may provide a framework for generating new ribozymes that catalyze redox reactions similar to those found in basic metabolic pathways.

HERA—A program to draw schematic diagrams of protein secondary structures

A program is described which generates hydrogen bonding diagrams of protein structures and optionally helical wheels and helical nets. The program can also beta-strands beta-strands and to automatically extract simple structural motifs such as hairpins or Greek keys. The program greatly reduces the effort required to produce these diagrams and offers considerable flexibility in the information which can be represented. The usefulness of the program is illustrated by several examples including comparing homologous families, correlating protein structure with attributes of individual residues, and extracting all examples of the psi-loop motif from the Brookhaven Data Bank.