Helical Elements Research Articles

Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

Single-molecule fluorescence resonance energy transfer studies of freely diffusing hairpin ribozymes with different combinations of helical junction and loop elements reveal striking differences in their folding behavior. We examined a series of six different ribozymes consisting of two-, three- and four-way junction variants, as well as corresponding constructs with one of the two loops removed. Our results highlight the varying contributions of preformed secondary structure elements to tertiary folding of the hairpin ribozyme. Of the three helical junction variants studied, the four-way junction strongly favored folding to a docked conformation of the two loops, required for catalytic activity. Moreover, the four-way junction was uniquely able to fold to a similar compact structure even in the absence of specific loop-loop docking interactions. A key feature of the data is the observation of broadening/tailing in the fluorescence resonance energy transfer histogram peak for a single-loop mutant of the four-way junction at higher Mg 2+ concentrations, not observed for any of the other single-loop variants. This feature is consistent with interconversion between compact and extended structures, which we estimate takes place on the 100- μs timescale using a simple model for the peak shape. This unique ability of the four-way junction ribozyme to populate an undocked conformation with native-like structure (a quasi-docked state) likely contributes to its greater tertiary structure stability, with the quasi-docked state acting as an intermediate and facilitating the subsequent formation of the specific hydrogen bonding network during docking of the two loops. The inability of two- and three-way junction ribozymes to fully populate a docked conformation reveals the importance of correct helical junction geometry as well as loop elements for effective ribozyme folding.

Evidence for Differing Roles for Each Lobe of the Calmodulin-like Domain in a Calcium-dependent Protein Kinase

Calcium-dependent protein kinases (CDPKs) are structurally unique Ser/Thr kinases found in plants and certain protozoa. They are distinguished by a calmodulin-like regulatory apparatus (calmodulin-like domain (CaM-LD)) that is joined via a junction (J) region to the C-terminal end of the kinase catalytic domain. Like CaM, the CaM-LD is composed of two globular EF structural domains (N-lobe, C-lobe), each containing a pair of Ca(2+) binding sites. Spectroscopic analysis shows that the CaM-LD is comprised of helical elements, but the isolated CaM-LD does not form a conformationally homogeneous tertiary structure in the absence of Ca(2+). The addition of substoichiometric amounts of Ca(2+) is sufficient to stabilize the C-terminal lobe in a construct containing J and CaM-LD (JC) but not in the CaM-LD alone. Moreover, as J is titrated into Ca(2+)-saturated CaM-LD, interactions are stronger with the C-lobe than the N-lobe of the CaM-LD. Measurements of Ca(2+) affinity for JC reveal two cooperatively interacting high affinity binding sites (K(d)(,mean) = 5.6 nm at 20 mm KCl) in the C-lobe and two weaker sites in the N-lobe (K(d,mean) = 110 nm at 20 mm KCl). The corresponding Ca(2+) binding constants in the isolated CaM-LD are lower by more than 2 orders of magnitude, which indicates that the J region has an essential role in stabilizing the structure of the CDPK regulatory apparatus. The large differential affinity between the two domains together with previous studies on a plasmodium CDPK (Zhao, Y., Pokutta, S., Maurer, P., Lindt, M., Franklin, R. M., and Kappes, B. (1994) Biochemistry 33, 3714-3721) suggests a model whereby even at normally low cytosolic levels of Ca(2+), the C-lobe interacts with the junction, but the kinase remains in an autoinhibited state. Activation then occurs when Ca(2+) levels rise to fill the two weaker affinity binding sites in the N-lobe, thereby triggering a conformational change that leads to release of the autoinhibitory region.

CHARMM fluctuating charge force field for proteins: II protein/solvent properties from molecular dynamics simulations using a nonadditive electrostatic model.

A fluctuating charge (FQ) force field is applied to molecular dynamics simulations for six small proteins in explicit polarizable solvent represented by the TIP4P-FQ potential. The proteins include 1FSV, 1ENH, 1PGB, 1VII, 1H8K, and 1CRN, representing both helical and beta-sheet secondary structural elements. Constant pressure and temperature (NPT) molecular dynamics simulations are performed on time scales of several nanoseconds, the longest simulations yet reported using explicitly polarizable all-atom empirical potentials (for both solvent and protein) in the condensed phase. In terms of structure, the FQ force field allows deviations from native structure up to 2.5 A (with a range of 1.0 to 2.5 A). This is commensurate to the performance of the CHARMM22 nonpolarizable model and other currently existing polarizable models. Importantly, secondary structural elements maintain native structure in general to within 1 A (both helix and beta-strands), again in good agreement with the nonpolarizable case. In qualitative agreement with QM/MM ab initio dynamics on crambin (Liu et al. Proteins 2001, 44, 484), there is a sequence dependence of average condensed phase atomic charge for all proteins, a dependence one would anticipate considering the differing chemical environments around individual atoms; this is a subtle quantum mechanical feature captured in the FQ model but absent in current state-of-the-art nonpolarizable models. Furthermore, there is a mutual polarization of solvent and protein in the condensed phase. Solvent dipole moment distributions within the first and second solvation shells around the protein display a shift towards higher dipole moments (increases on the order of 0.2-0.3 Debye) relative to the bulk; protein polarization is manifested via the enhanced condensed phase charges of typical polar atoms such as backbone carbonyl oxygens, amide nitrogens, and amide hydrogens. Finally, to enlarge the sample set of proteins, gas-phase minimizations and 1 ps constant temperature simulations are performed on various-sized proteins to compare to earlier work by Kaminsky et al. (J Comp Chem 2002, 23, 1515). The present work establishes the feasibility of applying a fully polarizable force field for protein simulations and demonstrates the approach employed in extending the CHARMM force field to include these effects.

Modifications and deletions of helices within the hairpin ribozyme-substrate complex: an active ribozyme lacking helix 1.

Within the hairpin ribozyme, structural elements required for formation of the active tertiary structure are localized in two independently folding domains, each consisting of an internal loop flanked by helical elements. Here, we present results of a systematic examination of the relationship between the structure of the helical elements and the ability of the RNA to form the catalytically active tertiary structure. Deletions and mutational analyses indicate that helix 1 (H1) in domain A can be entirely eliminated, while segments of helices 2, 3, and 4 can also be deleted. From these results, we derive a new active minimal ribozyme that contains three helical elements, an internal loop, and a terminal loop. A three-dimensional model of this truncated ribozyme was generated using MC-SYM, and confirms that the catalytic core of the minimized construct can adopt a tertiary structure that is very similar to that of the nontruncated version. A new strategy is described to study the functional importance of various residues and chemical groups and to identify specific interdomain interactions. This approach uses two physically separated and truncated domains derived from the minimal motif.

Structure of the conserved domain of ANAC, a member of the NAC family of transcription factors.

The structure of the DNA-binding NAC domain of Arabidopsis ANAC (abscisic-acid-responsive NAC) has been determined by X-ray crystallography to 1.9A resolution (Protein Data Bank codes 1UT4 and 1UT7). This is the first structure determined for a member of the NAC family of plant-specific transcriptional regulators. NAC proteins are characterized by their conserved N-terminal NAC domains that can bind both DNA and other proteins. NAC proteins are involved in developmental processes, including formation of the shoot apical meristem, floral organs and lateral shoots, as well as in plant hormonal control and defence. The NAC domain does not possess a classical helix-turn-helix motif; instead it reveals a new transcription factor fold consisting of a twisted beta-sheet surrounded by a few helical elements. The functional dimer formed by the NAC domain was identified in the structure, which will serve as a structural template for understanding NAC protein function at the molecular level.

INTERRELATION OF CURING AND BOTANY IN VANILLA (VANILLA PLANIFOLIA) BEAN

INTERRELATION OF CURING AND BOTANY IN VANILLA (VANILLA PLANIFOLIA) BEAN

In Vitro Protein Complex Formation with Cytoskeleton-anchoring Domain of Occludin Identified by Limited Proteolysis

Occludin is an essential membrane protein component of cellular tight junctions, participating in both cell-cell adhesion in the paracellular space and anchoring of the junctional complex to the cytoskeleton. The latter function is accomplished through binding of the C-terminal cytoplasmic region to scaffolding proteins that mediate binding to cytoskeletal actin. We isolated a structural domain from both the bacterial-expressed C-terminal cytoplasmic region of human occludin and native cellular occludin, extracted from epithelial (Madin-Darby canine kidney) or endothelial (human brain) cells, by limited proteolysis with trypsin. This human occludin domain contains the last 119 amino acids as identified by N-terminal sequencing and peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Based on the sequence and secondary structure prediction, this domain contains 4 of 5 alpha-helices in the C-terminal region and is linked to the fourth membrane-spanning region by a loosely structured tethering polypeptide. Comparison of circular dichroism spectra of recombinant proteins corresponding to the entire C-terminal region versus only the binding domain region also supports the interpretation that the helical structural elements are concentrated in that domain. Co-immunoprecipitation of this domain with ZO-2 demonstrated preservation of the specificity of the scaffolding protein-binding function, and binding studies with immobilized ZO-2 suggest the presence of multiple ZO-2 binding sites in this domain. These results provide a basis for development of a structural model of the ZO-binding site that can be used to investigate regulation of tight junction anchoring by intracellular signaling events.

A template-proximal RNA paired element contributes to Saccharomyces cerevisiae telomerase activity.

The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA. Here, we report the identification of a paired element located immediately 5' of the template region in the Saccharomyces cerevisiae telomerase RNA. Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro. Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides. Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template. Thus, the S. cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification. In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by telomerase.

Compensating for the mutual coupling effect in a normal-mode helical antenna array for adaptive nulling

A practical normal-mode helical antenna array together with a new method for the compensation of the mutual coupling effect is proposed for the efficient adaptive nulling of interferences in a mobile communication environment. The helical antenna array is shown to be able to generate deeper nulls than an equivalent monopole antenna array in the presence of the mutual coupling effect. The helical antenna elements are smaller in size and less susceptible to the mutual coupling effect than monopole antenna elements. The new method for compensation of the mutual coupling effect is shown to have a greater ability and flexibility than previous methods. A new definition of mutual impedance is introduced and used to find the coupling-free signal voltages across the antenna terminal loads. Numerical examples demonstrate the superior performance of the proposed adaptive array and the new compensation method.

Structure of the C-Terminal Domain of Human La Protein Reveals a Novel RNA Recognition Motif Coupled to a Helical Nuclear Retention Element

The La protein is an important component of ribonucleoprotein complexes that acts mainly as an RNA chaperone to facilitate correct processing and maturation of RNA polymerase III transcripts, but can also stimulate translation initiation. We report here the structure of the C-terminal domain of human La, which comprises an atypical RNA recognition motif (La225–334) and a long unstructured C-terminal tail. The central β sheet of La225–334 reveals novel features: the putative RNA binding surface is formed by a five-stranded β sheet and, strikingly, is largely obscured by a long C-terminal α helix that encompasses a recently identified nuclear retention element. Contrary to previous observations, we find that the La protein does not contain a dimerization domain.

Insulin Assembly Damps Conformational Fluctuations: Raman Analysis of Amide I Linewidths in Native States and Fibrils

The crystal structure of insulin has been investigated in a variety of dimeric and hexameric assemblies. Interest in dynamics has been stimulated by conformational variability among crystal forms and evidence suggesting that the functional monomer undergoes a conformational change on receptor binding. Here, we employ Raman spectroscopy and Raman microscopy to investigate well-defined oligomeric species: monomeric and dimeric analogs in solution, native T6 and R6 hexamers in solution and corresponding polycrystalline samples. Remarkably, linewidths of Raman bands associated with the polypeptide backbone (amide I) exhibit progressive narrowing with successive self-assembly. Whereas dimerization damps fluctuations at an intermolecular β-sheet, deconvolution of the amide I band indicates that formation of hexamers stabilizes both helical and non-helical elements. Although the structure of a monomer in solution resembles a crystallographic protomer, its encagement in a native assembly damps main-chain fluctuations. Further narrowing of a β-sheet-specific amide I band is observed on reorganization of insulin in a cross-β fibril. Enhanced flexibility of the native insulin monomer is in accord with molecular dynamics simulations. Such conformational fluctuations may initiate formation of an amyloidogenic nucleus and enable induced fit on receptor binding.

Active peptidic mimics of the second intracellular loop of the V(1A) vasopressin receptor are structurally related to the second intracellular rhodopsin loop: a combined 1H NMR and biochemical study.

Vasopressin (VP) receptors belong to the widespread G protein-coupled receptor family. The crucial role of VP receptor intracellular loops in the coupling with the heterotrimeric G proteins was previously demonstrated by construction of a vasopressin receptor chimera. Yet, no fine structural data are available concerning the receptor molecular determinants involved in their interactions with G proteins. In this study, we synthesized both a linear and a cyclic form of the second intracellular loop (i2) of the human V(1a) vasopressin receptor isoform that is important for the interaction between the alphaq/alpha11 G protein and the receptor. These two peptides are biologically active. They specifically inhibit vasopressin binding to the V(1a) receptor, suggesting that the corresponding endogenous peptides contribute to the structure of the vasopressin binding site via intra- or intermolecular interactions with the core of the V(1a) receptor. The i2 peptide structures were determined by (1)H NMR. Both exhibit a helix and helical elements in their N- and C-terminal parts, respectively, separated by a turn imposed by a proline residue. More interestingly, the central Pro-Leu motif conserved in many GPCRs and thought to be important for coupling to G proteins can adopt different conformations. The "U" shape structure of the i2 loop is compatible with its anchoring to transmembrane domains III and IV and is very similar to the shape of bovine rhodopsin i2. Altogether, these data contribute to a better understanding of the structure of a not yet crystallized GPCR using the mimetic peptide approach.

Homochiral Self Recognition in Gold-Phosphane-Helicates

Treatment of Au(SMe2)Cl with a mixture of (R*,S*)- and (R*,R*)-(±)-p4 (p4 = 1,1,4,7,10,10-hexaphenyl-1,4,7,10-tetraphosphadecane) and TlOTf produces [Au4(R,S-p4)2](OTf)4 (1) and a racemic compound of [Au4(R,R-p4)2](OTf)4 and [Au4(S,S-p4)2](OTf)4 (2). Compounds 1 and 2 were isolated as colorless crystals and characterized by single crystal X-ray diffraction. In both 1 and 2, four gold atoms are bridged by two p4 ligands. The structure of 1 is a meso-helicate composed of a P, an M and a central achiral domain. The ligands exhibit a head-to-head arrangement. The structures of 2 consist of a non-palindromic double helix. In both 1 and 2 the R centers induce a left-handed (M) and the S centers a right-handed (P) helicity. The helical elements of both 1 and 2 are unsaturated and show Au···Au interactions. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

The Giardia intestinalis filamentous cyst wall contains a novel beta(1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study.

Assembly of a protective cyst wall by Giardia is essential for the survival of the parasite outside the host intestine and for transmission among susceptible hosts. The structure of the G. intestinalis filamentous cyst wall was studied by chemical methods, mass spectrometry, and (1)H nuclear magnetic resonance spectroscopy. Isolated cyst wall material contains carbohydrate and protein in a ratio of 3:2 (w/w), and the carbohydrate moiety is composed of a beta(1-3)-N-acetyl-D-galactopyranosamine homopolymer. Conformational analysis by molecular dynamics and persistence length calculations of GalNAc oligomers in solution demonstrated a flexible structure consisting of left- and right-handed helical elements. It is most likely that in the solid state, the polysaccharide forms ordered helices or possibly multiple helical structures having strong interchain interactions. The highly insoluble nature of the Giardia cyst wall must be due to these strong interchain interactions and, probably, a strong association between the carbohydrate and the protein moiety.

A 15-Residue Bifunctional Element in d-AKAP1 Is Required for Both Endoplasmic Reticulum and Mitochondrial Targeting

The cAMP-dependent protein kinase anchoring protein, d-AKAP1, has two N-terminal splice variants. The shorter forms (N0, d-AKAP1a, and -1c) target to mitochondria, and the longer forms (N1, d-AKAP1b, and -1d) with 33 additional residues N-terminal to N0 target to the endoplasmic reticulum (ER) (Huang, L. J., Wang, L., Ma, Y., Durick, K., Perkins, G., Deerinck, T. J., Ellisman, M. H., and Taylor, S. S. (1999) J. Cell Biol. 145, 951-959). In d-AKAP1a, translation may initiate from both Met-34 or Met-49 producing two molecules both targeted to mitochondria. The shorter molecule contains the 15-residue targeting motif, homologous to the N-terminal mitochondrial targeting motif of hexokinase I. Extensive mutagenesis showed that one hydrophobic surface of the 15-residue hexokinase-homologous segment contained the key elements for mitochondrial targeting. The same 15 residues are also part of the ER-targeting signal, but for ER targeting multiple hydrophobic residues are required that encompass both surfaces of the helix. The different involvement of the same helical motif for targeting to the two organelles appears to reflect different modes of interaction with the two organelles. This is the first example of a bifunctional helical element that is required for both ER and mitochondrion targeting.

Cloning, heterologous expression, and substrate specificities of protein farnesyltransferases from Trypanosoma cruzi and Leishmania major

Chagas disease and leishmaniasis are tropical diseases caused by the protozoan parasites, Trypanosoma cruzi and Leishmania species, respectively. Protein farnesyltransferase (PFT) is being investigated as a target for anti-trypanosomatid agents because inhibitors of this enzyme are highly toxic to these parasites compared to mammalian cells. Here, we report the cloning of the α- and β-subunit genes of PFT from T. cruzi and Leishmania major. The proteins encoded by these genes are considerably larger than those of mammalian PFTs due to the presence of a number of inserts of >25 amino acids that map to junctions between helical structural elements. These inserts are not part of the active site or the interface between the two subunits. Northern blots demonstrate expression of messenger RNA for the PFT subunits in both mammalian and insect life-cycle stages of these parasites. The T. cruzi, Trypanosoma brucei, and L. major PFTs were overexpressed in the Sf9 cell/baculovirus system as active enzyme forms. Kinetic studies with a panel of CALX-containing peptides with all 20 amino acids in the X-position show that trypanosomatid PFTs have similar substrate specificities and these are different from the mammalian PFT substrate specificity patterns.

The Decay of Swirl in Horizontal Dispersed Oil-Water Flow

This paper reports experimental work to investigate the characteristics of swirl in dispersed liquid-liquid flow in a horizontal pipe. In this work the focus was on determining the pressure gradient in a horizontal 25.4 mm (nominal one-inch) tube and its spatial evolution in swirling liquid-liquid flow. Experimental results are presented for Kerosene (EXXOL D80) and tap water at room temperature. The swirl was introduced by Kenic helical static mixer elements at the pipe inlet. The pressure gradient was measured for different numbers of helical elements at three different positions (near the beginning, middle and end of the test section) for single-phase flows of water and oil and also for dispersed flows of oil and water. A cross-shaped de-swirl device was used to distinguish effects due to swirl from those due to the additional mixing and turbulence induced by the mixer elements. A gamma densitometer system was applied for measuring the chordal volume fraction distribution across the tube and to obtain tomographic results for phase distribution across the tube both with and without helical elements. It was concluded that the introduction of helical elements at the tube inlet has a considerable influence on the flow behaviour and produces a swirl flow in the pipe whose intensity decays with distance from the inlet. The rate of decay depends on the type of dispersion (oil or water continuous). The influence of mixing phenomena on pressure gradient is manifested in several ways, including a sharpening of the pressure gradient peak in dispersed flow and the occurrence of a secondary pressure gradient maximum over a range of conditions. Furthermore, the results obtained with the gamma densitometer indicate that the helical element is an efficient means of mixing. It can generate a mixing intensity equivalent to a turbulent flow at higher mixture velocity.

An inducible helix-Gly-Gly-helix motif in the N-terminal domain of histone H1e: a CD and NMR study.

Knowledge of the structural properties of linker histones is important to the understanding of their role in higher-order chromatin structure and gene regulation. Here we study the conformational properties of the peptide Ac-EKTPVKKKARKAAGGAKRKTSG-NH(2) (NE-1) by circular dichroism and (1)H-NMR. This peptide corresponds to the positively charged region of the N-terminal domain, adjacent to the globular domain, of mouse histone H1e (residues 15-36). This is the most abundant H1 subtype in many kinds of mammalian somatic cells. NE-1 is mainly unstructured in aqueous solution, but in the presence of the secondary-structure stabilizer trifluoroethanol (TFE) it acquires an alpha-helical structure. In 90% TFE solution the alpha-helical population is approximately 40%. In these conditions, NE-1 is structured in two alpha-helices that comprise almost all the peptide, namely, from Thr17 to Ala27 and from Gly29 to Thr34. Both helical regions are highly amphipathic, with the basic residues on one face of the helix and the apolar ones on the other. The two helical elements are separated by a Gly-Gly motif. Gly-Gly motifs at equivalent positions are found in many vertebrate H1 subtypes. Structure calculations show that the Gly-Gly motif behaves as a flexible linker between the helical regions. The wide range of relative orientations of the helical axes allowed by the Gly-Gly motif may facilitate the tracking of the phosphate backbone by the helical elements or the simultaneous binding of two nonconsecutive DNA segments in chromatin.

Fluid flow in tubes with helical elements

The flow patterns of fluids in convoluted channels were studied experimentally. The existence of secondary flow in tubes with helical static elements was proved. In such devices the magnitude of superimposed vortical motion is comparable to that of axial flow. The double helical flow persists downstream for certain distance, until dissipated by viscous friction. For the flow visualisation colour-band experiments and measurements using laser technique were applied. On the basis of the experiments several new statements were established on the microstructure of the flow. The laminar flow conditions were compared with the behaviour of flows in coiled tubes and also in tubes with twisted tape inserts. For easier comparison a modified Dean number was proposed, which took into account the curvature of the spherical line cut out by the helical element from the tubular housing.

An efficient ligation reaction promoted by a Varkud Satellite ribozyme with extended 5'- and 3'-termini.

The Neurospora Varkud Satellite (VS) RNA is capable of promoting a reversible self-cleavage reaction important for its replication pathway. In vivo the VS RNA performs a cis-cleavage reaction to generate monomeric length transcripts that are subsequently ligated to produce circular VS RNA. The predominant form of VS RNA observed in vivo is the closed circular form, though minimal VS ribozyme self-cleavage constructs lack detectable ligation activity. MFOLD analysis of the entire VS RNA sequence revealed an extended region 5' and 3' of the minimal self-cleaving region that could anneal to form a complementary helix, which we have termed helix 7. In full-length VS RNA, this helix appears to span over 40 bp of sequence and brings the 5'- and 3'-ends of the RNA into proximity for the ligation reaction. Here we report a variant of the VS ribozyme with an extended 5'- and 3'-terminus capable of forming a truncated helix 7 that promotes the ligation reaction in vitro. Through mutation and selection of this RNA we have identified a ribozyme containing two point mutations in the truncated helix 7 that ligates with >70% efficiency. These results show that an additional helical element absent in current VS ribozyme constructs is likely to be important for the ligation activity of VS RNA.