Polynucleotide folding in yeast tRNAPhe: elucidation of short-, medium-, and long-range interactions of sugar-phosphate-sugar backbone and base using a "blocked" nucleotide probe

It has recently been proposed that the repeating backbone nucleotide may be regarded as consisting of two blocks of equal magnitude representable by two virtual bonds. Implicit consideration of the nucleotide (ψ,ψ) and internucleotide (ω′,ω) geometry that generate variety in polynucleotide conformations, and of the constancy of the repeating structural moieties (P-C4′ and C4′-P) independent of the above rotations, has enabled us to utilize this scheme in the study of ordered structures such as di-, oligonucleotides and, most significantly, tRNA. The polynucleotide folding dictated by short-, intermediate-, and long-range interactions in the monoclinic and orthorhombic forms is described and compared through circular plots depicting the virtual bond torsions and distance plots constructed independently for backbone as well as bases. The torsions and the bond angles associated with the virtual bonds afford a clear distinction between ordered helical segments from loops and bends of tRNA. Lower virtual bond torsions (?60° to 60°) concomitant with higher values of virtual bond angles characterize various bend regions, while torsions around 160°–210° typify ordered helical strands. The distance plot elucidates the type of interaction associated with various sub-structures (helix–helix, helix–loop, and loop–loop) that form the constituents of different structural domains. Several other features such as the manifestation of the P₁₀ loop and the approximate twofold symmetry in the tRNA molecule are conspicuous on the distance plot.     

Secondary and tertiary structural foldings in tRNA. A diagonal plot analysis using the blocked nucleotide scheme.

A distance plot obtained using the blocked nucleotide concept, which regards the repeating nucleotide moieties to be made up of two blocks of nearly equal magnitude, has permitted us to visualize the polynucleotide backbone folding in yeast tRNAPhe. The plot clearly manifests medium- and long-range tertiary interactions involving various structural domains. Apart from the well known T psi-D loop interactions, other long-range interactions associated with the variable loop as well as the D loop are explicitly seen. Most importantly, the plot reveals an approximate two-fold symmetry in the molecule between the domains related to the tertiary interactions in addition to the symmetry between long helical domains. The different patterns on the plot are interpreted in terms of helix-helix, loop-helix and loop-loop interactions.     

Analysis of the possible helical structures of nucleic acids and polynucleotides. Application of (n-h) plots.

The two helical parameters n and h where n is the number of nucleotide residues per turn and h is the height per nucleotide residue have been evaluated for single stranded helical polynucleotide chains comprising C(3') -endo and C(2') endo class of nucleotides. The helical parameters are found to be especially sensitive to the C(4')-C(3') (sugar pucker) and the C(4')-C(5') torsions. The (n-h) plots display only one important helix forming domain for each class of nucleotides characterized by the sugar pucker and the C(4')-C(5') torsion. A correlation between the (n-h) plots and the known RNA (A,A') and DNA (A,B,C) helical forms has been established. It is found that all forms of helices except the C-DNA possess a favorable combination of P-O torsions. The analysis of the (n-h) plots suggests that C-DNA can have a conformation very similar to B-DNA. Although the (n-h) plots predict the stereochemical possibility of both right-handed and left-handed helices, nucleic acids apparently prefer right-handed conformation because of the energetics associated with the sugar-phosphate backbone and the base.     

Mechanisms of chain folding in nucleic acids. The (omega, omega) plot and its correlation to the nucleotide geometry in yeast tRNAPhe1.

The (omega', omega) polot depicting the internucleotide P-O bond rotation angles in yeast phenylalanyl transfer RNA has established the interdependence of the phosphodiesters and the nucleotide geometries in the folding of the polynucleotide backbone. The plot distinguishes the regions characteristic of secondary helical structures and tertiary structural loops and bends. The folding of the polynucleotide chain is accomplished either solely by rotations around the P-O bonds or in concert with rotations around the nucleotide C4'-C5' bond with or without changes in the sugar ring pucker. In spite of differences in nucleotide sequence and intraloop tertiary interactions in the anticodon and pseudouridine loops, a characteristic repeating structural unit is found for the sugar-phosphate backbone of the tetranucleotide segment around the sharp turns.     

Thermal stability of RNA hairpins containing a four-membered loop and a bulge nucleotide

Fourteen RNA hairpins containing a four-membered loop and a bulge nucleotide were synthesized and their thermal stabilities determined. The combined contribution of a four-membered loop and bulge A to the free energy of a hairpin is calculated to be 9.3 kcal/mol at 37 degrees C and successfully predicts the stability of an independent RNA hairpin. The introduction of a bulge nucleotide to the helical stem of an RNA hairpin destabilizes the molecule in a sequence-dependent manner. The individual thermodynamic contributions of a four-membered loop and bulge A, G, and U residues to the stability of an RNA hairpin loop are presented.     

Conformational feasibility of a hairpin with two purines in the loop. 5'-d-GGTACIAGTACC-3'

Structural feasibility and conformational requirements for the sequence 5'-d-GGTACIAGTACC-3' to adopt a hairpin loop with I6 and A7 in the loop are studied. It is shown that a hairpin loop containing only two nucleotides can readily be formed without any unusual torsional angles. Stacking is continued on the 5'-side of the loop, with the I6 stacked upon C5. The base A7, on the 3'-side of the loop, can either be partially stacked with I6 or stick outside without stacking. Loop closure can be achieved for both syn and anti conformations of the glycosidic torsions for G8 while maintaining the normal Watson-Crick base pairing with the opposite C5. All torsional angles in the stem fall within the standard B-family of DNA helical structures. The phosphodiesters of the loop have trans,trans conformations. Loop formation might require the torsion about the C4'-C5' bond of G8 to be trans as opposed to the gauche+ observed in B-DNA. These results are discussed in relation to melting temperature studies [Howard et al. (1991) Biochemistry (preceding paper in this issue)] that suggest the formation of very stable hairpin structures for this sequence.     

Evaluation of the conformational propensities of peptide isosteres as a basis for selecting bioactive pseudopeptides.

Our aim was to compare the repertoires of conformers formed by the model zwitterionic peptides AA and AAA in aqueous solution with the conformational profiles of a range of their peptide isosteres, so as to facilitate selection of isosteres for synthesis and testing as biologically stable surrogates of bioactive di- and tripeptides. Comparisons were based upon the results of conformational analysis using a random search approach implemented within the SYBYL molecular modelling package, using zwitterionic molecules, simulated aqueous solvation using a dielectric constant of 80 and allowing all torsions to vary. For each compound, individual conformers were grouped on the basis of specific combinations of psi, phi and omega torsions and, using their energies, the aggregated percentage for each group was calculated using a Boltzmann distribution and displayed using a 3D pseudo Ramachandran plot relating percentage conformer to psi and phi torsions. Retroamide, N-methylamide and thioamide isosteres showed the best match to natural peptides and to the molecular recognition parameters defined for substrates of peptide transporters. The results should aid rational design of therapeutic agents in various areas, e.g. oral delivery of drugs by peptide transporters and of peptidase inhibitors. This approach may usefully be applied to various biochemical and pharmaceutical topics.     

The movement of spermatozoa with helical head: theoretical analysis and experimental results.

The present work is concerned with the study of the swimming of flagellated microscopic organisms with a helical head and a helical pattern of flagellar beating, such as Xenopus sperms. The theoretical approach is similar to that taken by Chang and Wu (1971) in the study of helical flagellar movement. The model used in the present study allows us to determine the velocity of propulsion (U) and the frequency of rotation of the sperm head (fh) as a function of the frequency of the wave of motion (ft) traveling along the tail. The results relative to the case of helical and planar flagellar waves are compared. Our main finding is that the helical shape of the head seems to increase the efficiency of propulsion of the spermatozoon when compared with the more commonly shaped spherical head. Experimentally measured values of fh versus U may be fitted by a linear plot whose slope is much higher than that corresponding to the case of planar flagellar beating. This fact is consistent with an effectively three-dimensional (nonplanar) movement of the flagellar tail. However, the results do not fit those predicted from a circular helix, suggesting that a different shape of the flagellar beating should be considered.     

Structure and dynamics of a helical hairpin and loop region in annexin 12: a site-directed spin labeling study

A 30-residue nitroxide scan encompassing a helical hairpin and an extended loop in soluble annexin 12 (helices D and E in repeat 2; residues 134-163) has been analyzed in terms of nitroxide side chain mobility and accessibility to collision with paramagnetic reagents (Pi). Values of Pi for both O(2) and a Ni(II) metal complex (NiEDDA) are remarkably well correlated with the fractional solvent accessibility of the native side chains at the corresponding positions computed from the known crystal structure. This result demonstrates the utility of Pi as an experimental measure of side chain accessibility in solution, as well as the lack of structural perturbation due to the presence of the nitroxide side chain. The pattern of side chain mobility is also in excellent agreement with predictions from the crystal structure. The results presented here extend the correlations between mobility and structure described in earlier work on other helical proteins, and suggest their generality. The periodic dependence of Pi and mobility along the sequence of annexin 12 reveals the helical segments and their orientation in the fold, as expected for a nonperturbing nitroxide side chain. However, these data do not distinguish the helix-loop-helix motif from a continuous helix, because immobilized side chains in the short loop sequence maintain the periodicity. As shown here, the ratio of Pi values for O(2) and NiEDDA clearly delineates the loop region, due to size exclusion effects between the two reagents. A new feature evident in a nitroxide scan through multiple secondary elements is a modulation of the basic Pi and mobility patterns along the sequence, apparently due to differences in helix packing and backbone motion. Thus, in the short helix D, residues are consistently more mobile and accessible throughout the sequence compared to the residues in the longer, less-solvated and more ordered helix E.     

Backbone conformation in nucleic acids: an analysis of local helicity through heminucleotide scheme and a proposal for a unified conformational plot

A relationship has been established to express the local helicity of a polynucleotide backbone directly in terms of the virtual bonds spanning the conformationally equivalent heminucleotide repeats, with a view to provide a better understanding of the cumulative effects of all the chemical bond rotational variations on local helicity. Using this, an analysis made with a few oligodeoxynucleotide crystal structures clearly brings forth that it is the concerted movements manifested in the near neighbour correlations between the pair of chemical bonds C4'-C5' and P-O5' and C4'-C3' and P-O3' of the 5' and 3' heminucleotides respectively that are primarily responsible for the observed non-uniform helical twists both in A and B type helical backbones. That these need not be restricted to oligodeoxynucleotides but may be a feature of oligoribonucleotides backbone also is shown from an analysis of helical segments of yeast tRNA(Phe). A proposal of a unified or a grand two dimensional conformational plot which would help visualise succinctly the overall effect of the variations in all the repeating six chemical bonds of a polynucleotide backbone is made. Apart from considerable simplification, the plot affords identification on it regions characteristic of helical, and loop and bend conformations of nucleic acid backbone chain.     

Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7.

Helicases that unwind DNA at the replication fork are ring-shaped oligomeric enzymes that move along one strand of a DNA duplex and catalyze the displacement of the complementary strand in a reaction that is coupled to nucleotide hydrolysis. The helicase domain of the replicative helicase-primase protein from bacteriophage T7 crystallized as a helical filament that resembles the Escherichia coli RecA protein, an ATP-dependent DNA strand exchange factor. When viewed in projection along the helical axis of the crystals, six protomers of the T7 helicase domain resemble the hexameric rings seen in electron microscopic images of the intact T7 helicase-primase. Nucleotides bind at the interface between pairs of adjacent subunits where an arginine is near the gamma-phosphate of the nucleotide in trans. The bound nucleotide stabilizes the folded conformation of a DNA-binding motif located near the center of the ring. These and other observations suggest how conformational changes are coupled to DNA unwinding activity.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin.

In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.     

Anti-insulin antibody structure and conformation. I. Molecular modeling and mechanics of an insulin antibody.

A knowledge-based three-dimensional model of an anti-insulin antibody, 125, was constructed using the structures of conserved residues found in other known crystallographic immunoglobulins. Molecular modeling and mechanics were done with the 125 amino acid sequences using QUANTA and CHARMm on a Silicon Graphics 4D70GT workstation. A minimal model was made by scaffolding using crystallography coordinates of the antibody HyHEL-5, because it had the highest amino acid sequence homology with 125 (84% light chain, 65% heavy chain). The three hypervariable loop turns that are longer in 125 than in HyHEL-5 (L1, L3, and H3) were modeled separately and incorporated into the HyHEL-5 structure; then other amino acid substitutions were made and torsions optimized. The 125 model maintains all the structural attributes of an antibody and the structures conserved in known antibodies. Although there are many polar amino acids (especially serines) in this site, the overall van der Waals surface shape is determined by positions of aromatic side chains. Based on this model, it is suggested that hydrogen bonding may be key in the interaction between the human insulin A chain loop antigenic epitope and 125.     

Backbone Conformation in Nucleic Acids: An Analysis of Local Helicity through Heminucleotide Scheme and a Proposal for a Unified Conformational Plot

Abstract

A relationship has been established to express the local helicity of a polynucleotide backbone directly in terms of the virtual bonds spanning the conformationally equivalent heminucleotide repeats, with a view to provide a better understanding of the cumulative effects of all the chemical bond rotational variations on local helicity. Using this, an analysis made with a few oligodeoxynucleotide crystal structures clearly brings forth that it is the concerted movements manifested in the near neighbour correlations between the pair of chemical bonds C4′—C5′ and P—05′ and C4′-C3′ and P-03′ of the 5′ and 3′ heminucleotides respectively that are primarily responsible for the observed non-uniform helical twists both in A and B type helical backbones. That these need not be restricted to oligodeoxynucleotides but may be a feature of oligoribonucleotides backbone also is shown from an analysis of helical segments of yeast tRNA^Phe. A proposal of a unified or a grand two dimensional conformational plot which would help visualise succinctly the overall effect of the variations in all the repeating six chemical bonds of a polynucleotide backbone is made. Apart from considerable simplification, the plot affords identification on it regions characteristic of helical, and loop and bend conformations of nucleic acid backbone chain.     

Stereochemical effects of non-ionic methylphosphonates on nucleotide conformations.

The preferred conformations of deoxyribo and ribonucleoside 3'-methylphosphonates are analysed by minimizing the conformational energy as a function of all the major parameters including the sugar ring for both the S- and R-isomers. The results show that neither the substitution nor the nature of the diastereomer affects significantly the preferred conformations compared to the naturally occurring nucleoside 3'-phosphates. The preferred range of C3'-O3' bond torsions or the phase angles of pseudorotation (P) of the sugar are unaffected. The chiral substitution on the phosphate always adopts a conformation distal to the secondary C3' carbon atom in the minimum energy conformational state. Further, it introduces certain restrictions on the preferred range of P-O3' torsions depending on the methylphosphonate configuration. Methylphosphonate, especially the S-isomer, renders the normal gauche- range of P-O3' bond torsions responsible for the stacked helical duplexes to be energetically unfavourable besides introducing a high energy barrier between trans and gauche conformations. Therefore it is suggested that duplexes with S-methylphosphonate may favour extended phosphodiester conformations. These factors explain the observed lower melting temperature as well as the downfield shifts in the 31P signals in duplexes containing the S-isomer.     

Structural propensities in the heme binding region of apocytochrome b5. I. Free peptides

The water-soluble domain of rat microsomal cytochrome b5 is a small globular hemoprotein. Under native conditions, the apoprotein consists of a well-folded hydrophobic core and a 42-residue loop, which is substantially disordered in solution. Association with the heme cofactor causes the loop to organize into a second well-folded hydrophobic core encompassing four short helices, H2-H5. Of these, H3 and H4 are recognized as intrinsically disordered by algorithms that analyze primary structures for folding propensities. Three peptides, spanning H2-H5, H2-H3, and H4-H5, were designed, synthesized, and characterized to identify local structural preferences in the isolated loop. In addition, two replacements (D60R and N57P, which are known to stabilize holocytochrome b5) were introduced individually in the H4-H5 peptide. Helical content measured by nuclear magnetic resonance and far-UV circular dichroism spectroscopy in solutions of 2,2,2-trifluoroethanol revealed that H4 possessed a lower propensity to form the holoprotein structure than H3. Both replacements in H4 resulted in measurable changes in observed overall helical propensities. It was concluded that the prediction of intrinsic disorder was reliable. Furthermore, the stability of the holoprotein did not correlate simply with helical propensities in the disordered regions.     

The Effect of End Constraints on Protein Loop Kinematics

Despite the prevalent involvement of loops in function little is known about how the constraining of end groups influences their kinematics. Using a linear inverse-kinematics approach and assuming fixed bond lengths, bond angles, and peptide bond torsions, as well as ignoring molecular interactions to assess the effect of the end-constraint only, it is shown that the constraint creates a closed surface in torsion angle space. For pentapeptides, the constraint gives rise to inaccessible regions in a Ramachandran plot. This complex and tightly curved surface produces interesting effects that may play a functional role. For example, a small change in one torsion angle can radically change the behavior of the whole loop. The constraint also produces long-range correlations, and structures exist where the correlation coefficient is 1.0 or −1.0 between rotations about bonds separated by >30 Å. Another application allows some torsion angles to be targeted to specified values while others are constrained. When this application was used on key torsions in lactate dehydrogenase, it was found that the functional loop first folds forward and then moves sideways. For horse liver alcohol dehydrogenase, it was confirmed that the functional loop's Pro-Pro motif creates a rigid arm in an NAD-activated switch for domain closure.     

Design and initial characterization of a circular permuted variant of the potent HIV-inactivating protein cyanovirin-N.

A circular permuted variant of the potent human immunodeficiency virus (HIV)-inactivating protein cyanovirin-N (CV-N) was constructed. New N- and C-termini were introduced into an exposed helical loop, and the original termini were linked using residues of the original loop. Since the three-dimensional structure of wild-type cyanovirin-N is a pseudodimer, the mutant essentially exhibits a swap between the two pseudo-symmetrically related halves. The expressed protein, which accumulates in the insoluble fraction, was purified, and conditions for in vitro refolding were established. During refolding, a transient dimeric species is also formed that converts to a monomer. Similar to the wild-type CV-N, the monomeric circular permuted protein exhibits reversible thermal unfolding and urea denaturation. The mutant is moderately less stable than the wild-type protein, but it displays significantly reduced anti-HIV activity. Using nuclear magnetic resonance spectroscopy, we demonstrate that this circular permuted monomeric molecule adopts the same fold as the wild-type protein. Characterization of these two architecturally very similar molecules allows us to embark, for the first time, on a structure guided focused mutational study, aimed at delineating crucial features for the extraordinary difference in the activity of these molecules.     

Insect wax secretion the growth of tubular crystals

Filaments of a pure long-chain wax secreted by the wooly alder aphid are shown to represent a previously-uncharacterized, tubular, crystal habit for paraffins. Although this habit is never seen in solution or epitaxial growth of such materials, it requires only a circular closure at the wax secretion site, further growth behavior being controlled totally by van der Waal's intermolecular interactions. The linear paraffin-like wax molecules pack in the filament monolayer envelope with chain axes oriented radially from the filament axis. Elongation of the wax filaments corresponds to the strong interactions along the [110] vectors in the O_⊥ polymethylene chain packing, and stabilization of the filament occurs via strong interchain interactions along $\text{[1}\text{1}\text{0]}$ and [010] in helical paths of opposite chirality.