Helical Sequences Research Articles

An experimental approach to testing modular evolution: directed replacement of alpha-helices in a bacterial protein.

We have used oligonucleotide site-directed mutagenesis to ask whether certain structural motifs in proteins are determined mainly by local interactions among amino acids. Multiple consecutive amino acids in three alpha-helices in the alkaline phosphatase (EC 3.1.3.1) of Escherichia coli have been replaced with helical sequences from four other sources. Altogether, 12 distinct helical replacements were created, 9 of which retain enzymatic activity. Most short stretches of helical sequence can be replaced with unrelated helical sequences without eliminating enzyme activity. Replacements of the carboxyl half of an alpha-helix are less harmful than those of the amino half, and the two together are synergistic rather than additive. These results are consistent with the hypothesis that proteins originally evolved by the assembly of small functional folding units.

Hydrophobic organization of membrane proteins.

Membrane-exposed residues are more hydrophobic than buried interior residues in the transmembrane regions of the photosynthetic reaction center from Rhodobacter sphaeroides. This hydrophobic organization is opposite to that of water-soluble proteins. The relative polarities of interior and surface residues of membrane and water soluble proteins are not simply reversed, however. The hydrophobicities of interior residues of both membrane and water-soluble proteins are comparable, whereas the bilayer-exposed residues of membrane proteins are more hydrophobic than the interior residues, and the aqueous-exposed residues of water-soluble proteins are more hydrophilic than the interior residues. A method of sequence analysis is described, based on the periodicity of residue replacement in homologous sequences, that extends conclusions derived from the known atomic structure of the reaction center to the more extensive database of putative transmembrane helical sequences.

A molecular modeling study on binding of drugs to calmodulin

Computer-graphical methods have been used to study the interaction between a series of drugs and calmodulin. Based on the X-ray crystallographic coordinates of the alpha-C atoms of calmodulin, a molecular model of the helical sequences was built. The model has been used to derive two possible binding sites for phenothiazines and one binding site for penfluridol. The principal binding forces occur through contacts between acidic amino acids of calmodulin and the protonated side-chain nitrogen of the drugs as well as between a basic amino acid and the electronegative substituents of the aromatic rings. Calculated interaction energies show a good correlation with experimental inhibition data.

Electro-optical studies of various stereostructural forms of poly( N-vinylcarbazole)

The electro-optical Kerr effect has been measured for various stereostructural forms of poly( N-vinylcarbazole) (PVK) in solution in 1,4-dioxan at 298 K. The electro-optic behaviour of various types of PVK, prepared using different catalyst systems, may be explained in terms of 3 1 helical sequences of isotactic PVK and all- trans sequences of syndiotactic PVK. The Kerr effect of 2 1 and 4 1 helical forms of syndiotactic PVK has also been considered.

The binding of heparin to type IV collagen: domain specificity with identification of peptide sequences from the α1(IV) and α2(IV) which preferentially bind heparin

Three distinctive heparin-binding sites were observed in type IV collagen by the use of rotary shadowing: in the NC1 domain and at distances 100 and 300 nm from the NC1 domain. Scatchard analysis indicated different affinities for these sites. Electron microscopic analysis of heparin-type IV collagen interaction with increasing salt concentrations showed the different affinities to be NC1 greater than 100 nm greater than 300 nm. The NC1 domain bound specifically to chondroitin/dermatan sulfate side chains as well. This binding was observed at the electron microscope and in solid-phase binding assays (where chondroitin sulfate could compete for the binding of [3H]heparin to NC1-coated substrata). The triple helix-rich, rod-like domain of type IV collagen did not bind to chondroitin/dermatan sulfate side chains. In solid-phase binding assays only heparin could compete for the binding of [3H]heparin to this domain. In order to more precisely map potential heparin-binding sites in type IV collagen, we chemically synthesized 17 arginine- and lysine-containing peptides from the alpha 1(IV) and alpha 2(IV) chains. Three peptides from the known sequence of the alpha 1(IV) and alpha 2(IV) chains were shown to specifically bind heparin: peptide Hep-I (TAGSCLRKFSTM), from the alpha 1(NC1) chain, peptide Hep-II (LAGSCLARFSTM), a peptide corresponding to the same sequence in peptide Hep-I from the alpha 2 (NC1) chain, and peptide Hep-III (GEFYFDLRLKGDK) which contained an interruption of the triple helical sequence of the alpha 1(IV) chain at about 300 nm from the NC1 domain, were demonstrated to bind heparin in solid-phase binding assays and compete for the binding of [3H]heparin to type IV collagen-coated substrata. Therefore, each of these peptides may represent a potential heparin-binding site in type IV collagen. The mapping of the binding of heparin or related structures, such as heparan sulfate proteoglycan, to specific sequences of type IV collagen could help the understanding of several structural and functional properties of this basement membrane protein as well as interactions with other basement membrane and/or cell surface-associated macromolecules.

Covalent interactions of type IX collagen in cartilage.

The cross-linking of type IX collagen in fetal bovine cartilage was investigated. The main cross-link was dihydroxy-lysinonorleucine (borohydride-reduced) with a lesser amount of the mature cross-link, pyridinoline. Dihydroxylysinonorleucine was present in all three chains of the COL2 domain of the type IX molecule, but only two of them contained pyridinoline even in mature cartilage. Amino acid sequence analysis of individual tryptic peptides that contained 3H-labeled cross-links showed that they had derived from sites of covalent interaction between type IX collagen and the telopeptide sequences of type II collagen. One two-chained peptide was a helical sequence of alpha 2 (IX) COL2 linked to an alpha 1 (II) N-telopeptide. A second peptide was a different helical sequence from another type IX chain linked to an alpha 1(II) c-telopeptide. This latter helical sequence was also the principal site of pyridinoline cross-linking in type IX collagen.

Drosophila basement membrane procollagen alpha 1(IV). II. Complete cDNA sequence, genomic structure, and general implications for supramolecular assemblies.

A Drosophila melanogaster gene for a basement membrane procollagen chain was recently identified from the sequence homology of the carboxyl (NC1) end of the polypeptide that it encodes with the corresponding domain of human and murine collagens IV (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). This gene is at chromosome location 25C. Here we report the complete 6-kilobase cDNA sequence coding for a chain of 1775 amino acids, as well as the genomic structure. The gene is composed of nine relatively large exons separated by eight relatively small introns. This organization is different from the multiple small exons separated by large introns reported for mouse and human type IV collagens (Kurkinen, M., Bernard, M. P., Barlow, D. P., and Chow, L. T. (1985) Nature 317, 177-179. Sakurai, Y., Sullivan, M., and Yamada, Y. (1986) J. Biol. Chem. 261, 6654-6657. Soininen, R., Tikka, L., Chow, L., Pihlajaniemi, T., Kurkinen, M., Prockop, D. J., Boyd, C. D., and Tryggvason, K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1568-1572). Drosophila and human alpha 1(IV) procollagen chains share not only polypeptide domains near their amino and carboxyl ends for making specialized, intermolecular junctional complexes, but also 11 of 21 sites of imperfections of the collagen triple helix. However, neither the number nor the nature of the amino acids in these imperfections appear to have been conserved. These imperfections of the helical sequence may be important for the supramolecular assembly of basement membrane collagen. The 9 cysteine residues of the Drosophila collagen thread domain are arranged as several variations of a motif found in vertebrate collagens IV only near their amino ends, in their "7 S" junctional domains. The relative positions of these cysteine residues provide numerous opportunities for disulfide bonding between molecules in both parallel and antiparallel arrays. There is a pseudorepeat of one-third of the thread length, and there are numerous possibilities for disulfide-linked microfibrils and networks. We propose that collagen microfibrils, stabilized by disulfide segment junctions, are a versatile ancestral form from which specialized collagen fibers and networks arose.

Biologically active calcitonin analogs which have minimal interactions with phospholipids

A number of peptide hormones have been shown to contain amphipathic helical segments capable of binding to phospholipids. This conformational feature has been associated with increased biological activity of these hormones. We demonstrate, however, that two calcitonin analogs, [Gly8,Ala16]-des-Leu19 salmon calcitonin and des-1-amino-[Ala1,7,Gly8]-des-Leu19 salmon calcitonin have minimal interactions with phospholipids. Neither of these peptides acquire any increased helical content in the presence of dimyristoylphosphatidylglycerol and these peptides have only weak effects in altering the phase transition properties of this lipid. Therefore, although the presence of a phospholipid-induced amphipathic helical sequence may enhance the biological activity, it is not required for activity.

Phyllotactic patterns: Characterization by geometrical activity at the formative region

Patterns of leaves or florets, phyllotaxis, have long been well characterized using parameters that describe the patterns as lattices of points or arrangements of contiguous circles. The patterns are usually interpreted in terms of spiral lines drawn through the points or circles. For example, in a pine cone the number of visible spiral lines, in two sets with opposing spiral sense, is typically a pair of adjacent numbers in the Fibonacci series. This format for characterization, while precise and terse for spiral Fibonacci patterns, has four disadvantages or limitations when applied generally. First, when the spiral notation is applied to obviously orthogonal patterns, such as decussate, it implies developmental chirality even though the pattern has no intrinsic handedness. Second, the classification has led to no convenient subgrouping, of all patterns: i.e., do all forms with one organ per node constitute a natural group? Third, the characterization is of a static pattern based on an assumed constant center of symmetry. Characterization is not related to the mode of formation of the patterns. Finally, the notation is not convenient for describing gradual developmental transitions between patterns. We find that a broad range of phyllotactic patterns can be conveniently characterized by diagrams, each derived from a single measurement of the apical surface that produces the organs. During each developmental cycle, this area expands to a maximum and then is abruptly reduced to a minimum by demarcation of a new leaf (or leaves). The center of area is generally different before and after demarcation, hence its movement during the demarcation event is a vector. We present a method for juxtaposing these vectors over consecutive plastochrons. The resulting planar figure is diagnostic of the broad categories of phyllotaxis. In “balanced” forms, whorled apices, there is no net movement of the center of area so the resulting figure is a point. In the second category, “linear”, the vectors reverse, retracing a line segment. Leaves alternating in a plane, distichous phyllotaxy, arise from this pattern. In the third category, “polygonal”, the juxtaposed vectors advance so as to circumscribe a polygon. Such plants (some Euphorbias ) generate leaves in helical sequence but the leaves stand in vertical ranks. In the fourth category, “circular” the vectors circumscribe a circle. This behavior is found in plants with irrational divergence angles and hence applies to the common Fibonacci spiral forms. The vectorial mode of primary categorization appears to lack the four listed shortcomings.

Probes for double helical DNA sequence information: molecular mechanics study of a proposed model.

The biological activity of many molecules which bind to double helical DNA is related to the sequence specificity of their binding. The development of new substances of this type is challenging; a general solution to the problem does not exist. A new mechanism for small molecule-duplex DNA interaction termed intercalative displacement is proposed. The approach is promising for the design of new substances which will recognize sequence information on DNA. Molecular mechanics calculations in the absence of solvent and counterions predict that the proposed model is structurally and energetically closely related to the well known process of standard intercalation. The implications of these calculations for experimental studies are discussed.

Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins

Protein segments that form amphipathic α-helices in their native state have periodic variation in the hydrophobicity values of the residues along the segment, with a 3.6 residue per cycle period characteristic of the α-helix. The assignment of hydrophobicity values to amino acids (hydrophobicity scale) affects the display of periodicity. Thirty-eight published hydrophobicity scales are compared for their ability to identify the characteristic period of α-helices, and an optimum scale for this purpose is computed using a new eigenvector method. Two of the published scales are also characterized by eigenvectors. We compare the usual method for detecting periodicity based on the discrete Fourier transform with a method based on a least-squares fit of a harmonic sequence to a sequence of hydrophobicity values. The two become equivalent for very long sequences, but, for shorter sequences with lengths commonly found in α-helices, the least-squares procedure gives a more reliable estimate of the period. The analog to the usual Fourier transform power spectrum is the “least-squares power spectrum”, the sum of squares accounted for in fitting a sinusoid of given frequency to a sequence of hydrophobicity values. The sum of the spectra of the α-helices in our data base peaks at 97.5 °, and approximately 50% of the helices can account for this peak. Thus, approximately 50% of the α-helices appear to be amphipathic, and, of those that are, the dominant frequency at 97.5 ° rather than 100 ° indicates that the helix is slightly more open than previously thought, with the number of residues per turn closer to 3.7 than 3.6. The extra openness is examined in crystallographic data, and is shown to be associated with the C terminus of the helix. The alpha amphipathic index, the key quantity in our analysis, measures the fraction of the total spectral area that is under the 97.5 ° peak, and is a characteristic of hydrophobicity scales that is consistent for different sets of helices. Our optimized scale maximizes the amphipathic index and has a correlation of 0.85 or higher with nine previously published scales. The most surprising feature of the optimized scale is that arginine tends to behave as if it were hydrophobic; i.e. in the crystallographic data base it has a tendency to be on the hydrophobic face of the amphipathic helix. Although the scale is optimal only for predicting α-amphipathicity, it also ranks high in identifying β-amphipathicity and in distinguishing interior from exterior residues in a protein. We factor the expressions for the power spectra into a matrix product so that the helical sequence information is isolated from the hydrophobicity scale. The largest eigenvalue of the matrix containing only helical sequence information also identifies the 97.5 ° frequency, thus confirming a 3.7 residue per turn spacing independently of hydrophobicity scales.

An hypothesis on the binding of an amphipathic, alpha helical sequence in Ii to the desetope of class II antigens.

Abstract When we investigated the hypothesis that amphipathic alpha helical peptides digested from foreign antigen bind to class II major histocompatability complex (MHC) molecules' binding site (desetope) for foreign antigen to be presented to T cell receptors, we found such an extended amphipathic helix in Ii. This amphipathic helix was hypothesized to bind Ii to class II MHC antigens until release in endosomes containing digested foreign antigen. Then these amphipathic Ii polypeptides might polymerize so as not to compete with foreign antigen for binding to class II MHC molecules. Various structural models were consistent with these views and led to the suggestion of specific forms of polymeric interaction.

Sequences that adopt non- B-DNA conformation in form V DNA as probed by enzymic methylation

pBR322 form V DNA is a highly torsionally strained molecule with a linking number of zero. We have used sequence-specific DNA methylases as probes for B-DNA in this molecule, exploiting the inability of methylases to methylate single-stranded DNA and Z-DNA, both of which are known to occur in form V DNA. Some sequences in form V DNA were shown to be totally in the B-form, others were totally in an altered, unmethylatable conformation, while still other sites appeared to exist partly in altered and partly in normal B-conformation. Some potential Z-forming sequences (alternating pyrimidine/purine) of less than seven base-pairs were not in the Z conformation in form V DNA, whereas others did adopt an altered structure, indicating a modulating influence of flanking sequences. Furthermore, regions of imperfect alternating pyrimidine/purine structure were sometimes capable of adopting an altered structure. In addition, some regions of altered structure had no apparent Z-forming sequences, nor were they in polypurine stretches, which have also been proposed to form left-handed DNA. These non- B-DNA conformations may represent novel left-handed helical structures or sequences that become single stranded under torsional strain. Long regions of either altered (unmethylatable) DNA or B-DNA were not always observed. In fact, one region showed three transitions between B-like DNA and altered structure within 26 base-pairs.

Surface-viewed shoot apex ofAngiopteris lygodiifolia Ros. (Marattiaceae)

Marattian ferns are thought to be an exception to the rule that a single apical cell is always present in the shoot apex of ferns; the occurrence of plural apical initials has been generally accepted for these ferns. However, a contradicting conclusion was reached in this study which examined the apical organization of the shoot ofAngiopteris lygodiifolia Ros., using fresh materials which had not been fixed. Shoot apices were hand-sectioned transversely into thin sections, including the surface layer of the shoot apex, which were observed by differential interference contrast microscopy without staining.In contrast with the generally accepted view, the shoot apex ofA. lygodiifolia was found to usually possess a single apical cell with three cutting faces. The segments cut off from the apical cell are regularly arranged in a helical sequence. The apical cell seems to actually function as an initial cell of the whole shoot apex. The shoot apices, particularly those of plants cultivated in a greenhouse, sometimes show somewhat irregular organization. In extreme cases, no apical cell is recognizable. However, even in these exceptional cases of such apparently irregular shoot apices, plural apical initials are not found.

Molecular organization of glycophorin A: implications for membrane interactions.

AbstractPhysical studies and conformational analysis of human glycophorin A suggest a revised model for its molecular organization, self‐association, and interactions with the erythrocyte membrane. Intrinsic viscosity has been used to study, under more physiological conditions, the monomer–dimer equilibrium demonstrated previously by polyacrylamide–SDS gel electrophoresis. The results show that the equilibrium persists in the absence of detergent and support earlier indications that the dimer is probably the physiologically relevant form and that it is promoted by salt, inhibited by conventional denaturants, and abolished by carboxymethylation.Combined application of CD, fitted to the poly‐(L‐lysine) model spectra of Greenfield and Fasman, and conformational prediction, by the statistical method of Chou and Fasman and the stereochemical approach of Lim, suggests five helical sequences in glycophorin A: Arg‐39 to Tyr‐52 (A); Gln‐63 to Glu‐70 (B); Glu‐72 to Leu‐89 (C); Ile‐95 to Lys‐101 (D); and Leu‐118 to Asn‐125 (E). Sequence A occurs only at low pH and may be stabilized by favorable noncovalent interactions of O‐linked tetrasaccharide side chains. The other four helices all occur in the dimeric form of glycophorin A at physiological pH and ionic strength. Sequence D is destroyed by trypsin, and is also lost on conversion to the monomeric form of the glycoprotein at low ionic strength. Sequence E is denatured by 6M guanidine hydrochloride/4M urea. Sequences B and C, which are separated by a single proline residue, are stable under all these conditions.Dimerization of the major, hydrophobic helical sequence, (C) may be promoted and directed by an adjacent short sequence of intermolecular parallel β‐sheet (Leu‐90 to Tyr‐93). It is proposed that these two structures span the lipid bilayer in vivo, and that helices B and D lie, respectively, along the outer and inner surfaces of the membrane. Molecular organization in the N‐ and C‐terminal regions of the molecule is discussed in terms of evidence from the present work and from other recent investigations.

Evolution of chick type I procollagen genes.

Although the major types of vertebrate collagen have a number of structural properties in common, significant DNA sequence homologies have not been detected between different portions of the helical coding domains within the same gene or between different genes. However, under non-stringen hybridization conditions we found considerable cross-homology within and between alpha 1(I) and alpha 2(I) chick cDNAs in the coding regions for helical sequences. Detailed analyses at the DNA sequence level have led us to propose that the gene for chick pro alpha 2(I) collagen arose from a 9-bp primordial sequence. A consensus sequence for the 9-bp repeat was derived: GGTCCTCCT, which codes for a Gly-Pro-Pro triplet. The primordial ancestor of this 9-bp unit, GGTCCTXCT, apparently underwent duplication and divergence. Each resulting 9-bp sequence was triplicated to form a 27-bp domain, and a condensation event produced a 54-bp domain. This genetic unit then underwent multiple rounds of amplification to form the ancestral gene for the full-length helical section of alpha 2(I). A different 9-bp consensus sequence (GGTCCCCCC) seems to have been the basis of the chick pro alpha 1(I) gene.

The specificity of antibodies elicited by poly(dG).poly(dC).

Antibodies have been raised to the synthetic DNA polymer poly(dG).poly(dC). These antibodies have the ability to distinguish this right-handed polymer from natural mixed sequence DNA, as well as from other right- and left-handed synthetic DNA polymers. They show reduced but measurable binding to synthetic polymers which contain various combinations of guanine and cytosine polynucleotides suggesting that both helical shape and sequence are recognized by this antiserum.

Scattering functions of poly(amino acid) in helix-coil transition

Scattering functions of poly(amino acid) in the helix-coil transition region have been calculated on the basis of the Zimm-Bragg theory, to examine the effects of the helical content on the scattering function and on the cross-section factor, from which the radius of gyration of the cross section and the mass per unit length of the helical sequence can be obtained. The molecule is assumed to contain only one α-helical sequence of any length, leaving unfolded coils at the chain ends. The statistical mechanical averages of the interference among scattering units, apart from the interference within the helical sequence, are approximated by the expansion in terms of the even moments up to the eighth moment, which are calculated by the generator matrix method based on the conformation energies computed for poly ( l-alanine). The scattering function at intermediate angles varies systematically according to the variation of the helical content, demonstrating the sensitivity of intermediate-angle scattering to the conformational changes in the helix-coil transition. The comparison of the Guinier plots of the cross-section factors, calculated from the scattering functions at various helical contents, has indicated that the radius of gyration of the cross section of the helical sequence can be determined accurately from the plots even when the coil content is as much as 30%, and that the mass per unit length is determinable to within an error of 6% unless the coil content exceeds 10%.

Dimensional Properties of Polypeptides in the Helix–Coil Transition Region I. Molecular Dimension

An expression was derived to correlate the unperturbed mean-square radius of gyration of an intermediate polypeptide chain ‹S2›oI with helical content fH (see eq 10). An unperturbed state corresponding to the α-helix was obtained by converting the helix to a tortuous random-coil with a bond-rotation angle of +100° or −100° (in cis-convention) per residue, or per “virtual bond,” and a bond angle satisfying the experimental value of 8.8 for the characteristic ratio of the random-coil of PBLG. The “quasi” linear expansion factor αh of a helical sequence in an intermediate chain was evaluated (see eq 18) using the Zimm–Bragg–Nagai theory in collaboration with our expression for ‹S2›o. The intermediate chain may be a kind of swollen random-coil owing to the existence of helical sequences in the chain, and thus, the linear expansion factor αsI of the entire intermediate chain was approximately formulated as αsI=αsCαh where αsC is the linear expansion factor for the radius of gyration of a random-coil. With αsI and ‹S2›oI, the mean-square radius of gyration ‹S2›I of polypeptides in the transition region was calculated (eq 12) and compared with that obtained experiment (see Figure 4). Our theoretical results were found to agree well with experiment.

The effect of cooling rate upon the morphology of quenched melts of isotactic polypropylenes

Experimental data are provided on the molecular structure of paracrystalline samples of quenched melts (glasses). We propose that rods of helical molecules are present in the ‘glasses’, their length being sensitive to the temperature of the melt from which the ‘glass’ is produced, the amount of time held in the melt and the rate of quenching. A comparison is made between samples crystallized from the melt and those crystallized via the glassy phase. By measuring the lamellar thickness, from Raman spectroscopy, and calculating the absorbance ratio A 998 A 973 from infra-red spectroscopy, longer helical sequences were shown to exist to a greater extent in the samples crystallized from the melt. Samples crystallized from the glassy phase contained shorter helical sequences. In investigating the effect of the surrounding temperature on solid polypropylenes, we have shown that the density of the material can be related to the temperature of storage. We comment on the relevance of this observation to dimensional stability in mouldings.