Alpha-helical Secondary Structure Research Articles

The T‐Lymphocyte Proliferative Response to Synthetic Peptide Antigens of Defined Secondary Structure

Immunodominant sites in proteins recognized by T lymphocytes are segments consisting of at least 7-8 amino acids. It has previously been proposed that these sites in proteins are alpha-helical and amphipatic structures. We synthesized and investigated the immunogenicity of three synthetic peptides (MP7, MP8, and MP9), each consisting of the same 15 amino acids, but differing with respect to sequence. Based on information analysis and circular dichroism measurements, MP7 was shown to have an alpha-helical secondary structure and, based on previously assigned hydrophilicity indices, was also strongly longitudinally amphipatic. MP8 also was conformed as an alpha-helix, but was amphipatic in the sense that the N-terminal half of the molecule was hydrophilic and the C-terminal half hydrophobic. MP9 had neither an amphipatic nor alpha-helical structure. All three peptides were immunogenic in some strains of mice but none was immunogenic in all strains. This supports other studies concluding that amphipaticity per se is neither a necessary nor sufficient requirement for immunogenicity of a peptide. On the other hand, the present experimental data suggest that longitudinally amphipatic alpha-helical peptides may function better as T-cell determinants than the other peptides investigated.

CDNA sequence, predicted primary structure, and evolving amphiphilic helix of human aspartyl-tRNA synthetase

Eight of the mammalian aminoacyl-tRNA synthetases associate as a multienzyme complex, whereas prokaryotic and low eukaryotic synthetases occur only as free soluble enzymes. Association of the synthetases may result in effective compartmentalization of synthetases and suggests the association of the entire protein biosynthetic machinery. To elucidate the structural elements and the nature of the molecular interactions involved in the association of the synthetases, we have cloned and sequenced the complementary DNA coding human aspartyl-tRNA synthetase. The full length cDNA encodes an open reading frame of 500 amino acids with 56% identity with yeast aspartyl-tRNA synthetase. The similarity with yeast aspartyl-tRNA synthetase is unevenly distributed with a high percent of identity at the C-terminus and relatively low identity at the N-terminus. The N-terminal sequence strongly prefers an alpha-helical secondary structure and shows amphiphilic characteristics. Further comparison with the yeast synthetases showed that the basic positively charged helixes in yeast synthetases are evolved to a neutral amphiphilic helix in this mammalian synthetase. The mammalian neutral amphiphilic helix is so far unique among all known sequences of bacterial, yeast, and mammalian synthetases and may account for the association of synthetases in the synthetase complex.

Terminal regions of flagellin are disordered in solution

Limited proteolysis of flagellin from Salmonella typhimurium SJW1103 by subtilisin, trypsin and thermolysin results in homologous degradation patterns. The terminal regions of flagellin are very sensitive to proteolysis. These parts are degraded into small oligopeptides at the very early stage of a mild digestion that yields a relatively stable fragment with a molecular weight of 40,000. Further proteolytic degradation results in a stable 27,000 Mr fragment. The 40,000 Mr tryptic fragment has been identified as residues 67 to 446 of the flagellin sequence, while the 27,000 Mr fragment involves the 179 to 418 segment. The NH2-terminal sequence positions for the corresponding fragments produced by subtilisin are 60 and 174 for the 40,000 Mr and 27,000 Mr fragments, respectively. The fragments lost their polymerizing ability. Structural properties of flagellin and its 40,000 Mr tryptic fragment were compared by circular dichroism spectroscopy and differential scanning calorimetry. Analysis of the calorimetric melting profiles suggests that terminal parts of flagellin have no significant internal stability and they are in extensive contact with water. However, these regions contain some secondary structure, probably alpha-helices, as revealed by comparison of the circular dichroic spectra in the far-ultraviolet region. Our results indicate that, although the terminal regions of flagellin may contain some alpha-helical secondary structure of marginal stability, they have no compact ordered tertiary structure in solution. On the contrary, the central region of the molecule involves at least two compact structural units.

Functional analysis of the antigenic structure of a minor T cell determinant from pigeon cytochrome C. Evidence against an alpha-helical conformation.

A minor T cell determinant from pigeon cytochrome c, composed of residues 43 to 58 (p43-58), was synthesized along with a series of 48 analogs containing amino or carboxyl-terminal deletions or single amino acid substitutions. These peptides were analyzed functionally for their ability to elicit unique T cell populations on immunization of C57BL/10 mice and to stimulate a degenerate T cell clone capable of recognizing p43-58 in association with two different Ia molecules, A beta b:A alpha b and A beta d:A alpha d. These experiments allowed us to identify the residues in the determinant that are critical for T cell activation. Residues 50 and 52 had the dominant influence on T cell specificity, and residues 47, 48, 49, 51, and 53 had weak effects. Residues 46 and 54 were hardly recognized by the TCR at all, but appeared to influence the potency of the determinant by interacting with the Ia molecule. Finally, substitutions at positions 55 to 58 had no effect, but removal of these residues reduced the potency of the peptide, suggesting a contribution from the peptide backbone of this part of the molecule during T cell activation. An analysis of the spatial relationship of these dominant epitopic and agretopic residues suggests that this determinant does not assume a pure alpha-helical secondary structure when bound to the Ia molecule.

Evolutionary relationship between the TonB-dependent outer membrane transport proteins: nucleotide and amino acid sequences of the Escherichia coli colicin I receptor gene

The nucleotide sequence of the Escherichia coli colicin I receptor gene (cir) has been determined. The predicted mature protein consists of 599 amino acids and has a molecular weight of 67,169. Several previously noted characteristics of other E. coli outer membrane protein sequences were also identified in the sequence of Cir. These include an overall acidic nature, the absence of long hydrophobic stretches of amino acids, and a lack of predicted alpha-helical secondary structure. Because two classes of outer membrane proteins (the TonB-dependent transport proteins and the porins) share some structural features, protein sequences from both of these groups were aligned pairwise and scored for sequence similarity. Statistical evidence suggested that the porins were not related to the proteins in the TonB-dependent group; however, there was a significant relationship between the proteins in the TonB-dependent group. On the basis of the multiple progressive sequence alignment and the similarity scores derived from it, a tree representing evolutionary distance between five TonB-dependent outer membrane transport proteins was generated.

Biosynthesis of winter flounder antifreeze proprotein in E.coli.

A semisynthetic winter flounder antifreeze proprotein (proAFP) coding region was constructed and inserted into a lacZ expression vector. ProAFP was produced from the vector in Escherichia coli as a C-terminal fusion to the first 289 amino acids of beta-galactosidase (beta-gal). The proAFP and beta-gal domains of the beta-gal-proAFP fusion protein were separated by the recognition signal for the blood coagulation protease, factor Xa. Upon induction with isopropylthio-beta-D-galactoside the fusion protein accumulated to levels of 15% of the total protein. The beta-gal-proAFP fusion protein was partially purified by differential centrifugation, but required solubilization prior to factor Xa digestion. The solubilized fusion protein was efficiently and correctly cleaved by factor Xa, after which the proAFP was purified by gel permeation. Bacterial proAFP was indistinguishable from natural proAFP by the criteria of antifreeze activity, amino-terminal sequence (15 cycles), reverse-phase HPLC and SDS-polyacrylamide gel electrophoresis. Circular dichroism measurements showed that proAFP is a composite of random coil and alpha-helical secondary structure, with an alpha-helix content of 44% at 0 degrees C. It seems probable that the C-terminal region of proAFP, which corresponds to the mature AFP protein, is mainly alpha-helical, and that the N-terminal pro-segment is random coiled.

Biological properties of two models of calcitonin gene related peptide with idealized amphiphilic alpha-helices of different lengths.

Previous studies on calcitonin gene related peptide (CGRP) have demonstrated that it has the characteristics of an amphiphilic peptide, and from an examination of the sequence, we have proposed that it contains an amphiphilic alpha-helix. We have synthesized two analogues of CGRP which have different lengths of idealized amphiphilic alpha-helical secondary structure. The first model, CGRM-1, has been substituted with residues generating an idealized amphiphilic alpha-helix in the region between residues 8 and 25, equivalent to approximately five turns of an alpha-helix. This peptide is not an agonist in any of our bioassays, but it does bind with low affinity to rCGRP receptors in crude liver membranes. Our second model, CGRM-2, has an idealized amphiphilic alpha-helix between residues 8 and 18, which is equivalent to approximately three turns of an alpha-helix. In an in vitro rat vas deferens assay, this peptide is an agonist with a potency one-fourth that of the native hormone. However, the potency of CGRM-2 in an adenylate cyclase assay is much lower, only 1/140th the potency of CGRP. Both model peptides display amphiphilic characteristics commensurate with their design. We conclude that there is an amphiphilic alpha-helix in rCGRP between residues 8 and 18 and that this helix terminates in the vicinity of residue 18.

The activation of pigeon cytochrome c-specific T cell hybridomas by antigenic peptides is influenced by non-native sequences at the amino terminus of the determinant.

We recently demonstrated that the sequence 95-104 contains all the residues necessary for direct recognition of the I-Ek restricted pigeon cytochrome c determinant but that residues located in sequences to the amino-terminal side of residue 95 improve the ability of peptides containing the sequence 95-104 to stimulate Ag-specific T cell clones. In this study we use synthetic peptides with amino-terminal leader sequences containing residues that differ with respect to their conformational stabilizing effects, charge, and hydrophilicity to examine the mechanism by which they modulate T cell recognition. Our findings indicate that the role of these residues in T cell stimulation is not related to their ability to stabilize alpha-helical secondary structure, nor do they appear to be processed differently. The leader sequences do not differentially influence the ability of the peptides to be presented by APC displaying Ia molecules of related haplotype, i.e., E alpha kE beta k, E alpha kE beta b, and E alpha kE beta s, to T cells which recognize the pigeon cytochrome c determinant on such presenting cells. Because antigenic potency correlates with the inclusion of hydrophobic residues and positively charged residues in the leader sequences, we discuss our findings with reference to the possibility that they non-specifically enhance the interaction of the antigenic peptides with the APC membrane.

Attenuated total reflectance Fourier transform infrared studies of the interaction of melittin, two fragments of melittin, and delta-hemolysin with phosphatidylcholines.

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystals and a low resolution structure of interleukin-2.

Recombinant derived human interleukin-2 and an analog in which cysteine 125 has been replaced with alanine have been crystallized in a form suitable for x-ray diffraction. The crystals are triclinic, space group P1, with two protein molecules in the unit cell; unit cell parameters are a = 55.8 A, b = 40.1 A, c = 33.7 A, alpha = 90.0 degrees, beta = 109.3 degrees, gamma = 93.2 degrees. The interleukin-2 structure has been solved to 5.5 A resolution using heavy atom isomorphous replacement methods. The resultant low resolution model reveals a significant fraction of alpha helical secondary structure and outlines the overall tertiary structure of the molecule.

Carboxyl-terminal sequence of entactin deduced from a cDNA clone.

Entactin is a widely distributed basement membrane sulfated glycoprotein of approximately equal to 150 kDa. The entactin gene is expressed early in mouse embryogenesis. Two cDNA clones complementary to rat entactin mRNA were isolated by antibody screening of an oligo(dT)-primed cDNA library constructed in the lambda gt11 expression vector. One of the clones, lambda 1E, was subcloned into plasmid pBR322 and further characterized. The clone contained sequences complementary to an mRNA species 6 kilobases in length. This mRNA was translated in rabbit reticulocyte lysates to yield a polypeptide of 143 kDa that was precipitated with anti-entactin antiserum. The cDNA insert, 1328 base pairs long, was sequenced and found to contain an open reading frame of 729 base pairs that coded for 243 amino acids at the carboxyl terminus of entactin. Analysis of the peptide revealed no extended alpha-helical or beta-sheet secondary structures. Radiolabeled probes prepared by nicktranslation of p lambda 1E were used to monitor the steady-state levels of entactin mRNA in F9 embryonal carcinoma cells that were induced to differentiate by exposure to retinoic acid and dibutyryl cyclic AMP. The increase in steady-state levels of entactin mRNA lagged behind the increase in mRNA for the B2 chain of laminin, suggesting that laminin and entactin are independently rather than coordinately regulated.

Repetition as the essence of life on this earth: music and genes.

In prebiotic nucleic acid replication, templates appear to have been in short supply. A single round of tandem duplication of existing oligomers assured progressive extension of templates to the length adequate for encoding of polypeptide chains. Thus, the first set of coding sequences had to be repeats of base oligomers encoding polypeptide chains of various periodicities. On one hand, the readiness of these periodical polypeptide chains to assume alpha-helical and/or beta-sheet secondary structures contributed to the extremely rapid initial functional diversification of these polypeptide chains. It would be recalled that most, if not all, of the sugar-metabolizing enzymes had already achieved the inviolable functional competence before the division of prokaryotes from eukaryotes. On the other hand, a certain (dipeptidic?) of the peptidic periodicities was apparently chosen as the timekeeping unit by the biological clock. Musical compositions too apparently evolved originally as a timekeeping device. Accordingly, repetitiousness is evident in all musical compositions. Evolution of musical compositions from the early Baroque to the late Romantic parallels that of coding sequences from rather exact repeats of base oligomers to more complex modern coding sequences in which repetitious elements are less conspicuous and more varied. Inasmuch as the earth is governed by the hierarchy of periodicities (days, months and years), such reliance on periodicities is rather expected.

Structural analysis of acute-phase α2-macroglobulin

High resolution images of rat acute-phase alpha 2-macroglobulin (AP alpha 2M) have been obtained by using dark-field electron microscopy. No staining or artifact-inducing procedures were used. Analysis of unfiltered electron microscope plates, exposed to minimal electron beam radiation, revealed highly contrasted particles of variable morphology with dimensions of approx. 19 nm X 14 nm. An electron-dense core with four to six projections could be seen. Two-fold symmetry was evident in selected images, supporting the four-subunit composition of the protein. Image processing and filtering confirmed the presence and configuration of the projections by demonstrating exact molecular dimensions of 16 nm X 9.5 nm and a shape with six projections like that of the Russian letter zh. SDS/polyacrylamide-gel electrophoresis revealed that this molecule was in the proteinase-bound form. C.d. data revealed a surprisingly low content of alpha-helical secondary structure (12%) and an atypically large content of beta-form structure (33%). Comparison of the amino acid compositions of AP alpha 2M and human alpha 2-macroglobulin indicated a high degree of homology between the two molecules. It is concluded that the conformation of rat AP alpha 2M, both at the molecular and secondary structural levels, is strikingly similar to that of human alpha 2-macroglobulin.

Affinity of nonhomologous amphiphilic peptides toward a monoclonal antibody raised against apolipoprotein A-I.

Monoclonal antibodies against human apolipoprotein A-I (apoA-I) were generated by the hybridoma technique. Clone G-10 was selected on the basis of its highest titer. The affinity of this antibody toward a series of synthetic peptides differing in length, amino acid composition, and amphiphilicity was tested by using both the indirect and the competitive enzyme-linked immunosorbent techniques (ELISA). From these measurements we calculated dissociation constants of the complexes of the antibody with apoA-I bound to the surface of the microtiter plate, apoA-I in solution, and any of the several peptides in solution. The dissociation constant (Kd) of the immobilized apoA-I/anti-apoA-I-complex, Kd = 2 x 10(-9) M, was significantly lower than that of the complex resulting from the interaction between anti-apoA-I and either apoA-I in solution or any of the several amphiphilic helical peptides in solution. Peptides devoid of amphiphilic secondary structure were inert. These data are consistent with the proposal that monoclonal G-10 recognizes in antigenic peptides an alpha-helical secondary structure of defined hydrophilic-lipophilic balance and comparatively less the specific amino acid side chains. We propose that the highest contribution to the free energy of binding (8 Kcal/mole) is derived from the docking of the helix to the antibody. It follows that in probing the specificity of a monoclonal antibody the conformation and the physical environment of the interacting antigen must be taken into account.

Alpha-helical coiled-coil structures of Trypanosoma brucei variable surface glycoproteins.

We have used electron microscopy to examine purified intact variable surface glycoproteins (VSGs) from clones derived from two distinct stocks of Trypanosoma brucei. The VSG molecule from MITat 1.2 has a large elongated domain consistent with the shape of the dimeric N-terminal domain determined by X-ray analysis (see preceding paper), and a heretofore unseen short, thin fibrous tail presumed to be the C-terminal domain. Electron microscopy on DiTat 1.3, however, indicates a morphology quite distinct from that of MITat 1.2. Analysis of four VSG amino acid sequences reveals 7-fold periodicities (heptad repeats) which indicate that alpha-helical coiled-coil secondary structure elements occur in all of these VSGs, consistent with the observation of helical bundles in one VSG. These results suggest the possibility that VSG antigenic diversity may be related to a diversity in length and disposition of alpha-helical bundles and coiled-coil domains.

Repeats of base oligomers as the primordial coding sequences of the primeval earth and their vestiges in modern genes.

Three outstanding properties uniquely qualify repeats of base oligomers as the primordial coding sequences of all polypeptide chains. First, when compared with randomly generated base sequences in general, they are more likely to have long open reading frames. Second, periodical polypeptide chains specified by such repeats are more likely to assume either alpha-helical or beta-sheet secondary structures than are polypeptide chains of random sequence. Third, provided that the number of bases in the oligomeric unit is not a multiple of 3, these internally repetitious coding sequences are impervious to randomly sustained base substitutions, deletions, and insertions. This is because the recurring periodicity of their polypeptide chains is given by three consecutive copies of the oligomeric unit translated in three different reading frames. Accordingly, when one reading frame is open, the other two are automatically open as well, all three being capable of coding for polypeptide chains of identical periodicity. Under this circumstance, a frame shift due to the deletion or insertion of a number of bases that is not a multiple of 3 fails to alter the down-stream amino acid sequence, and even a base change causing premature chain-termination can silence only one of the three potential coding units. Newly arisen coding sequences in modern organisms are oligomeric repeats, and most of the older genes retain various vestiges of their original internal repetitions. Some of the genes (e.g., oncogenes) have even inherited the property of being impervious to randomly sustained base changes.