Helix Of Subunit Research Articles

Conformational switching in the flagellar filament of Salmonella typhimurium

The flagellum is the bacterial organelle of motility. It is constructed from a basal body (rotary motor) a hook (universal joint), and a filament (propeller). The filament is a self-assembling helical structure usually composed of one protein species – flagellin. Flaqellins from different species differ in their molecular weight and amino acid composition. However, the amino and carboxy termini of all flagellin are highly homologous. The outer flagellin domain, comprising the central part of the primary sequence, is variable and not obligatory for motility. To function as a propeller, the straight helical flagellin assembly, in which all subunits ace equivalent, needs to supercoil into a corkscrew-shaped rigid structure in which the subunits are quasiequivalent. The filament can be viewed as an assembly of 11 helical protofilaments or subunit strands. Supercoiling is believed to be possible due to the coexistence of flagellin subunits in two stable and switchable conformations. Switching is co-operative and each protofilament acts as a unit. Switched protofilaments differ in length from unswitched ones, the result of which is an introduction of twisting and bending forces which can be relieved by deformation or supercoiling of the filament. If all subunits are either in the R- or L- conformation, the filament is straight and either right-handed or left-handed, respectively. Progressive switching of protofilaments results in a total of 12 polymorphic states.

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.

Mutations in three of the putative transmembrane helices of subunit a of the Escherichia coli F 1F 0-ATPase disrupt ATP-driven proton translocation

Three missense mutants in subunit a of the Escherichia coli F 1F 0-ATPase were isolated and characterized after hydroxylamine mutagenesis of a plasmid carrying the uncB (subunit a) gene. The mutations resulted in Asp 119 → His, Ser 152 → Phe, or Gly 197 → Arg substitutions in subunit a Function was not completely abolished by any of the mutations. The F 0 membrane sector was assembled in all three cases as judged by restoration of dicyclohexylcarbodiimide sensitivity to the F 1F 0-ATPase. The H + translocation capacity of F 0 was reduced in all three mutants. ATP-driven H +-translocation was also reduced, with the response in the Gly 197 → Arg mutant being almost nil and that in the Asp 119 → His and Ser 152 → Phe mutants less severely affected. The substituted residues are predicted to lie in the second, third, and fourth transmembrane helices suggested in most models for subunit a. The Gly 197 → Arg mutation lies in a very conserved region of the protein and the substitution may disrupt a structure that is critical to function. The Asp 119 → His and Ser 152 → Phe mutations also lie in areas with sequence conservation. A further analysis of randomly generated mutants may provide more information on regions of the protein that are crucial to function. Heterodiploid transformants, carrying plasmids with either the wild-type uncB gene or mutant uncB genes in an uncB (Trp 231 → stop) background, were characterized biochemically. The truncated subunit a was not detected in membranes of the background strain by Western blotting, and the uncB + plasmid complemented strain showed normal biochemistry. The uncB mutant genes were shown to cause equivalent defects in either the heterodiploid background configuration, or after incorporation into an otherwise wild-type unc operon. The subunit a (Trp 231 → stop) background strain was shown to bind F 1-ATPase nearly normally despite lacking subunit a in its membrane.

Where is the oxygen binding site of cytochrome c oxidase? Transmembrane helices of subunits I and II

Where is the oxygen binding site of cytochrome c oxidase? Transmembrane helices of subunits I and II

Substructure in exines of Artemisia vulgaris (Asteraceae)

Unit and subunit structure is exposed in exines of Artemisia vulgaris after partial oxidation methods which include 2-aminoethanol followed by exine expansion and exposure to potassium permanganate. Tectal bacules traced ontogenetically from a plasma membrane—glycocalyx unit-complex (“tuft”) consist of a super-coiled helical subunit (binder) wound around several axially straight helical subunits (core subunits). Fracturing occurs between tuft units when partial oxidation includes contraction of exines in HCL or ethanol prior to potassium permanganate. Tuft units are radial in the non-tectal bacules and throughout the nexine. Partial oxidation was controlled to give an end-product having a reduced although sufficient presence of sporopollenin to conserve the diagnostically taxon-distinct exine. The sporopolleninous remnant resisted acetolysis although without support (SiO) exines disintegrated into helical subunit-sized structures, transparent to electrons and unreactive to stains (including osmium tetroxide). Components of the partially oxidized exine having a binder and core unit structure, stain positively for acid polyanions (polysaccharides?) and protein. Osmophilia (unsaturated lipids + ?), extinguished during potassium permanganate treatment, had been located largely in what is interpreted to be the core region of tuft units.

X-ray diffraction and electron microscope studies on the structure of bacterial F pili

F pili are hollow cylinders with 80 Å outer diameter and 20 Å inner diameter. Both X-ray fibre diffraction and optical diffraction of electron micrographs show a strong layer-line corresponding to a spacing of 32 Å, to which a J 4 Bessel function is assigned on the basis of the optical diffraction. X-ray diffraction patterns show near-meridional intensity on a layer-line corresponding to a spacing of 12.8 Å, to which a J 1 Bessel function is assigned. Mass per length measurements on unstained specimens in the scanning transmission electron microscope give 3000 daltons/Å, indicating that the 11,200 dalton pilin subunits are 3.7 Å apart along the axial direction of the pili. These observations show that the pilus structure can be represented as four coaxial helices of pitch 128 Å with the pilin subunits elongated and overlapping along the line of these helices. Each of these helices of subunits is translated axially with respect to its neighbour, to give a basic helix of 3.6 units per turn of 12.8 Å pitch. Radial electron density calculations indicate a 50 Å diameter girdle of hydrophobic amino acids between the inner and outer diameters of the protein shell. A molecular model of the structure at low resolution is presented.

The origin of the tyrosyl circular dichroism of tropomyosin

1. 1.|The near ultraviolet circular dichroism of tropomyosin is due to tyrosine and to disulphide bonds. The optical activity of these chromophores can be distinguished by oxidising and reducing the protein. The circular dichroism due to tyrosine is exceptionally intense and is anomalous in that the shape of the spectrum and the wavelength of the maximum are different from those of the absorption spectrum. 2. 2.|The intense tyrosyl circular dichroism and the mismatch between circular dichroism and absorption spectra are likely to be due to tyrosine-tyrosine interactions at distances of less than 8 Å. 3. 3.|The results are analysed in terms of the coiled model for tropomyosin and lead to the conclusion that the tyrosines of one helical subunit interact with those of the other. The in-register alignment of the helical chains, with five tyrosines of one chain opposite those of the other, accounts for the tyrosine-tyrosine interactions. 4. 4.|The disulphide circular dichroism of oxidized tropomyosin is intense and is consistent with intra-molecular disulphide bonds between helical subunits which can form in the non-staggered model for tropomyosin.

Proteolytic degradation of myosin and the meromyosins by a water-insoluble polyanionic derivative of trypsin: Properties of a helical subunit isolated from heavy meromyosin

The hydrolysis of myosin by a water-insoluble polyanionic derivative of trypsin (IMET) has been studied with the pH-stat and by sedimentation velocity experiments. As with soluble trypsin, the kinetic data can be interpreted in terms of two first-order reaction classes. However, in the case of IMET, the fast reaction no longer parallels the formation of the meromyosins, and the total number of peptide bonds available for enzymic hydrolysis is markedly reduced. The implication of these findings for the conformation of the linkage between the meromyosins is discussed. Meromyosins formed by IMET have distinctive properties: Heavy meromyosin shows a 3 s component even when isolated from partially digested myosin. Light meromyosin can form lattice structures instead of fibrous aggregates. The 3 s component, heavy meromyosin subfragment 2, has been isolated by alcohol fractionation, followed by isoelectric precipitation. This water-soluble protein has a molecular weight of 61,000 and a helix content of 80%. These values, together with the sedimentation velocity and viscosity data, suggest a length of about 400for the molecule. Heavy meromyosin subfragment 2 forms paracrystals with a 145periodicity.

Erratum: Myosin Substructure: Isolation of a Helical Subunit from Heavy Meromyosin

In the report "Myosin substructure: isolation of a helical subunit from heavy meromyosin" by Susan Lowey (7 Aug. 1964), lines 5-7 of column 1, p. 599, should have read: ". . . an ionic strength of 0.1 and 0.03 M . As expected, when S 20,w was plotted. . . ."

MYOSIN SUBSTRUCTURE: ISOLATION OF A HELICAL SUBUNIT FROM HEAVY MEROMYOSIN.

A highly alpha-helical subunit has been isolated from the heavy-mero-myosin portion of myosin. This molecule has a helix content of about 73 percent and an amino acid composition similar to that of light meromyosin. The presence of this subunit supports the current view that the myosin molecule consists of a long helical rod with a globular region at one end.