scholarly journals P15 and P3, the Tail Completion Proteins of Bacteriophage T4, Both Form Hexameric Rings

2003 ◽  
Vol 185 (5) ◽  
pp. 1693-1700 ◽  
Author(s):  
Li Zhao ◽  
Shuji Kanamaru ◽  
Chatree'chalerm Chaidirek ◽  
Fumio Arisaka
Keyword(s):  
Electron Microscopy ◽  
Gene Product ◽  
Analytical Ultracentrifugation ◽  
Bacteriophage T4 ◽  
Sedimentation Equilibrium ◽  
Gene Products ◽  
Oligomeric State ◽  
Polypeptide Chains ◽  
Circular Dichroïsm

ABSTRACT Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail. gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube. In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism. Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains. Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube. The differential roles between gp15 and gp3 upon completion of the tail are discussed.

scholarly journals Structure of the Haemophilus influenzae HMW1B Translocator Protein: Evidence for a Twin Pore

2007 ◽  
Vol 189 (20) ◽  
pp. 7497-7502 ◽  
Author(s):  
Huilin Li ◽  
Susan Grass ◽  
Tao Wang ◽  
Tianbo Liu ◽  
Joseph W. St. Geme
Keyword(s):  
Electron Microscopy ◽  
Haemophilus Influenzae ◽  
Translocator Protein ◽  
Size Exclusion ◽  
Oligomeric State ◽  
Secretion Pathway ◽  
Exclusion Chromatography ◽  
Large Conformational Change ◽  
Negative Staining Electron Microscopy

ABSTRACT Secretion of the Haemophilus influenzae HMW1 adhesin occurs via the two-partner secretion pathway and requires the HMW1B outer membrane translocator. HMW1B has been subjected to extensive biochemical studies to date. However, direct examination of the structure of HMW1B has been lacking, leaving fundamental questions about the oligomeric state, the membrane-embedded β-barrel domain, the approximate size of the β-barrel pore, and the mechanism of translocator activity. In the current study, examination of purified HMW1B by size exclusion chromatography and negative staining electron microscopy revealed that the predominant species was a dimer. In the presence of lipid, purified HMW1B formed two-dimensional crystalline sheets. Examination of these crystals by cryo-electron microscopy allowed determination of a projection structure of HMW1B to 10 Å resolution. The native HMW1B structure is a dimer of β-barrels, with each β-barrel measuring 40 Å by 50 Å in the two orthogonal directions and appearing largely occluded, leaving only a narrow pore. These observations suggest that HMW1B undergoes a large conformational change during translocation of the 125-kDa HMW1 adhesin.

scholarly journals Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki Characterization of the molecule and determination of the number of polypeptide chains

1979 ◽  
Vol 183 (2) ◽  
pp. 325-330 ◽  
Author(s):  
E Ilan ◽  
E Daniel
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Guanidinium Chloride ◽  
Tadpole Shrimp ◽  
Polypeptide Chains

Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki, was found to have a sedimentation coefficient (s020,w) of 19.3 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 798000 +/- 20000. The amino acid composition showed the lack of cysteine and cystine residues. A haem content of 3.55 +/- 0.03% was determined, corresponding to a minimal mol.wt. of 17400 +/- 200. The pH-independence in the range pH 5-11 of the sedimentation coefficient indicates a relatively high stability of the native molecule. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a mol.wt. of 34000 +/- 1500. The molecular weight of the polypeptide chain was determined to be 32800 +/- 800 by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol. The findings indicate that Lepidurus haemoglobin is composed of 24 identical polypeptide chains, carrying two haem groups each.

scholarly journals Structural Analysis of Tobacco Etch Potyvirus HC-Pro Oligomers Involved in Aphid Transmission

2005 ◽  
Vol 79 (6) ◽  
pp. 3758-3765 ◽  
Author(s):  
Virginia Ruiz-Ferrer ◽  
Jasminka Boskovic ◽  
Carlos Alfonso ◽  
Germán Rivas ◽  
Oscar Llorca ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Analytical Ultracentrifugation ◽  
Protein A ◽  
Three Dimensional ◽  
Aphid Transmission ◽  
Sedimentation Equilibrium ◽  
Active Protein ◽  
High Biological Activity ◽  
Tobacco Etch Potyvirus ◽  
Oligomeric Forms

ABSTRACT Oligomeric forms of the HC-Pro protein of the tobacco etch potyvirus (TEV) have been analyzed by analytical ultracentrifugation and single-particle electron microscopy combined with three-dimensional (3D) reconstruction. Highly purified HC-Pro protein was obtained from plants infected with TEV by using a modified version of the virus that incorporates a histidine tag at the HC-Pro N terminus (hisHC-Pro). The purified protein retained a high biological activity in solution when tested for aphid transmission. Sedimentation equilibrium showed that the hisHC-Pro preparations were heterogenous in size. Sedimentation velocity confirmed the previous observation and revealed that the active protein solution contained several sedimenting species compatible with dimers, tetramers, hexamers, and octamers of the protein. Electron microscopy fields of purified protein showed particles of different sizes and shapes. The reconstructed 3D structures suggested that the observed particles could correspond to dimeric, tetrameric, and hexameric forms of the protein. A model of the interactions required for oligomerization of the HC-Pro of potyviruses is proposed.

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific β recombinase, and identification of a folding intermediate

2006 ◽  
Vol 387 (5) ◽  
pp. 525-533 ◽  
Author(s):  
Anshul Bhardwaj ◽  
Karin Welfle ◽  
Rolf Misselwitz ◽  
Silvia Ayora ◽  
Juan C. Alonso ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Ionic Strength ◽  
Analytical Ultracentrifugation ◽  
Size Exclusion ◽  
Sedimentation Equilibrium ◽  
Folding Intermediate ◽  
Guanidinium Chloride ◽  
Site Specific ◽  
Exclusion Chromatography ◽  
Circular Dichroïsm

Abstract Solution properties of β recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) β recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold β recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2→2I→2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to ΔG tot=17.9 kcal/mol, with ΔG N2→2I=4.2 kcal/mol for the first transition and ΔG I→U=6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of β recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.

scholarly journals The C-Terminal Fragment of the Precursor Tail Lysozyme of Bacteriophage T4 Stays as a Structural Component of the Baseplate after Cleavage

1999 ◽  
Vol 181 (9) ◽  
pp. 2739-2744 ◽  
Author(s):  
Shuji Kanamaru ◽  
Nadine C. Gassner ◽  
Nanzhang Ye ◽  
Shigeki Takeda ◽  
Fumio Arisaka
Keyword(s):  
Circular Dichroism ◽  
Structural Component ◽  
Cleavage Site ◽  
Analytical Ultracentrifugation ◽  
Bacteriophage T4 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Precursor Protein ◽  
Terminal Fragment ◽  
Circular Dichroïsm

ABSTRACT Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in β-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.

scholarly journals 1D1815 Structural analysis of the connector complex, P15, of bacteriophage T4 by analytical ultracentrifugation and electron microscopy

2000 ◽  
Vol 40 (supplement) ◽  
pp. S33
Author(s):  
Li Zhao ◽  
Chartree'chalerm Chaidirek ◽  
Yoko Takeda ◽  
Shuji Kanamaru ◽  
Fumio Arisaka
Keyword(s):  
Electron Microscopy ◽  
Structural Analysis ◽  
Analytical Ultracentrifugation ◽  
Bacteriophage T4

scholarly journals Tert-butyldimethylsilyl chitosan synthesis and characterization by analytical ultracentrifugation, for archaeological wood conservation

2020 ◽  
Vol 49 (8) ◽  
pp. 781-789 ◽  
Author(s):  
Jennifer M. K. Wakefield ◽  
Susan Braovac ◽  
Hartmut Kutzke ◽  
Robert A. Stockman ◽  
Stephen E. Harding
Keyword(s):  
Molar Mass ◽  
Analytical Ultracentrifugation ◽  
Monomer Unit ◽  
Sedimentation Equilibrium ◽  
Archaeological Wood ◽  
Water As Solvent ◽  
Stage Process ◽  
Future Work ◽  
Different Sources

AbstractThe Oseberg ship is one of the most important archaeological testimonies of the Vikings. After excavation in 1904, the wooden gravegoods were conserved using alum salts. This resulted in extreme degradation of a number of the objects a hundred years later through acid depolymerisation of cellulose and lignin. The fragile condition of the artefacts requires a reconsolidation which has to be done avoiding water as solvent. We synthesized tert-butyldimethylsilyl (TBDMS) chitosan which is soluble in a 50:50 solution of ethyl acetate and toluene. Measurement of its molecular weight, to anticipate its penetration, provided a challenge as the density difference of the polymer and solvent was too small to provide adequate solute redistribution under a centrifugal field, so a two-stage process was implemented (i) determination of the weight-average molar mass of the aqueous soluble activated precursor, chitosan mesylate, Mw,mc using sedimentation equilibrium with the SEDFIT-MSTAR algorithm, and determination of the degree of polymerisation DP; (ii) measurement of the average degree of substitution DSTBDMS of the TBDMS group on each chitosan monosaccharide monomer unit using NMR, to augment the Mw,mc value to give the molar mass of the TBDMS-chitosan. For the preparation, we find Mw = 9.8 kg·mol−1, which is within the acceptable limit for penetration and consolidation of degraded wood. Future work will test this on archaeological wood from different sources.

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