scholarly journals A new replication medium enables a rapid identification of φx-174 virus by synchronous fluorescence of Tryptophan.

2021 ◽  
Author(s):  
Yair Farber ◽  
Yaniv Shlosberg ◽  
Noam Adir ◽  
Israel Schechter ◽  
Robert Armon
Keyword(s):  
Rapid Identification ◽  
Intrinsic Fluorescence ◽  
Synchronous Fluorescence ◽  
Strong Fluorescence ◽  
Cultivation Medium ◽  
Rapid Methods ◽  
Fluorescence Measurements ◽  
Tryptophan Residues ◽  
Tryptophan Content ◽  
Amino Acid Tryptophan

Development of rapid methods for identification of bacteriophages based on their intrinsic fluorescence is challenging. Pure bacteriophages may be detected based on the strong fluorescence of the amino acid Tryptophan that exist in their proteins. Nevertheless, Tryptophan is a molecule that also exist in high quantities in the bacterial hosts and their cultivation media. In this work, we show that simple separation of the bacteriophage φx-174 from its E.coli host (grown on standard cultivation medium) by filtration is not sufficient for its identification based on the intrinsic fluorescence of its Tryptophan content. This is mostly because of the tryptophan residues that derive from the cultivation medium. We fabricate a new cultivation medium that does not have any significant fluorescence overlap with Tryptophan. By utilization of this new cultivation medium, we can identify φx174 based on the spectral fingerprint of its intrinsic Tryptophan content by synchronous fluorescence measurements.

Contribution of separate tryptophan residues to intrinsic fluorescence of actin. Analysis of 3D structure

FEBS Letters ◽  
1999 ◽  
Vol 452 (3) ◽  
pp. 205-210 ◽  
Author(s):  
I.M. Kuznetsova ◽  
T.A. Yakusheva ◽  
K.K. Turoverov
Keyword(s):  
3D Structure ◽  
Intrinsic Fluorescence ◽  
Tryptophan Residues

scholarly journals Membrane insertion of the N-terminal α-helix of equinatoxin II, a sea anemone cytolytic toxin

2004 ◽  
Vol 384 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Ion GUTIÉRREZ-AGUIRRE ◽  
Ariana BARLIČ ◽  
Zdravko PODLESEK ◽  
Peter MAČEK ◽  
Gregor ANDERLUH ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Lipid Membrane ◽  
Model Systems ◽  
Tryptophan Residue ◽  
Sea Anemones ◽  
Fluorescence Measurements ◽  
Tryptophan Residues ◽  
Equinatoxin Ii ◽  
Α Helix ◽  
Pore Forming Proteins

Equinatoxin II (Eqt-II) is a member of the actinoporins, a unique family of cytotoxins comprising 20 kDa pore-forming proteins isolated from sea anemones. Actinoporins bind preferentially to lipid membranes containing sphingomyelin, and create cation-selective pores by oligomerization of three to four monomers. Previous studies have shown that regions of Eqt-II crucial for its cytolytic mechanism are an exposed aromatic cluster and the N-terminal region containing an amphipathic α-helix. In the present study, we have investigated the transfer of the N-terminal α-helix into the lipid membrane by the use of three mutants containing an additional tryptophan residue in different positions within the amphipathic α-helix (Ile18→Trp, Val22→Trp and Ala25→Trp). The interaction of the mutants with different model systems, such as lipid monolayers, erythrocytes and ghost membranes, was extensively characterized. Intrinsic fluorescence measurements and the use of vesicles containing brominated phospholipids indicated a deep localization of the N-terminal amphipathic helix in the lipid bilayer, except for the case of Val22→Trp. This mutant is stabilized in a state immediately prior to final pore formation. The introduction of additional tryptophan residues in the sequence of Eqt-II has proved to be a suitable approach to monitor the new environments that surround defined regions of the molecule upon membrane interaction.

Intrinsic fluorescence as a probe of structure-function relationships in Ca2+-transport ATPases

1996 ◽  
Vol 16 (2) ◽  
pp. 87-106 ◽  
Author(s):  
Sérgio T. Ferreira ◽  
Tatiana Coelho-Sampaio
Keyword(s):  
Structure Function ◽  
Conformational Changes ◽  
Fluorescence Emission ◽  
Intrinsic Fluorescence ◽  
Future Prospects ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Measurements ◽  
Transport Atpases ◽  
Functional States

Applications of intrinsic fluorescence measurements in the study of Ca2+-transport ATPases are reviewed. Since the initial reports showing that the fluorescence emission was sensitive to Ca2+ binding, a substantial amount of work has focused on the use of both steady-state and time-resolved fluorescence spectroscopy to investigate structure-function relationships in sarcoplasmic reticulum and plasma membrane Ca2+-ATPases. These studies have revealed ligand-induced conformational changes, as well as provided information on protein-protein, protein-solvent and/or protein-lipid interactions in different functional states of these proteins. The main results of these studies, as well as possible future prospects are discussed.

Chemische Analyse und Struktur des Poliovirus. I. Cystein/Cystin Gehalt, vollständige Aminosäureanalyse und Hydrophobizität von Poliovirus und seinen natürlichen leeren Kapsiden / Chemical Analysis and Structure of Poliovirus. I. Cysteine/Cystine Content, Complete Amino Acid Analysis and Hydrophobicity of Poliovirus and Its Naturally Occurring Empty Capsids

1981 ◽  
Vol 36 (1-2) ◽  
pp. 164-172 ◽  
Author(s):  
Jochen Heukeshoven ◽  
Rudolf Demick
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Sulfhydryl Groups ◽  
Poliovirus Type ◽  
Nitrobenzoic Acid ◽  
Chemische Analyse ◽  
Tryptophan Residues ◽  
Tryptophan Content ◽  
Type 3

Abstract The cysteine content of poliovirus particles and naturally occuring empty capsids was determined by two methods: (1) reaction with vinylpyridine and subsequent amino acid analyses and (2) treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and measurement in the change of absorption. Both methods were performed under dissociating conditions in order to expose all sulfhydryl groups. Poliovirus, type 1, strain Mahoney, contains 10-11 cysteine/cystine residues per protomer, irrespective of the use of virus particles or empty capsids. Poliovirus, type 3, strain Saukett, contains 12-13 cysteine/cystine residues per protomer. Poliovirus particles are completely free of disulfide bridges, whereas empty capsids contain 2-4 cystine residues/protomer. No sulfhydryl groups are present on the surface of the virus particle, because of lack of reaction with DTNB. The tryptophan content was determined to be 13 ± 1 residues/protomer. By amino acid analysis under controlled hydrolyzing conditions 12 residues/protomer were found, whereas formylation in hydrochloric acid/formic acid revealed 14 residues/protomer; 13 tryptophan residues were calculated from the tyrosine-tryptophan relation and the optical density at 293.5 nm and 280 nm. The following parameters of poliovirus particles were calculated from the improved and complete amino acid analysis and the cysteine and tryptophan content: 1. The molecular weight of a protomer to be 92 700 ± 900 Dalton and of the poliovirus particle, type 1, strain Mahoney, to be 7.97 × 106 Dalton. 2. The relative hydrophobicity of the poliovirus polypeptide to be 1.18. 3. The extinction coefficients of poliovirus E1%260 = 7 4 and of empty capsids E1%280 = 16.2 ± 0.2.

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