Thiol protection in membrane protein purifications: A study with phage holins

Dewey, Jill S.; Struck, Douglas K.; Young, Ry

doi:10.1016/j.ab.2009.04.031

Cited by 3 publications

(4 citation statements)

References 15 publications

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“…The state of the sulfhydryls in purified sRI and sT‐sRI complex was assessed with Ellman's reagent, also known as 5,5′‐dithio‐bis(2‐nitrobenzoic acid) (DTNB, Sigma, St. Louis, MO), as previously described 41. Briefly, solutions of reduced glutathione, cystine, and purified sRI (40, 20, and 10 μ M ) for sRI analysis or sT‐sRI complex (20, 10, and 5 μ M ) for complex analysis were prepared in a volume of 90 μL in purification buffer.…”

Section: Methodsmentioning

confidence: 99%

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Protein determinants of phage T4 lysis inhibition

et al. 2012

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Genetic studies have established that lysis inhibition in bacteriophage T4 infections occurs when the RI antiholin inhibits the lethal hole-forming function of the T holin. The T-holin is composed of a single N-terminal transmembrane domain and a~20 kDa periplasmic domain. It accumulates harmlessly throughout the bacteriophage infection cycle until suddenly causing permeabilization of the inner membrane, thereby initiating lysis. The RI antiholin has a SAR domain that directs its secretion to the periplasm, where it can either be inactivated and degraded or be activated as a specific inhibitor of T. Previously, it was shown that the interaction of the soluble domains of these two proteins within the periplasm was necessary for lysis inhibition. We have purified and characterized the periplasmic domains of both T and RI. Both proteins were purified in a modified host that allows disulfide bond formation in the cytoplasm, due to the functional requirement of conserved disulfide bonds. Analytical centrifugation and circular dichroism spectroscopy showed that RI was monomeric and exhibited~80% alpha-helical content. In contrast, T exhibited a propensity to oligomerize and precipitate at high concentrations. Incubation of RI with T inhibits this aggregation and results in a complex of equimolar T and RI content. Although gel filtration analysis indicated a complex mass of 45 kDa, intermediate between the predicted 30 kDa heterodimer and 60 kDa heterotetramer, sedimentation velocity analysis indicated that the predominant species is the former. These results suggest that RI binding to T is necessary and sufficient for lysis inhibition.

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Section: Methodsmentioning

confidence: 99%

“…Ten μL of 10 m M DTNB, dissolved in ethanol, was added to each reaction, samples mixed, and incubated in the dark at room temperature for 30 min. Samples were assayed for the presence of the anionic by‐product 2‐nitro‐5‐thiobenzoate at 412 nm on a UV–vis spectrophotometer (Hitachi, Tokyo) 41…”

Section: Methodsmentioning

confidence: 99%

Protein determinants of phage T4 lysis inhibition

et al. 2012

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“…The upper and lower rings are suggested to have a head to tail arrangement. Very recently the use of thiol specific reagents (2,2 0 -dithiodipyridine) in S105 and S 21 68 purification has enabled the generation of purified protein with its cysteine residue in native thiol state [18]. Recently, the possible structure of pinholins (S 21 68) has been examined by combining the results of negative-stain transmission electron-microscopy and structure modeling [19].…”

Section: Introductionmentioning

confidence: 99%

Characterization of DLP12 Prophage Membrane Associated Protein: HolinGFP

Srividhya

Krishnaswamy

2012

Indian J Microbiol

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Lysis cassette genes from phages determine the final lytic event of the host cells. The lysis cassette genes are conserved in phages and prophages. The membrane associated holin from DLP12 prophage, available as a GFP fusion construct, was shown to be overexpressed, using confocal microscopy analysis, in bacterial cells. The protein expression caused cell death in E. coli AG1 strain suggesting the protein was functional. The His-tag Hol-inGFP protein was purified using cobalt affinity column and was eluted in the presence of different non-ionic detergents DDM (n-dodecyl-ß-D-maltoside), LDAO (Lauryldimethylamine-oxide), OG (n-octyl b-D-glucopyranoside) and C 12 E 9 (dodecyl nonaoxyethylene ether). HolinGFP existed predominantly as a dimer in LDAO in Superdex S200 gel filtration chromatography. Circular dichroism and fluorescence spectroscopy of the fluorescent HolinGFP in all four detergents (C 12 E 9 , DDM, LDAO, and OG) confirmed the folded state. Both dithiobis succinimidyl propionate and gluteraldehyde crosslinking revealed the existence of higher order oligomers and dimers. Hol-inGFP has been functionally and biophysically characterised and is being explored for crystallographic structure determination.

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“…The sensitivity is higher than that of DTNB due to less steric hindrance in accessing the free protein thiols. 32 A study by Kazunori Maruyama et al propose that 2,2'-Dithiopyridine was able to oxidise cysteines into an intramolecular S-S bond. 33 Two differences in the methodology were explored.…”

Section: Protection Of Cysteines By Reversible Thiolation With 22'-dithiodipyridinementioning

confidence: 99%

Characterisation of a bundling protein involved in the self-assembly of bioelastomeric snail egg cases

Loke¹

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The carnivorous marine snail Pugilina cochlidium is found predominantly on the sea shores of the South-East Asia region. This sea creature lays a string of egg capsules up to a metre in length which houses its embryos. The egg capsule material must be able to withstand and resist the harsh and relentless tidal forces of the ocean in order for the off-springs to develop properly and survive.The egg capsule was found to be made up of egg capsule protein precursors which domains are predominately filamentous made of coiled-coils. Four laminar protein sheets were also found to be oriented perpendicular to each other. Mechanical V Volts xg Centrifugal gravitational force βME 2-Mercaptoethanol, Beta-Mercaptoethanol

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Thiol protection in membrane protein purifications: A study with phage holins

Cited by 3 publications

References 15 publications

Protein determinants of phage T4 lysis inhibition

Protein determinants of phage T4 lysis inhibition

Characterization of DLP12 Prophage Membrane Associated Protein: HolinGFP

Characterisation of a bundling protein involved in the self-assembly of bioelastomeric snail egg cases

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