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Deoxycholate induced tetramer of αA-crystallin and sites of phosphorylation: Fluorescence correlation spectroscopy and femtosecond solvation dynamics
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10.1063/1.3702810
/content/aip/journal/jcp/136/15/10.1063/1.3702810
http://aip.metastore.ingenta.com/content/aip/journal/jcp/136/15/10.1063/1.3702810

Figures

Image of Scheme 1.
Scheme 1.

(a) Schematic representation of phosphorylation of amino acid. (b) Schematic structure of A-crystallin subunit (obtained from I-Tasser web server 60,61 ). Cystein is represented in yellow and tryptophan is in Green. The backbone structure of A-crystallin subunit is represented as ribbon, while both the Cys and Trp residues are showed as spacefill display.

Image of Scheme 2.
Scheme 2.

(a) Acrylodan (6-acrylolyl-2-dimethylaminonaphtalene). (b) 1,5-IAEDANS [5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]. (c) Protein labeling reaction scheme.

Image of Scheme 3.
Scheme 3.

Schematic representation of amino acid sequence and possible phosphorylation sites of human A-crystallin subunit (a) in oligomeric form (in absence of NaDC) and (b) in tetrameric form (in presence of NaDC). 1-62 amino acid residues represent N-terminal region (blue), while 63-150 residues represent “central -crystallin domain” (orange) and 151-173 residues exhibit the C-terminal tail (green). 54

Image of FIG. 1.
FIG. 1.

Normalized auto-correlation functions of acrylodan-A-crystallin system in the absence (red) and presence (blue) of 1 wt. % NaDC. Actual autocorrelation curves are shown in the inset.

Image of FIG. 2.
FIG. 2.

(a) Emission spectra (at = 405 nm) of acrylodan labeled A-crystallin (∼10 μM labeled protein in 50 mM Tris-HCl, H-7.4) in native state (red) and in presence of NaDC (blue). The inset represents intensity normalized emission. (b) Normalized tryptophan emission spectra (at = 295 nm) of A-crystallin in native state (red) and in presence of NaDC (blue). The protein (20 M) was in 50 mM Tris-HCl, H-7.4.

Image of FIG. 3.
FIG. 3.

(a) Stern-Volmer plot (at = 295 nm) showing quenching of Trp 9 of A-crystallin by acrylamide in native A crystalline (red) and in presence of 1 wt. % NaDC (blue). The protein (20 M) was in 50 mM Tris-HCl, H-7.4. Figure represents average of three experiments. (b) Stern-Volmer plot (at = 340 nm) showing quenching of 1,5-IAEDANS by acrylamide in native A-crystallin (red) and in presence of 1 wt. % NaDC (blue). The protein (∼13 M labeled protein) was in 50 mM Tris-HCl, H-7.4.

Image of FIG. 4.
FIG. 4.

Far-UV CD spectra of A-crystallin (5 M) in the absence (red) and in the presence of 1 wt. % NaDC (blue).

Image of FIG. 5.
FIG. 5.

Picosecond transients of acrylodan -A-crystallin system (at = 405 nm) in the absence (red) and presence (blue) of NaDC at (A) = 540 nm and (B) = 470 nm.

Image of FIG. 6.
FIG. 6.

Femtosecond transients of acrylodan -A-crystallin system (at = 405 nm) in the absence (red) and presence (blue) of NaDC at (A) = 540 nm and (B) = 470 nm.

Image of FIG. 7.
FIG. 7.

Time-resolved emission spectra (TRES) of acrylodan -A-crystallin system ( = 405 nm) (a) in the absence of NaDC at 0 ps (blue), 20 ps (olive), 200 ps (wine red), and 3500 ps (red) (b) in the presence of NaDC at 0 ps (blue), 10 ps (orange), 40 ps (green), 800 ps (olive), and 3500 ps (red). The steady state emission spectra were shown in the black dotted line.

Image of FIG. 8.
FIG. 8.

Decay of the solvent response function, C(t) of A-crystallin system in the absence (red) and presence (blue) of NaDC. The points denote the actual values of C(t) and the solid line denotes the best fit.

Image of FIG. 9.
FIG. 9.

Fluorescence anisotropy decay of acrylodan -A-crystallin system (at = 405 nm) along with a fitted curve in the absence (red) and presence (blue) of NaDC at = 480 nm.

Tables

Generic image for table
Table I.

Decay parameters of C(t) of acrylodan-A-crystallin system in the absence and presence of NaDC.

Generic image for table
Table II.

Anisotropy decay of acrylodan-A-crystallin system in the absence and presence of NaDC.

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/content/aip/journal/jcp/136/15/10.1063/1.3702810
2012-04-16
2014-04-17
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Deoxycholate induced tetramer of αA-crystallin and sites of phosphorylation: Fluorescence correlation spectroscopy and femtosecond solvation dynamics
http://aip.metastore.ingenta.com/content/aip/journal/jcp/136/15/10.1063/1.3702810
10.1063/1.3702810
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