Protein splicing and tryptophan fluorescence of salt‐dependent inteins from Haloquadratum walsbyi

AbstractThe Pacific oyster, Crassostrea gigas, is a traditional food worldwide. The soft body of the oyster can easily accumulate heavy metals such as cadmium (Cd). To clarify the molecular mechanism of Cd accumulation in the viscera of C. gigas, we identified Cd-binding proteins. 5,10,15,20-Tetraphenyl-21H,23H-porphinetetrasulfonic acid, disulfuric acid, tetrahydrate, and Cd-binding competition experiments using immobilized metal ion affinity chromatography revealed the binding of water-soluble high molecular weight proteins to Cd, including C. gigas protein disulfide isomerase (cgPDI). Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses revealed two CGHC motifs in cgPDI. The binding between Cd and rcgPDI was confirmed through a Cd-binding experiment using the TPPS method. Isothermal titration calorimetry (ITC) revealed the binding of two Cd ions to one molecule of rcgPDI. Circular dichroism (CD) spectrum and tryptophan fluorescence analyses demonstrated that the rcgPDI bound to Cd. The binding markedly changed the two-dimensional or three-dimensional structures. The activity of rcgPDI measured by a PDI Activity Assay Kit was more affected by the addition of Cd than by human PDI. Immunological analyses indicated that C. gigas contained cgPDI at a concentration of 1.0 nmol/g (viscera wet weight). The combination of ITC and quantification results revealed that Cd-binding to cgPDI accounted for 20% of the total bound Cd in the visceral mass. The findings provide new insights into the defense mechanisms of invertebrates against Cd.

Download Full-text

Resolution of the lifetimes and correlation times of the intrinsic tryptophan fluorescence of human hemoglobin solutions using 2 GHz frequency-domain fluorometry.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)68591-6 ◽

1988 ◽

Vol 263 (15) ◽

pp. 6972-6977

Author(s):

E Bucci ◽

H Malak ◽

C Fronticelli ◽

I Gryczynski ◽

J R Lakowicz

Keyword(s):

Frequency Domain ◽

Tryptophan Fluorescence ◽

Human Hemoglobin ◽

Correlation Times ◽

Intrinsic Tryptophan Fluorescence ◽

Frequency Domain Fluorometry

Download Full-text

Hydrophobic interactions in human casein micelle formation: β-casein aggregation

Journal of Dairy Research ◽

10.1017/s0022029900028909 ◽

1989 ◽

Vol 56 (3) ◽

pp. 427-433 ◽

Cited By ~ 22

Author(s):

Charles W. Slattery ◽

Satish M. Sood ◽

Pat Chang

Keyword(s):

Light Scattering ◽

Conformational Transition ◽

Hydrophobic Interactions ◽

Micelle Formation ◽

Tryptophan Fluorescence ◽

Phosphate Esters ◽

Casein Micelle ◽

Protein Association ◽

P Protein ◽

Micelle Size

SummaryThe association of non-phosphorylated (0-P) and fully phosphorylated (5-P) human β-caseins was studied by fluorescence spectroscopy and laser light scattering. The tryptophan fluorescence intensity (FI) level increased between 20 and 35 °C, indicating a change in the environment of that residue. A similar transition occurred when ANS was used as a probe. Transition temperatures were slightly lower in 10 mM-CaCl2 but were not affected by an equivalent increase in ionic strength caused by NaCl. The magnitude of the FI change was less for the 5-P than the 0-P protein but was increased for both by CaCl2 addition. These FI data were characteristic of a conformational change and this was supported by fluorescence polarization which indicated that with CaCl2, tryptophan and ANS mobility increased at the transition temperature even though the extent of protein association also increased. Light scattering suggested that protein association proeeeded with the primary formation of submicellar aggregates containing 20–30 monomers which then associated further to form particles of minimum micelle size (12–15 submicelles), and eventually larger. The temperature of precipitation of the 5-P form in the presence of CaCl2 was lower than the conformational transition and suggested that both hydrophobic interactions and Ca bridges between phosphate esters on adjacent molecules are important in micelle formation.

Download Full-text