Trehalose influence on beta-lactoglobulin stability and hydration by time resolved fluorescence.
The stabilizing role of the disaccharide trehalose on beta-lactoglobulin (BLG) against its chemical denaturation both at native and acidic pH has been explored by means of time-resolved fluorescence of the probe acrylodan covalently bound to the unique free cysteine of BLG. The changes in acrylodan fluorescence lifetime with guanidinium chloride concentration reveal BLG sigmoidal denaturation profiles which depend upon the amount of trehalose in solution. When adding trehalose the transition midpoint shifts towards higher denaturant concentration. This effect has been measured by fitting the data with a two-state model whose parameters indicate that an almost 60% increase in the denaturation free energy is induced independently of trehalose concentrations and pH values. Fluorescence anisotropy measurements performed in the same conditions reveal that the internal dynamics are largely affected by the sugar, which makes the acrylodan environment more rigid, and by the denaturant that acts in the opposite way. The overall rotational diffusion of BLG suggests that trehalose affects the hydrodynamic properties of the solution in the proximity of the protein; tentative mechanisms are discussed.
D'Alfonso
Laura
L
Dipartimento di Fisica, Università degli Studi di Milano-Bicocca, and Istituto Nazionale per la Fisica della Materia, Milano, Italy.
Collini
Maddalena
M
Baldini
Giancarlo
G
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D016880:Anisotropy; D002417:Cattle; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015233:Models, Statistical; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013997:Time Factors; D014199:Trehalose / Q000737:chemistry*; D014867:Water / Q000378:metabolism
D007782:Lactoglobulins; D014867:Water; C038518:acrylodan; D014199:Trehalose; D015081:2-Naphthylamine; D019791:Guanidine