Unfolding kinetics of beta-lactoglobulin induced by surfactant and denaturant: a stopped-flow/fluorescence study.
The beta-->alpha transition of beta-lactoglobulin, a globular protein abundant in the milk of several mammals, is investigated in this work. This transition, induced by the cationic surfactant dodecyltrimethylammonium chloride (DTAC), is accompanied by partial unfolding of the protein. In this work, unfolding of bovine beta-lactoglobulin in DTAC is compared with its unfolding induced by the chemical denaturant guanidine hydrochloride (GnHCl). The final protein states attained in the two media have quite different secondary structure: in DTAC the alpha-helical content increases, leading to the so-called alpha-state; in GnHCl the amount of ordered secondary-structure decreases, resulting in a random coil-rich final state (denatured, or D, state). To obtain information on both mechanistic routes, in DTAC and GnHCl, and to characterize intermediates, the kinetics of unfolding were investigated in the two media. Equilibrium and kinetic data show the partial accumulation of an on-pathway intermediate in each unfolding route: in DTAC, an intermediate (I(1)) with mostly native secondary structure but loose tertiary structure appears between the native (beta) and alpha-states; in GnHCl, another intermediate (I(2)) appears between states beta and D. Kinetic rate constants follow a linear Chevron-plot representation in GnHCl, but show a more complex mechanism in DTAC, which acts like a stronger binding species.
Viseu
Maria Isabel
MI
Centro de Química Estrutural, and Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Technical University of Lisbon, Lisbon, Portugal. iviseu@mail.ist.utl.pt
Melo
Eduardo P
EP
Carvalho
Teresa Isabel
TI
Correia
Raquel F
RF
Costa
Sílvia M B
SM
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry*; D008823:Micelles; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013501:Surface-Active Agents / Q000737:chemistry*
D007782:Lactoglobulins; D008823:Micelles; D000644:Quaternary Ammonium Compounds; D013501:Surface-Active Agents; C022806:dodecyltrimethylammonium; D019791:Guanidine
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