Kinetics of chaperoning of dithiothreitol-denatured alpha-lactalbumin by alpha-crystallin.
Molecular chaperones prevent the aggregation of partially folded or misfolded forms of protein. alpha-Crystallin performs such a function in the ocular lens. Dynamic light scattering (DLS) measurements were performed to gain insight into the kinetics and mechanism of alpha-crystallin chaperoning. Experiments were conducted as a function of alpha-lactalbumin concentration as well as the alpha-crystallin/alpha-lactalbumin ratio over a 24 h period. In the particle distribution patterns the lactalbumin concentration was partitioned into three compartments: (a) monomeric free lactalbumin; (b) lactalbumin in the chaperoning complex; and (c) lactalbumin aggregates. DLS intensities were converted to molar concentrations by assuming a model of a spherical chaperoning complex. In the model, alpha-crystallin is the central core and alpha-lactalbumin molecules occupy a ring surrounding the core. The kinetics of chaperoning was studied by proposing a simple scheme with four rate constants. The reversible reaction of the formation of the chaperoning complex is characterized by rate constants k(1) and k(2). The rate constants k(3) and k(4) govern the irreversible aggregation of lactalbumin: the former from the free monomeric lactalbumin pool and the latter describing the aggregation of the denatured lactalbumin released from the chaperoning complex. The rate constants, k(3) and k(4) are four magnitudes larger than k(1) and k(2). The equilibrium constant of chaperoning complex formation lies in favor of the reactants. k(4) is somewhat faster than k(3) and it is three times faster than k(s) governing the self-aggregation of lactalbumin in the absence of alpha-crystallin.
Bettelheim
Frederick A
FA
Chemistry Department, Adelphi University, Garden City, NY 11530, USA. bettelhe@adelphi.edu
D004229:Dithiothreitol / Q000737:chemistry; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D012542:Scattering, Radiation; D038201:alpha-Crystallins / Q000737:chemistry* / Q000378:metabolism*
D018832:Molecular Chaperones; D038201:alpha-Crystallins; D007768:Lactalbumin; D004229:Dithiothreitol