Energetics of Domain−Domain Interactions and Entropy Driven Association of β-Crystallins

• Published in: Biochemistry (Easton) 2004-01, Vol.43 (2), p.415-424
• Main Authors: Sergeev, Y. V, Hejtmancik, J. F, Wingfield, P. T
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; beta-Crystallin B Chain - chemistry; beta-Crystallin B Chain - genetics; beta-Crystallins - chemistry; beta-Crystallins - genetics; Crystallins - chemistry; Crystallins - genetics; Dimerization; Entropy; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Structure, Tertiary - genetics; Recombinant Proteins - chemistry; Sequence Deletion; Temperature; Thermodynamics; Ultracentrifugation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi034617f

β-Crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. They undergo modification as the lens ages, including cleavage of their terminal extensions. The energetics of βA3- and βB2-crystallin association was studied using site-directed mutagenesis and analytical ultracentrifugation. Recombinant (r) murine wild type βA3- and βB2-crystallins were modified by removal of either the N-terminal extension of βA3 (rβA3Ntr) or βB2 (rβB2Ntr), or both the N- and C-terminal extensions of βB2 (rβB2NCtr). The proteins were expressed in Sf9 insect cells or Escherichia coli and purified by gel-filtration and ion-exchange chromatography. All β-crystallins studied demonstrated fast reversible monomer−dimer equilibria over the temperature range studied (5−35 °C) with a tendency to form tighter dimers at higher temperatures. The N-terminal deletion of rβA3 (rβA3Ntr) significantly increases the enthalpy (+10.9 kcal/mol) and entropy (+40.7 cal/deg mol) of binding relative to unmodified protein. Removal of both N- and C-terminal extensions of rβB2 also increases these parameters but to a lesser degree. Deletion of the βB2-crystallin N-terminal extension alone (rβB2Ntr) gave almost no change relative to rβB2. The resultant net negative changes in the binding energy suggest that βΑ3- and βB2-crystallin association is entropically driven. The thermodynamic consequences of the loss of βΑ3-crystallin terminal extensions by in vivo proteolytic processing could increase their tendency to associate and so promote the formation of higher order associates in the aging and cataractous lens.