Kinetic Coupling of Folding and Prolyl Isomerization of β2-Microglobulin Studied by Mutational Analysis

beta(2)-Microglobulin (beta 2-m), a protein responsible for dialysis-related amyloidosis, adopts a typical immunoglobulin domain fold with the N-terminal peptide bond of Pro32 in a cis isomer. The refolding of beta 2-m is limited by the slow trans-to-cis isomerization of Pro32, implying that intermediates with a non-native trans-Pro32 isomer are precursors for the formation of amyloid fibrils. To obtain further insight into the Pro-limited folding of beta 2-m, we Studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V beta 2-ms) as well as the wild-type beta 2-m. W39 beta 2-m is a triple mutant in which both of the authentic Trip residues (Trp60 and Trp95) are replaced by Phe and a buried Trp common to other immunoglobulin domains is introduced at the position of Leu39 (i.e., L39W/W60F/W95F). W39 Q-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V beta 2-m is a mutant in which Pro32 is replaced by Val, useful for probing the kinetic role of the trans-to-cis isomerization of Pro32. A comparative analysis of the unfolding/refolding kinetics of these Mutants including three types of double-jump experiments revealed the prolyl isomerization to be coupled with the conformational transitions, leading to apparently unusual kinetics, particularly for the unfolding. We suggest that careful consideration of the kinetic Coupling Of unfolding/refolding and prolyl isomerization which has tended to be neglected in recent Studies, is essential for clarifying the mechanism of protein folding and, moreover, its biological significance. (C) 2008 Elsevier Ltd. All rights reserved.