Kinetic coupling of folding and prolyl isomerization of beta2-microglobulin studied by mutational analysis.
Beta(2)-microglobulin (beta2-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 beta2-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 beta2-m, we studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V beta2-ms) as well as the wild-type beta2-m. W39 beta2-m is a triple mutant in which both of the authentic Trp 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 beta2-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V beta2-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.
Sakata
Michiko
M
Institute for Protein Research, Osaka University and CREST, Japan Science and Technology Agency, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Chatani
Eri
E
Kameda
Atsushi
A
Sakurai
Kazumasa
K
Naiki
Hironobu
H
Goto
Yuji
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D001613:beta 2-Microglobulin; D014364:Tryptophan; D011392:Proline