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

Sakata, Michiko; Chatani, Eri; Kameda, Atsushi; Sakurai, Kazumasa; Naiki, Hironobu; Goto, Yuji

doi:10.1016/j.jmb.2008.08.003

Cited by 43 publications

(66 citation statements)

References 44 publications

(79 reference statements)

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“…Previous studies with WT β2m showed that trans→cis prolyl bond isomerization of Pro32 is the rate-limiting step in the protein-folding mechanism (33,34). As a consequence, the apparent folding rate of WT β2m is independent of denaturant concentration (SI Appendix, Fig.…”

Section: Discussionmentioning

confidence: 88%

Both the cis - trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly

Torbeev

Hilvert

2013

Proc. Natl. Acad. Sci. U.S.A.

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The human protein β2-microglobulin (β2m) aggregates as amyloid fibrils in patients undergoing long-term hemodialysis. Isomerization of Pro32 from its native cis to a nonnative trans conformation is thought to trigger β2m misfolding and subsequent amyloid assembly. To examine this hypothesis, we systematically varied the free-energy profile of proline cis-trans isomerization by replacing Pro32 with a series of 4-fluoroprolines via total chemical synthesis. We show that β2m's stability, (un)folding, and aggregation properties are all influenced by the rate and equilibrium of Pro32 cistrans isomerization. As anticipated, the β2m monomer was either stabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the stereoisomeric (2S,4R)-fluoroproline, which disfavors this conformation. However, substitution of Pro32 with 4,4-difluoroproline, which has nearly the same cis-trans preference as proline but an enhanced isomerization rate, caused pronounced destabilization of the protein and increased oligomerization at neutral pH. More remarkably, these subtle alterations in chemical compositionincorporation of one or two fluorine atoms into a single proline residue in the 99 amino acid long protein-modulated the aggregation properties of β2m, inducing the formation of polymorphically distinct amyloid fibrils. These results highlight the importance of conformational dynamics for molecular assembly of an amyloid cross-β structure and provide insights into mechanistic aspects of Pro32 cis-trans isomerism in β2m aggregation.protein conformation | polymorphism | amyloidogenesis | native chemical ligation

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Section: Discussionmentioning

confidence: 88%

Both the cis - trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly

Torbeev

Hilvert

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…It follows that the presence of a trans peptide bond between residues 31 and 32 within the native structure of ␤2m is not a sufficient condition to induce fibrillar aggregation; rather, some partial unfolding, perhaps associated with oligomerization, is needed to trigger amyloid formation. This inference was further reinforced by Sakata et al (7), who showed that, in contrast to P32G, the mutant P32V does not undergo any amyloid transition. They also pointed out the necessity of introducing a coupling between the trans-cis isomerization and the adjacent kinetic step and suggested a minimal folding model (7).…”

mentioning

confidence: 84%

“…This inference was further reinforced by Sakata et al (7), who showed that, in contrast to P32G, the mutant P32V does not undergo any amyloid transition. They also pointed out the necessity of introducing a coupling between the trans-cis isomerization and the adjacent kinetic step and suggested a minimal folding model (7). The apparently monoexponential unfolding pattern of the spectroscopic traces was thus rationalized as the consequence of the occurrence of two processes with similar timing: unfolding and trans-cis peptidyl-prolyl isomerization between positions 31 and 32.…”

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confidence: 84%

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein β2-Microglobulin Revealed by Real-time Two-dimensional NMR

Corazza

Rennella

Schanda

et al. 2010

Journal of Biological Chemistry

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␤2-microglobulin (␤2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified ␤2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G ␤2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G ␤2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the ␤2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type ␤2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G ␤2m mutant.Among the amyloidogenic proteins that have been most frequently studied over the last years, there is ␤ 2 -microglobulin (␤2m) 4 that is responsible for dialysis-related amyloidosis.␤2m, the non-polymorphic light chain of class I major histocompatibility complex, is a small protein that converts into fibrils without the necessity of any chemical modification. Fibril formation occurs in vitro, and most probably also in vivo, through the intact protein. This allows extending experimental conclusions from in vitro studies to the natural process. More importantly, ␤2m recapitulates exquisitely the paradigm of the partially unfolded intermediate, bridging the native fold and the fibrillar conformation, as traditionally invoked to explain the conformational transition from the native fold to the amyloid. Therefore, a detailed characterization of all intermediate states occurring along the folding pathway is of great importance to understand the process of fibril formation. A kinetic scheme of ␤2m refolding entailing two fast steps, burst phase and fast phase, prior to reaching a slow conversion phase from the intermediate to the native state, was first reported by Chiti et al. (1). This long-lived ␤2m refolding intermediate, termed I 2 or I T by different authors, has long been recognized as an effective fibril-competent species, formerly regarded as an ensemble of species (1) and later as a single species (2-4). It has been shown that I T contains a non-native trans peptide bond between His-31 and Pro-32 that slowly converts into cis conformation during the final refolding step (3). The isomerization occurs with minor rearrangements of the protein toward the native structure from an already native-lik...

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“…Previous refolding studies by Jahn et al 25 and Sakata et al 48 suggested that the putative amyloidogenic state, the I T state, is less stable than the native conformation and in equilibrium with a small amount of the unfolded state. The distribution of the unfolded and I T states was 2.4%/97.6% for Jahn et al 25 and 0.5%/99.5% for Sakata et al, 48 respectively, immediately after the initiation of the refolding. The formation of the fibril during the refolding experiment 10 also seems explicable by our notion that the unfolded state is amyloidogenic.…”

Section: The Time Scale Of Each Step In the Formation Of Fibrilsmentioning

confidence: 99%

The Monomer–Seed Interaction Mechanism in the Formation of the β2-Microglobulin Amyloid Fibril Clarified by Solution NMR Techniques

Yanagi

Sakurai

Yoshimura

et al. 2012

Journal of Molecular Biology

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

Cited by 43 publications

References 44 publications

Both the cis - trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly

Both the cis - trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein β2-Microglobulin Revealed by Real-time Two-dimensional NMR

The Monomer–Seed Interaction Mechanism in the Formation of the β2-Microglobulin Amyloid Fibril Clarified by Solution NMR Techniques

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