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

Abstract

␤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...

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

“…7b). 22,46 This observation indicates that these residues might prefer to form the native structure even at low pH, deviating from the ideal two-state transition between the acid-denatured state and urea-unfolded state. Thus, the correlations of these residues do not gather on the diagonal line.…”

“…It is reported that the folding intermediate with transPro32 (denoted as I T ) is in an amyloidogenic state at this pH. [22][23][24][25][26] Eichner et al reported the structure of ΔN6-β2m, which is thought to be a mimic of I T . 24 The deletion of the N-terminal residues induces restructuring around Pro32 and subsequent structural fluctuations.…”

Effect of polyethylene glycols on the alkaline-induced molten globule intermediate of bovine serum albumin

“…Characterization of these structures and the factors involved in their stability would provide an insight to understand how and when various forces come into play in directing protein folding [1][2][3][4]. The development of a broad range of techniques has led to the identification and characterization of stable intermediates in several proteins [5][6][7][8].…”

Inhibition of β2-Microglobulin Amyloid Fibril Formation by α2-Macroglobulin

“…The ANS fluorescence was measured using a Hitachi F-7000 spectrofluorometer at 25°C with excitation at 350 nm and emission at 400 -600 nm. 3 ) and Western Blot Analysis-The reaction mixture containing 0 or 12.5 M ␤2-m, 0 or 0.63 M ␣2M, 50 mM citrate buffer (pH 4 -5) or phosphate buffer (pH 6 -7.5), 100 mM NaCl, and 0 -0.5 mM SDS or 0 -100 g/ml heparin was incubated for 1 h at 25°C. BS 3 (Thermo Fisher Scientific), an amine-reactive cross-linking reagent, was then added at a final concentration of 5 mM to the mixture.…”

“…amyloid fibrils is thought to be induced by partial unfolding of ␤2-m (3,4). Several groups have established conditions under which ␤2-m amyloid fibril formation occurs at a neutral pH (5)(6)(7)(8)(9)(10)(11).…”