Molten Globule Precursor States Are Conformationally Correlated to Amyloid Fibrils of Human β-2-Microglobulin

Abstract

Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. However, their experimental characterization is challenging due to the transient nature and conformational heterogeneity of the involved states. Here, we demonstrate that direct carbon-detected NMR experiments allow observation, assignment, and structural analysis of molten globule amyloid intermediates that are severely broadened by conformational exchange. The method is used to characterize the structure and dynamics of partially unfolded intermediates of the 99-residue protein -2-microglobulin, which is the major component of insoluble aggregates occurring in dialysis-related amyloidosis. Comparison of the conformational properties of the molten globule-like intermediates with levels of deuterium incorporation into amyloid fibrils of -2-microglobulin revealed a close relationship between the conformational properties of the metastable intermediates and the -sheet-rich insoluble aggregates of -2-microglobulin.Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. 1,2 However, the experimental characterization of the conformation of amyloid intermediates is challenging due to their transient nature and conformational heterogeneity. 3,4 This severely limits our knowledge about the relation of the conformation of the precursor states to the structure of amyloid fibrils.Dialysis-related amyloidosis is a protein misfolding disease resulting from deposition of amyloid aggregates in skeletal tissue that contain fibrils of the 99-residue-long protein -2-microglobulin (b2m). 5 Amyloid formation of b2m is strongly enhanced in conditions that destabilize its globular structure. 6 This can be achieved in Vitro by acid denaturation, with two distinct intermediate states being formed under acidic conditions. The highest population of the partially unfolded intermediate occurs at pH 3.6, where also the rate of fibril formation reaches a maximum. 7 However, long and straight amyloid fibrils resembling those extracted from patients are formed from a precursor state formed at pH 2.5. 8,9 Here, we compare the conformational properties of these two partially unfolded intermediates with single-residue resolution and demonstrate that a close relationship exists between the structure and dynamics of the amyloid precursor species and the morphology of mature fibrils of b2m.At pH 2.5 b2m is highly unfolded, the majority of resonances are observed in 1 H-15 N HSQC spectra (Supporting Information, Figure 1) and can be assigned to individual residues. 10 In contrast, only about 40-60 resolved peaks on top of a broad hump of unresolved signal intensity (visible at lower contour levels) could be observed at pH 3.6 ( Figure 1a). 11 To obtain sequence-specific information about the pH 3.6 intermediate, we tested direct 13 C detection. 12-14 Direct carbon detection was previously successfully used fo...

Direct observation of minimum‐sized amyloid fibrils using solution NMR spectroscopy

“…Solution NMR has been used to characterize the β2m molecule in the native and acid‐denatured state 35–37. It has been also applied for the investigations of fibril formation and folding process of β2m 38–42. As for β2m fibrils, Platt et al 24.…”

The βγ-crystallins: Native state stability and pathways to aggregation

“…In almost all cases closely investigated, the aggregated states turn out to be polymers of specific partially folded or unfolded intermediates. Examples include the domain swapped polymers formed from mutants of α 1 -antitrypsin in liver cells (Lomas and Parfrey, 2004; Yamasaki et al, 2011); the amyloid fibers formed from mutant transthyretin molecules, probably within cells (Colon and Kelly, 1992); β 2 -microglobulin fibers in blood (Skora et al, 2010); the amyloid fibers formed from α-synuclein (Fink, 2006); the inclusion bodies formed from P22 tailspike and coat proteins chains (King et al, 1996); and polymers of a number of other proteins (Horwich, 2002). In fact the classic example the polymerization of sickle hemoglobin into fibers is an exception, since the precursor is a native state of the mutant protein.…”

A six-dimensional alpha proton detection-based APSY experiment for backbone assignment of intrinsically disordered proteins

“…For example, a combination of five-, six-and sevendimensional HN-detected experiments, which were recorded using automated projection spectroscopy (APSY; Hiller et al 2005) in combination with the assignment software MARS (Jung and Zweckstetter 2004), allowed automatic assignment of 92 % of the assignable residues of the 441-residue IDP Tau (Narayanan et al 2010). In addition, 13 C-detection is a very powerful approach to increase signal dispersion and reduce line broadening, which is either caused by solvent exchange or conformational exchange (Felli and Brutscher 2009;Skora et al 2010). 13 C-detected NMR experiments (Bermel et al 2006a(Bermel et al , b, 2012aBertini et al 2004;Pervushin and Eletsky 2003;Shimba et al 2004;Takeuchi et al 2010), in particular when combined with non-uniform sampling techniques (Barna et al 1987;Bermel et al 2012bBermel et al , 2013Chylla and Markley 1995;Coggins and Zhou 2006;Holland et al 2011;Kazimierczuk et al 2006;Kazimierczuk and Orekhov 2011;Korzhneva et al 2001;Novacek et al 2011;Orekhov et al 2001), can therefore enable the resonance assignment and structural characterization of large IDPs (Csizmok et al 2008;Narayanan et al 2010;Novacek et al 2013;Zawadzka-Kazimierczuk et al 2012a).…”