The Controlling Roles of Trp60 and Trp95 in β2-Microglobulin Function, Folding and Amyloid Aggregation Properties

Esposito, Gennaro; Ricagno, Stéfano; Corazza, Alessandra; Rennella, Enrico; Gumral, Devrim; Mimmi, Maria Chiara; Betto, Elena; Pucillo, Carlo; Fogolari, Federico; Viglino, Paolo; Raimondi, Sara; Giorgetti, Sofia; Bolognesi, Benedetta; Merlini, Giampaolo; Stoppini, Monica; Bolognesi, M.; Bellotti, Vittorio

doi:10.1016/j.jmb.2008.03.002

Cited by 84 publications

(167 citation statements)

References 49 publications

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“…Thermal unfolding of tertiary structures monitored by near-UV CD [ Fig. 2(A)] is in complete accord with previous results, [21][22][23] showing that the W60G mutation substantially increases thermal stability of the b2m fold (T m shift of about þ8 C). On the opposite, the W60V mutation does not have any measurable effect on the T m (Table I).…”

supporting

confidence: 91%

“…b2m. 21,23 For D59P, instead, stability data were acquired by thermal denaturation, monitoring the extrinsic fluorescence of the Sypro Orange probe. This mutant showed distinctly lower thermal stability than w.t.…”

Section: Thermal Stabilitymentioning

confidence: 99%

“…Its role in intermolecular interactions likely justifies its strong evolutionary conservation through vertebrates. The b2m W60G mutant, was produced and characterized in a previous work, 21 showing marked differences from the w.t. protein.…”

Section: Introductionmentioning

confidence: 99%

“…This mutant is more stable to chemical unfolding, does not aggregate into amyloid fibrils under mild conditions, has a more regular DE-loop conformation and shows a reduced tendency to form oligomers. 21 It has been suggested that such increased stability might be the result of a more acceptable backbone conformation within the DE-loop. 21 Such observations prompted the design of other DE-loop mutants.…”

Section: Introductionmentioning

confidence: 99%

“…21 It has been suggested that such increased stability might be the result of a more acceptable backbone conformation within the DE-loop. 21 Such observations prompted the design of other DE-loop mutants. The D59P mutant confirmed that mutations affecting the backbone conformational strain of the DE-loop may have long-range effects.…”

Section: Introductionmentioning

confidence: 99%

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DE‐loop mutations affect β2 microglobulin stability, oligomerization, and the low‐pH unfolded form

et al. 2010

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b2 microglobulin (b2m) is the light chain of class-I major histocompatibility complex (MHC-I). Its accumulation in the blood of patients affected by kidney failure leads to amyloid deposition around skeletal joints and bones, a severe condition known as Dialysis Related Amyloidosis (DRA). In an effort to dissect the structural determinants of b2m aggregation, several b2m mutants have been previously studied. Among these, three single-residue mutations in the loop connecting strands D and E (W60G, W60V, D59P) have been shown to affect b2m amyloidogenic properties, and are here considered. To investigate the biochemical and biophysical properties of wild-type (w.t.) b2m and the three mutants, we explored thermal unfolding by Trp fluorescence and circular dichroism (CD). The W60G mutant reveals a pronounced increase in conformational stability. Protein oligomerization and reduction kinetics were investigated by electrospray-ionization mass spectrometry (ESI-MS). All the mutations analyzed here reduce the protein propensity to form soluble oligomers, suggesting a role for the DE-loop in intermolecular interactions. A partially folded intermediate, which may be involved in protein aggregation induced by acids, accumulates for all the tested proteins at pH 2.5 under oxidizing conditions. Moreover, the kinetics of disulfide reduction reveals specific differences among the tested mutants. Thus, b2m DE-loop mutations display long-range effects, affecting stability and structural properties of the native protein and its low-pH intermediate. The evidence presented here hints to a crucial role played by the DE-loop in determining the overall properties of native and partially folded b2m.

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supporting

confidence: 91%

Section: Thermal Stabilitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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