The effect of mutation on an aggregation-prone protein: An in vivo, in vitro, and in silico analysis

Abstract

Significance Protein aggregation is a major problem for human health. However, our understanding of how folded proteins aggregate into amyloid lags behind. Using the tripartite β-lactamase assay (TPBLA) with our test protein, β 2 -microglobulin (β 2 m), we show the ability to differentiate the behavior of single-point variants and highlight the remarkable sensitivity to the identity of the residue at position 76. After evolving the aggregation-prone protein, D76N-β 2 m, the only mutations able to improve D76N-β 2 m behavior in vivo involve residues in a single 7-residue sequence of the protein. Further characterization in vitro shows that a single-point mutant in this region can abolish D76N-β 2 m aggregation.

Dimers of D76N-β2-microglobulin display potent antiamyloid aggregation activity

“…To initiate our studies of D76N-β 2 m assembly, we measured the rate of aggregation of the protein into amyloid using thioflavin T (ThT) fluorescence and compared the behavior of the protein with that of WT-β 2 m. The experiments were performed at pH 6.2 in 25 mM sodium phosphate buffer supplemented with 115 mM NaCl—conditions identical to those used previously to determine the aggregation mechanism of ΔN6-β 2 m ( 27 ). Consistent with our previous results ( 16 , 17 ), D76N-β 2 m aggregates into amyloid rapidly under these conditions, with a T half (time to reach 50% of the maximum ThT signal) of 17.3 ± 2.4 h and reaching a plateau after ∼20 h, while WT-β 2 m did not form ThT-positive fibrils within the 42 h timescale of this experiment ( Fig. 1 B ).…”

“…The new Cys residues were inserted into solvent-exposed loops in the protein, in order to avoid alterations in the structure of the native protein. Sequence changes in the major aggregation-prone region of the protein (residues 60–66), shown previously to be the only region to affect the rate of aggregation into amyloid using mutational scanning, were also avoided ( 16 ). Indeed, control experiments in which each Cys-containing protein was modified with (diamagnetic) MTSL showed that all variants are natively folded at the temperature of the NMR PRE experiments (25 °C), despite being thermodynamically destabilized ( Figs.…”

“…We demonstrate the structural integrity of labeled proteins herein using CD, NMR, and thermal stability measurements. Importantly, previous studies of 56 variants of D76N-β 2 m, obtained using random mutagenesis and screening for sequences with altered aggregation potential, have revealed that the presence of a single region, spanning residues 60 to 66, alongside Asn at residue 76, is essential for amyloid formation, with mutations elsewhere in the protein having little effect ( 16 ). Importantly, the inhibitory dimers identified here do not involve this critical region (which forms the E-strand in the native structure ( Fig.…”

“…1 A ). The D76N-β 2 m variant retains a native Ig fold (RMSD compared with WT-β 2 m of 0.59 Å (C α atoms) ( 14 )) is less stable than WT-β 2 m (ΔΔG° = 8.36 kJ/mol at pH 7.4 ( 15 , 16 )) and aggregates more rapidly than the normally highly aggregation-resistant WT protein in vitro and in vivo ( 14 , 15 , 17 ). The variant also changes the phenotype of the disease, resulting in the deposition of D76N-β 2 m amyloid fibrils in the liver, spleen, salivary glands, and heart without renal dysfunction ( 14 ).…”

Disease-relevant β2-microglobulin variants share a common amyloid fold

“…1). Despite substantial research effort 44 , however, including recent design and selection of >60 variants 45 , why this single amino acid substitution (but not similar single substitutions elsewhere in the protein) has such a dramatic effect on the amyloid potential of the protein remains unclear. Patients bearing the β 2 m-D76N mutation are heterozygous, and the serum concentration of β 2 m is not increased 33 .…”