Competition between Intramolecular and Intermolecular Interactions in an Amyloid-Forming Protein

Abstract

Despite much progress in understanding the folding and the aggregation processes of proteins, the rules defining their interplay have yet to be fully defined. This problem is of particular importance since many diseases are initiated by protein unfolding and hence the propensity to aggregate competes with intramolecular collapse and other folding events. Here, we describe the roles of intramolecular and intermolecular interactions in defining the length of the lag time and the apparent rate of elongation of the 100-residue protein human β2-microglobulin at pH 2.5, commencing from an acid-denatured state that lacks persistent structure but contains significant non-random hydrophobic interactions. Using a combination of site-directed mutagenesis, quantitative kinetic analysis and computational methods, we show that only a single region of about 10 residues in length, determines the rate of fibril formation, despite the fact that other regions exhibit a significant intrinsic propensity for aggregation. We rationalise these results by analysing the effect of incorporating the conformational properties of acid-unfolded β2-microglobulin and its variants at pH 2.5 as measured by NMR spectroscopy into the Zyggregator aggregation prediction algorithm. These results demonstrate that residual structure in the precursor state modulates the intrinsic propensity of the polypeptide chain to aggregate and that the algorithm developed here allows the key regions for aggregation to be more clearly identified and the rates of their self-association to be predicted. Given the common propensity of unfolded chains to form non-random intramolecular interactions as monomers and to self-assemble subsequently into amyloid fibrils, the approach developed should find widespread utility for the prediction of regions important in amyloid formation and their rates of self-assembly.

Nucleation of amyloid fibrils

“…Equation (32) parallels that in Ref. 13 and, as can be readily verified, it holds true also for the CNT n* and J from Eqs.…”

“…34,35 So far, a considerable effort has been devoted to understanding how the amino acid sequence of proteins and the experimental conditions affect the kinetics of amyloid fibril formation. 30,32,33,[36][37][38][39][40][41][42][43][44] Experiments that investigate the physiochemical properties of the natural amino acids (such as -propensity, hydrophobicity, aromatic content and charge) have been used to substantiate phenomenological models able to predict changes in the aggregation rate upon mutation as well as to predict amino acid sequences of proteins, so-called hot spots, that are likely to belong to the fibril core. [45][46][47][48][49][50] Although both the experimental studies and the theoretical models show that the kinetic parameters of aggregation depend strongly on the specificity of the amino acid sequence of the protein, it may be expected that this specificity is a particular expression of a common fibril nucleation/growth mechanism which could be treated in the framework of existing general theories of nucleation and growth of new phases.…”

Converting the Highly Amyloidogenic Human Calcitonin into a Powerful Fibril Inhibitor by Three-dimensional Structure Homology with a Non-amyloidogenic Analogue

“…In fact, in vitro fibril formation of globular proteins and of short peptides can be modulated by changes in the amino acid sequence (38,(65)(66)(67)(68). A "structural" view of fibrillation suggests that a way to prevent it is to stabilize an ␣/␤ discordant helix (i.e.…”

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

“…Noteworthy, truncation of b2m after residue 83 impairs aggregation. 19 To gain insight into the correlation between the conformational properties of the precursors with the morphology of resulting amyloid fibrils ( Supporting Information, Figure 6), we determined the amide hydrogen/deuterium (H/D) exchange profile of amyloid fibrils of b2m at pH 2.5 using NMR spectroscopy. Fibrils formed in H 2 O were subjected to solvent exchange in D 2 O for 7 days at 4°C , pD 2.5.…”

“…Importantly, reduction of the disulfide bridge leads to formation of fibrils with different morphology. 10 A very good correlation was found between the solvent protection pattern of the fibrils and the exchange broadening of the molten globule-like intermediate represented by signal intensities in the carbon-detected spectra ( Figure 2): (i) residues 1-20 and 88-99 are least protected in amyloid fibrils of b2m and show the highest signal intensities in the pH 2.5 20 as well as pH 3.6 precursor ( Figures 1 and 2) 10 Many hydrophobic residues are found in these regions, and mutagenesis experiments designed to decrease the hydrophobicity resulted in slower aggregation kinetics, 19,21 suggesting the formation of hydrophobic clusters. Two peptides (residues 21-40, the so-called K3 peptide, and residues 59-71), which overlap with the regions that are strongly exchange broadened in the pH 3.6 intermediate, were reported to self-associate in Vitro.…”