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Fibril growth kinetics reveal a region of beta2-microglobulin important for nucleation and elongation of aggregation.

Platt GW, Routledge KE, Homans SW, Radford SE - J. Mol. Biol. (2008)

Bottom Line: We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type beta(2)m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62-70) that has been predicted to be highly amyloidogenic.The results reveal the importance of aromatic residues in this part of the beta(2)m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation.No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

ABSTRACT
Amyloid is a highly ordered form of aggregate comprising long, straight and unbranched proteinaceous fibrils that are formed with characteristic nucleation-dependent kinetics in vitro. Currently, the structural molecular mechanism of fibril nucleation and elongation is poorly understood. Here, we investigate the role of the sequence and structure of the initial monomeric precursor in determining the rates of nucleation and elongation of human beta(2)-microglobulin (beta(2)m). We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type beta(2)m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62-70) that has been predicted to be highly amyloidogenic. The results reveal the importance of aromatic residues in this part of the beta(2)m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation. Structural analysis of the conformational properties of the unfolded monomer for each variant using NMR relaxation methods revealed that all variants contain significant non-random structure involving two hydrophobic clusters comprising regions 29-51 and 58-79, the extent of which is critically dependent on the sequence. No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state. Together, the data suggest a model for beta(2)m amyloid formation in which structurally specific interactions involving the highly hydrophobic and aromatic-rich region comprising residues 62-70 provide a complementary interface that is key to the generation of amyloid fibrils for this protein at acidic pH.

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Average R2 rates for all of the variants studied here. ‘Trip’ refers to the triple mutant F62AY63AY67A. (a) R2 values for the hydrophobic cluster centred at residue 70 for each variant, obtained from average values for residues 61, 68, 73 and 75. (b) R2 values for the hydrophobic cluster centred at residue 40 for each variant (based on average R2 values for residues 33, 34, 41 and 43 that lie in this cluster). (c) Interaction between the two clusters demonstrated by plotting the average R2 values for each cluster in each variant against each other.
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fig3: Average R2 rates for all of the variants studied here. ‘Trip’ refers to the triple mutant F62AY63AY67A. (a) R2 values for the hydrophobic cluster centred at residue 70 for each variant, obtained from average values for residues 61, 68, 73 and 75. (b) R2 values for the hydrophobic cluster centred at residue 40 for each variant (based on average R2 values for residues 33, 34, 41 and 43 that lie in this cluster). (c) Interaction between the two clusters demonstrated by plotting the average R2 values for each cluster in each variant against each other.

Mentions: To determine the effect of the amino acid substitutions on the structure and dynamics of the unfolded state of β2m at pH 2.5 (the initial starting point of the aggregation reactions described below), 1H–15N heteronuclear single quantum coherence (HSQC) spectra of wild-type β2m and the 12 variants were acquired, and the relaxation properties of individual resonances were determined. The resulting spectra indicated that all 13 proteins are highly unfolded at pH 2.5, with the majority of residues giving rise to intense resonances with little chemical shift dispersion (Fig. 2a–c). Consistent with previous studies of the wild-type protein at this pH,11,21 measurement of the R2 relaxation rates revealed a number of resonances that are significantly broadened, whilst others are broadened beyond detection. These cluster into two regions of the sequence involving residues ∼ 29–51 and ∼ 58–79, consistent with these regions deviating from a random structure under these conditions (Fig. 2d–f). Akin to the properties of the wild-type protein (Fig. 2a and d),21 residues 62–70 in all of the variants (corresponding to β-strand E in native β2m; Fig. 1a) show the most broadened resonances, whilst the second group of residues (29–51) also displays significant broadening, consistent with the presence of two hydrophobic clusters in the acid-unfolded state. Importantly, substitution of residues in the region 62–70 not only affects the dynamics within its own cluster but also perturbs those of the residues in the cluster centred on residue 40, indicating that these two regions are mutually interacting (Fig. 3a–c). By contrast, Ile7, a residue that is highly dynamic in the acid-unfolded state and is not involved in the residual structure, has little effect on the dynamics of either cluster. Finally, and by contrast with the small to moderate changes in R2 observed when single point mutants are introduced into the region 62–70, reducing the aromatic content of this region substantially by replacing three aromatic residues with alanine simultaneously (F62AY63AY67A, hereinafter referred to as the triple mutant) results in the greatest reduction in R2 values in both clusters (Fig. 3a and b). Therefore, over the range of amino acid changes introduced, hydrophobic clustering involving residues 62–70 has been titrated from a behaviour similar to that of wild-type β2m (e.g., I7A), through proteins with partially disrupted clusters (as is the case with most of the single point variants in the region 62–70), to the triple mutant that is the most highly dynamic species of this set (Fig. 3a), enabling the importance of residual structure in this region for amyloid assembly to be explored.


Fibril growth kinetics reveal a region of beta2-microglobulin important for nucleation and elongation of aggregation.

Platt GW, Routledge KE, Homans SW, Radford SE - J. Mol. Biol. (2008)

Average R2 rates for all of the variants studied here. ‘Trip’ refers to the triple mutant F62AY63AY67A. (a) R2 values for the hydrophobic cluster centred at residue 70 for each variant, obtained from average values for residues 61, 68, 73 and 75. (b) R2 values for the hydrophobic cluster centred at residue 40 for each variant (based on average R2 values for residues 33, 34, 41 and 43 that lie in this cluster). (c) Interaction between the two clusters demonstrated by plotting the average R2 values for each cluster in each variant against each other.
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Show All Figures
getmorefigures.php?uid=PMC2627305&req=5

fig3: Average R2 rates for all of the variants studied here. ‘Trip’ refers to the triple mutant F62AY63AY67A. (a) R2 values for the hydrophobic cluster centred at residue 70 for each variant, obtained from average values for residues 61, 68, 73 and 75. (b) R2 values for the hydrophobic cluster centred at residue 40 for each variant (based on average R2 values for residues 33, 34, 41 and 43 that lie in this cluster). (c) Interaction between the two clusters demonstrated by plotting the average R2 values for each cluster in each variant against each other.
Mentions: To determine the effect of the amino acid substitutions on the structure and dynamics of the unfolded state of β2m at pH 2.5 (the initial starting point of the aggregation reactions described below), 1H–15N heteronuclear single quantum coherence (HSQC) spectra of wild-type β2m and the 12 variants were acquired, and the relaxation properties of individual resonances were determined. The resulting spectra indicated that all 13 proteins are highly unfolded at pH 2.5, with the majority of residues giving rise to intense resonances with little chemical shift dispersion (Fig. 2a–c). Consistent with previous studies of the wild-type protein at this pH,11,21 measurement of the R2 relaxation rates revealed a number of resonances that are significantly broadened, whilst others are broadened beyond detection. These cluster into two regions of the sequence involving residues ∼ 29–51 and ∼ 58–79, consistent with these regions deviating from a random structure under these conditions (Fig. 2d–f). Akin to the properties of the wild-type protein (Fig. 2a and d),21 residues 62–70 in all of the variants (corresponding to β-strand E in native β2m; Fig. 1a) show the most broadened resonances, whilst the second group of residues (29–51) also displays significant broadening, consistent with the presence of two hydrophobic clusters in the acid-unfolded state. Importantly, substitution of residues in the region 62–70 not only affects the dynamics within its own cluster but also perturbs those of the residues in the cluster centred on residue 40, indicating that these two regions are mutually interacting (Fig. 3a–c). By contrast, Ile7, a residue that is highly dynamic in the acid-unfolded state and is not involved in the residual structure, has little effect on the dynamics of either cluster. Finally, and by contrast with the small to moderate changes in R2 observed when single point mutants are introduced into the region 62–70, reducing the aromatic content of this region substantially by replacing three aromatic residues with alanine simultaneously (F62AY63AY67A, hereinafter referred to as the triple mutant) results in the greatest reduction in R2 values in both clusters (Fig. 3a and b). Therefore, over the range of amino acid changes introduced, hydrophobic clustering involving residues 62–70 has been titrated from a behaviour similar to that of wild-type β2m (e.g., I7A), through proteins with partially disrupted clusters (as is the case with most of the single point variants in the region 62–70), to the triple mutant that is the most highly dynamic species of this set (Fig. 3a), enabling the importance of residual structure in this region for amyloid assembly to be explored.

Bottom Line: We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type beta(2)m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62-70) that has been predicted to be highly amyloidogenic.The results reveal the importance of aromatic residues in this part of the beta(2)m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation.No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

ABSTRACT
Amyloid is a highly ordered form of aggregate comprising long, straight and unbranched proteinaceous fibrils that are formed with characteristic nucleation-dependent kinetics in vitro. Currently, the structural molecular mechanism of fibril nucleation and elongation is poorly understood. Here, we investigate the role of the sequence and structure of the initial monomeric precursor in determining the rates of nucleation and elongation of human beta(2)-microglobulin (beta(2)m). We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type beta(2)m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62-70) that has been predicted to be highly amyloidogenic. The results reveal the importance of aromatic residues in this part of the beta(2)m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation. Structural analysis of the conformational properties of the unfolded monomer for each variant using NMR relaxation methods revealed that all variants contain significant non-random structure involving two hydrophobic clusters comprising regions 29-51 and 58-79, the extent of which is critically dependent on the sequence. No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state. Together, the data suggest a model for beta(2)m amyloid formation in which structurally specific interactions involving the highly hydrophobic and aromatic-rich region comprising residues 62-70 provide a complementary interface that is key to the generation of amyloid fibrils for this protein at acidic pH.

Show MeSH
Related in: MedlinePlus