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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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Schematic diagram of a possible mechanism for β2m aggregation at pH 7.0 and pH 2.5. Aggregation at pH 7.0 proceeds via a native-like intermediate.44 At pH 2.5, β2m is highly unfolded, with the residues in the region 62–70 participating in a non-native hydrophobic cluster. This is shown in the figure by circles coloured as in Fig. 5. A single disulphide bond (yellow) links residues 25–80 and is essential for fibril formation.12 In the initial stages of fibril formation, we propose that residues 62–70 form one key complementing surface involved in both the nucleation and the elongation phases of fibril growth and may provide a target for therapeutic molecules to disrupt amyloid formation. Since the fibrils formed from a native-like state at neutral pH and those formed from an acid-denatured state are morphologically indistinguishable,47 the fibril formation pathways presumably must converge, although the point at which they do so remains uncertain. The precise details of the size of the nucleus and the structure of the fibrils remain unresolved.
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fig7: Schematic diagram of a possible mechanism for β2m aggregation at pH 7.0 and pH 2.5. Aggregation at pH 7.0 proceeds via a native-like intermediate.44 At pH 2.5, β2m is highly unfolded, with the residues in the region 62–70 participating in a non-native hydrophobic cluster. This is shown in the figure by circles coloured as in Fig. 5. A single disulphide bond (yellow) links residues 25–80 and is essential for fibril formation.12 In the initial stages of fibril formation, we propose that residues 62–70 form one key complementing surface involved in both the nucleation and the elongation phases of fibril growth and may provide a target for therapeutic molecules to disrupt amyloid formation. Since the fibrils formed from a native-like state at neutral pH and those formed from an acid-denatured state are morphologically indistinguishable,47 the fibril formation pathways presumably must converge, although the point at which they do so remains uncertain. The precise details of the size of the nucleus and the structure of the fibrils remain unresolved.

Mentions: It has previously been observed that β2m forms amyloid-like fibrils at pH 7 via an on-pathway folding intermediate (Fig. 7).44 This aggregation-prone species possesses a highly native-like structure according to chemical shift analysis of NMR spectra, but contains a trans His31–Pro32 peptide bond compared with the cis isomer found in native β2m.39 Despite analysis of this early aggregation-prone species, the mechanism of further assembly steps remains elusive. Interestingly, a key role for the region encompassing residues 62–70 in the aggregation of β2m at acidic pH is consistent with the increased amyloidogenicity observed under physiological conditions for the derivative containing a single cleavage of the polypeptide chain at Lys58.48 In this variant, the aromatic-rich region of the protein involving residues 62–70 may be less conformationally constrained, increasing the opportunity for it to participate in intermolecular interactions. How the aggregation mechanism under acidic conditions relates to that under physiological conditions is currently unresolved, and further experiments will be needed to determine directly the role of residues 62–70 in this pathway. Interestingly, there are a large number of amino acids bearing carboxyl groups within the 58–79 region (Asp59, Glu69, Glu74, Asp76 and Glu77) that, whilst most likely neutral under the acidic conditions of this study, are presumably charged under physiological conditions. Thus, electrostatic repulsion of these side chains may account for the slower aggregation rates of β2m at neutral pH.


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)

Schematic diagram of a possible mechanism for β2m aggregation at pH 7.0 and pH 2.5. Aggregation at pH 7.0 proceeds via a native-like intermediate.44 At pH 2.5, β2m is highly unfolded, with the residues in the region 62–70 participating in a non-native hydrophobic cluster. This is shown in the figure by circles coloured as in Fig. 5. A single disulphide bond (yellow) links residues 25–80 and is essential for fibril formation.12 In the initial stages of fibril formation, we propose that residues 62–70 form one key complementing surface involved in both the nucleation and the elongation phases of fibril growth and may provide a target for therapeutic molecules to disrupt amyloid formation. Since the fibrils formed from a native-like state at neutral pH and those formed from an acid-denatured state are morphologically indistinguishable,47 the fibril formation pathways presumably must converge, although the point at which they do so remains uncertain. The precise details of the size of the nucleus and the structure of the fibrils remain unresolved.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2627305&req=5

fig7: Schematic diagram of a possible mechanism for β2m aggregation at pH 7.0 and pH 2.5. Aggregation at pH 7.0 proceeds via a native-like intermediate.44 At pH 2.5, β2m is highly unfolded, with the residues in the region 62–70 participating in a non-native hydrophobic cluster. This is shown in the figure by circles coloured as in Fig. 5. A single disulphide bond (yellow) links residues 25–80 and is essential for fibril formation.12 In the initial stages of fibril formation, we propose that residues 62–70 form one key complementing surface involved in both the nucleation and the elongation phases of fibril growth and may provide a target for therapeutic molecules to disrupt amyloid formation. Since the fibrils formed from a native-like state at neutral pH and those formed from an acid-denatured state are morphologically indistinguishable,47 the fibril formation pathways presumably must converge, although the point at which they do so remains uncertain. The precise details of the size of the nucleus and the structure of the fibrils remain unresolved.
Mentions: It has previously been observed that β2m forms amyloid-like fibrils at pH 7 via an on-pathway folding intermediate (Fig. 7).44 This aggregation-prone species possesses a highly native-like structure according to chemical shift analysis of NMR spectra, but contains a trans His31–Pro32 peptide bond compared with the cis isomer found in native β2m.39 Despite analysis of this early aggregation-prone species, the mechanism of further assembly steps remains elusive. Interestingly, a key role for the region encompassing residues 62–70 in the aggregation of β2m at acidic pH is consistent with the increased amyloidogenicity observed under physiological conditions for the derivative containing a single cleavage of the polypeptide chain at Lys58.48 In this variant, the aromatic-rich region of the protein involving residues 62–70 may be less conformationally constrained, increasing the opportunity for it to participate in intermolecular interactions. How the aggregation mechanism under acidic conditions relates to that under physiological conditions is currently unresolved, and further experiments will be needed to determine directly the role of residues 62–70 in this pathway. Interestingly, there are a large number of amino acids bearing carboxyl groups within the 58–79 region (Asp59, Glu69, Glu74, Asp76 and Glu77) that, whilst most likely neutral under the acidic conditions of this study, are presumably charged under physiological conditions. Thus, electrostatic repulsion of these side chains may account for the slower aggregation rates of β2m at neutral pH.

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