Modulation of S6 fibrillation by unfolding rates and gatekeeper residues

TY - JOUR

T1 - Modulation of S6 fibrillation by unfolding rates and gatekeeper residues

AU - Pedersen, J. S.

AU - Christensen, Gunna

AU - Otzen, Daniel Erik

PY - 2004/8/6

Y1 - 2004/8/6

N2 - We present a protein engineering analysis of the fibrillation of a protein from a thermophilic organism, the 101 residue S6 from Thermus thermophilus. When agitated, S6 fibrillates at pH 2.0 in 0.4 M NaCl. Under these solvent conditions, S6 has native-like secondary structure and also unfolds and refolds cooperatively. However, its tertiary structure appears to be more plastic than at neutral pH, and some regions of the protein may be partially unstructured. At 42°C, there is a lag phase of several days after which fibrillation takes place over several hours. Data from the fibrillation behaviour of a comprehensive series of single and double mutants of S6 suggests that several factors control the onset of fibrillation. Firstly, there appears to be a contiguous region of "gatekeeper" residues that inhibit fibrillation, since their truncation significantly reduces the duration of the lag phase. This region overlaps extensively with the partially unstructured region of the protein, suggesting that residues with enhanced flexibility and solvent-accessibility are important for the initiation of fibrillation. Secondly, longer lag phases correlate with faster rates of unfolding. We interpret this to mean that kinetic stability also controls fibrillation but in the sense that the quasi-native state, rather than the denatured state, is the species that participates in nucleation. This implies that fibrillation can also occur from a quasi-native state as opposed to an ensemble of highly fluctuating structures, and highlights the delicate balance between flexibility and structure required to form organized assemblies of polypeptide chains.

AB - We present a protein engineering analysis of the fibrillation of a protein from a thermophilic organism, the 101 residue S6 from Thermus thermophilus. When agitated, S6 fibrillates at pH 2.0 in 0.4 M NaCl. Under these solvent conditions, S6 has native-like secondary structure and also unfolds and refolds cooperatively. However, its tertiary structure appears to be more plastic than at neutral pH, and some regions of the protein may be partially unstructured. At 42°C, there is a lag phase of several days after which fibrillation takes place over several hours. Data from the fibrillation behaviour of a comprehensive series of single and double mutants of S6 suggests that several factors control the onset of fibrillation. Firstly, there appears to be a contiguous region of "gatekeeper" residues that inhibit fibrillation, since their truncation significantly reduces the duration of the lag phase. This region overlaps extensively with the partially unstructured region of the protein, suggesting that residues with enhanced flexibility and solvent-accessibility are important for the initiation of fibrillation. Secondly, longer lag phases correlate with faster rates of unfolding. We interpret this to mean that kinetic stability also controls fibrillation but in the sense that the quasi-native state, rather than the denatured state, is the species that participates in nucleation. This implies that fibrillation can also occur from a quasi-native state as opposed to an ensemble of highly fluctuating structures, and highlights the delicate balance between flexibility and structure required to form organized assemblies of polypeptide chains.

KW - [GdmCl} , midpoint GdmCl concentration of denaturation

KW - D, denatured state

KW - fibrillation

KW - flexibility

KW - GdmCl, guanidinium chloride

KW - kinetics

KW - protein engineering

KW - TFE, trifluoroethanol

KW - unfolding

UR - http://www.scopus.com/inward/record.url?scp=3342902033&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2004.06.020

DO - 10.1016/j.jmb.2004.06.020

M3 - Journal article

C2 - 15276845

AN - SCOPUS:3342902033

SN - 0022-2836

VL - 341

SP - 575

EP - 588

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 2

ER -