Bergerac-SH3: "frustation" induced by stabilizing the folding nucleus.
The influence of an inserted exogenous independent folding element on the thermodynamics and folding properties of SH3 domain from alpha-spectrin has been investigated by creating a fused form between this small all-beta domain and a stable beta-hairpin (BH19). NMR analysis of synthetic peptides shows that insertion of BH19 nucleates formation of the original natural beta-hairpin (distal loop) that is part of the SH3 folding nucleus. The resulting protein (Bergerac-SHH) is more stable, folds faster and contains an elongated hairpin protruding from the globular domain as determined by 2D-NMR. "Protein engineering" analysis of the inserted region shows that it is folded in the transition state. Interestingly, stabilisation by insertion of the distal loop region results in the appearance of a compact intermediate revealed by a curved chevron plot at low denaturant concentration. This effect is eliminated at low salt concentrations by a single mutation of a hydrophobic residue within BH19 sequence, which is most probably involved in non-native interactions. Local stabilisation by enlargement and reinforcement of the folding nucleus, global compaction by the addition of salt and non-native interactions are shown to contribute to the observed deviation from the two-state behaviour.
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Unidad de Biofisica-CSIC-UPV, Apdo. 644, Bilbao, 48080, Spain.
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D000595:Amino Acid Sequence; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D009994:Osmolar Concentration; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D018909:src Homology Domains / Q000235:genetics
D010455:Peptides; D011993:Recombinant Fusion Proteins; D013049:Spectrin