Contributions of a Surface Hydrophobic Cluster to the Folding and Structural Stability of Ubiquitin

• Published in: Journal of the Chinese Chemical Society (Taipei) 2008-08, Vol.55 (4), p.772-781
• Main Authors: Chen, Rita P.-Y., Liang, Fu-Cheng, Lee, Chung-Tien, Zerella, Rosa, Chan, Sunney I.
• Format: Article
• Language: English
• Subjects: Hairpin; Hydrophobic interaction; Kinetics; Protein folding; Ubiquitin
• ISSN: 0009-4536; 2192-6549
• DOI: 10.1002/jccs.200800116

The role of the small exterior hydrophobic cluster (SEHC) in the strand region of the N‐terminal β‐hairpin of ubiquitin on the structural stability and the folding/unfolding kinetics of the protein have been examined. We introduce a Phe→Ala substitution at residue 4 in the strand region of the N‐terminal β‐hairpin of the ubiquitin. A peptide with the same amino acid sequence as the first 21 residues of the mutated ubiquitin has also been synthesized. The F4A mutation unfolds the hairpin structure of the peptide segment without disruption of the turn. The same mutation does not seem to affect the overall structure, but the stability of the mutated full‐length protein decreases by approx. 2 kcal/mol. Kinetically, the entire hairpin structure is implicated in the transition state during folding of the wild type protein. The rate of refolding is retarded by the F4A mutation in ∼80% of the protein molecules. The F4A substitution also increases the unfolding rate of the protein by 10 fold. Thus the hydrophobic side‐chain of Phe‐4 not only contributes to the stability of the hairpin, but also to the stability of the entire protein by forming a cluster together with the hydrophobic residues on the C‐terminal strand. The role of the small exterior hydrophobic cluster (SEHC) in the strand region of the N‐terminal b‐hairpin of ubiquitin on the structural stability and the folding/unfolding kinetics of the protein have been examined. We introduce a Phe®Ala substitution at residue 4 in the strand region of the N‐terminal b‐hairpin of the ubiquitin. A peptide with the same amino acid sequence as the first 21 residues of the mutated ubiquitin has also been synthesized. The F4A mutation unfolds the hairpin structure of the peptide segment without disruption of the turn. The same mutation does not seem to affect the overall structure, but the stability of the mutated full‐length protein decreases by approx. 2 kcal/mol. Kinetically, the entire hairpin structure is implicated in the transition state during folding of the wild type protein. The rate of refolding is retarded by the F4A mutation in ∼80% of the protein molecules. The F4A substitution also increases the unfolding rate of the protein by 10 fold. Thus the hydrophobic side‐chain of Phe‐4 not only contributes to the stability of the hairpin, but also to the stability of the entire protein by forming a cluster together with the hydrophobic residues on the C‐terminal strand.