Probing the Channel-Bound Shaker B Inactivating Peptide by Stereoisomeric Substitution at a Strategic Tyrosine Residue

• Published in: Biochemistry (Easton) 2003-07, Vol.42 (29), p.8879-8884
• Main Authors: Encinar, J. A, Fernández, A. M, Poveda, J. A, Molina, M. L, Albar, J. P, Gavilanes, F, Gonzalez-Ros, J. M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Calorimetry, Differential Scanning; CHO Cells; Cricetinae; Crystallography, X-Ray; Lipid Bilayers; Models, Molecular; Molecular Sequence Data; Mutation; Peptides - chemistry; Protein Conformation; Sequence Homology, Amino Acid; Stereoisomerism; Temperature; Tyrosine - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0343121

A synthetic peptide patterned after the sequence of the inactivating ball domain of the Shaker B K+ channel, the ShB peptide, fully restores fast inactivation in the deletion Shaker BΔ6−46 K+ channel, which lacks the constitutive ball domains. On the contrary, a similar peptide in which tyrosine 8 is substituted by the secondary structure-disrupting d-tyrosine stereoisomer does not. This suggests that the stereoisomeric substitution prevents the peptide from adopting a structured conformation when bound to the channel during inactivation. Moreover, characteristic in vitro features of the wild-type ShB peptide such as the marked propensity to adopt an intramolecular β-hairpin structure when challenged by anionic phospholipid vesicles, a model target mimicking features of the inactivation site in the channel protein, or to insert into their hydrophobic bilayers, are lost in the d-tyrosine-containing peptide, whose behavior is practically identical to that of noninactivating peptide mutants. In the absence of high resolution crystallographic data on the inactivated channel/peptide complex, these latter findings suggest that the structured conformation required for the peptide to promote channel inactivation, as referred to above, is likely to be β-hairpin.