Probing the channel-bound shaker B inactivating peptide by stereoisomeric substitution at a strategic tyrosine residue.
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 BDelta6-46 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 beta-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 beta-hairpin.
Encinar
J A
JA
Instituto de Biologia Molecular y Celular, Universidad Miguel Hernández, Elche, 03202 Alicante, Spain.
Fernández
A M
AM
Poveda
J A
JA
Molina
M L
ML
Albar
J P
JP
Gavilanes
F
F
Gonzalez-Ros
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002152:Calorimetry, Differential Scanning; D006224:Cricetinae; D018360:Crystallography, X-Ray; D047908:Intracellular Signaling Peptides and Proteins; D008051:Lipid Bilayers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry*
D047908:Intracellular Signaling Peptides and Proteins; D008051:Lipid Bilayers; D010455:Peptides; C076103:Shaker B inactivating peptide; D014443:Tyrosine