Biophysical Journal 72: 554-566 (1997)
© 1997 the Biophysical Society

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Conformation of a Protein Kinase C Substrate, NG(28-43), and Its Analog in Aqueous and Sodium Dodecyl Sulfate Micell Solutions

Institute of Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China

ABSTRACT

A peptide corresponding to the neuronal protein neurogranin (NG) residues 28-43, NG(28-43), and its analog, [A³⁵]NG(28-43), have been investigated by NMR, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies. The peptides existed in aqueous solution predominantly in radom form. However, a nascent helical structure was detected in the central region of the parent peptide from NMR data. Furthermore, a helical structure can be detected for both peptides with greater induced secondary structure for the parent peptide in the presence of sodium dodecyl sulfate (SDS) micelle. The formation of micelles for SDS was confirmed by results from EPR as well as ¹³C NMR. As shown by CD experiments, helical conformer was induced for NG(28-43) in vesicular solution containing phosphatidyl serine (PS), whereas no helix can be discerned for the peptide in phosphatidyl choline (PC)-containing vesicular solution. Together with the induction of the peptide into helix in SDS micellar solution as suggested by both NMR and CD data, these results underscored the electrostatic contribution to the interaction of the PKC substrate peptides and proteins with membrane. According to NMR and CD data, a dynamic equilibrium existed between free and micelle-bound states for the peptide. Moreover, proton-deuterium exchange results and SDS-induced linewidth broadening of proton resonances allowed delineation of the orientation of the amphipathic helix on the surface of SDS micelle. The result was supported by spin label experiments that indicated F35 of NG(28-43) interacted strongly with the hydrocarbon interior of micelle. Based on the experimental findings, a working model was proposed that attempted to partly explain the roles played by the nonpolar amino acid near the phosphorylation site, by the negatively charged phospholipids, and by the basic amino acids of the substrate.