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

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

Backbone dynamics of a biologically active human FGF-1 monomer, complexed to a hexasaccharide heparin-analogue, by N NMR relaxation methods

The binding site and backbone dynamics of a bioactive complex formed by the acidic fibroblast growth factor (FGF-1) and a specifically designed heparin hexasaccharide has been investigated by HSQC and relaxation NMR methods. The comparison of the relaxation data for the free and bound states has allowed showing that the complex is monomeric, and still induces mutagenesis, and that the protein backbone presents reduced motion in different timescale in its bound state, except in certain points that are involved in the interaction with the fibroblast growth factor receptor (FGFR)