Organized polymeric assemblies that incorporate bioactive sequences and structures are finding important applications for the study of protein structure-function relationships. We have recently described a heteropolymeric peptide-amphiphile system that forms organized structures in solution and on surfaces. While the overall three-dimensional features of peptide-amphiphiles have been studied previously, the precise environment of specific residues, particularly those within biologically active regions, have not been examined in detail. In the present study, we have used heteronuclear single quantum coherence (HSQC) and inverse-detected 1H-15N NMR spectroscopy to examine the structure and dynamics of a peptide and peptide-amphiphile that incorporate the α1(IV)1263-1277 ([IV-H1]) amino acid sequence from type IV collagen. Three variants of the sequence (Gly-Pro- Hyp)4-[IV-H1]-(Gly-Pro-Hyp)4 were constructed with a single 15N-labeled Gly placed in the middle of the N-terminal (Gly-Pro-Hyp)4 region (residue Gly7), in the middle of the [IV-H1] sequence (residue Gly19), or in the middle of the C-terminal (Gly-Pro-Hyp)4 region (residue Gly34). These peptides were also N-terminally acylated with hexanoic acid to create an analogous series of 15N-labeled peptide-amphiphiles. HSQC spectra indicated that both the peptide and the peptide-amphiphile were in triple-helical conformation at low temperature, supporting prior circular dichroism (CD) spectroscopic results. The intensities of the triple-helical cross-peaks were stronger for the peptide-amphiphile, consistent with an enhanced triple- helical thermal stability within the peptide-amphiphile construct compared to that of the peptide alone. Relative relaxation values for the peptide- amphiphile monomeric and trimeric species were consistent with those reported previously for other triple-helical peptides. Relaxation measurements indicated that the triple-helical [IV-H1] region did not appear to be dramatically more flexible than the Gly-Pro-Hyp regions. The angle between Gly N-H bonds and the helix dyad axis, determined from the relaxation data, was within the range expected for triple helices. Overall, the peptide headgroup of the C6-(Gly-Pro-Hyp)4-[IV-H1]-(Gly-Pro-Hyp)4 peptide- amphiphile appears to form a continuous triple helix that behaves similarly, in a dynamic sense, to a triple-helical peptide. The enhanced thermal stability of the peptide-amphiphile compared to the analogous triple-helical peptide, along with the multitude of organized structures formed by lipidlike compounds, suggest that peptide-amphiphiles could be utilized as targeted liposomes, sensors, receptors, or enzymes.