Modular Helix–Coil–Helix Architecture in an Aib-Rich Lipidated Peptide: Biomacromolecular Insights from High-Field NMR

Tirzepatide is a next-generation dual GLP-1/GIP agonist whose therapeutic performance has been attributed to its engineered amphiphilicity, α-aminoisobutyric acid (Aib)–stabilized segments, and site-specific lipid anchoring. However, its molecular conformational landscape in solution has remained unresolved. Here, we combine high-field 1.2 GHz NMR spectroscopy, comprehensive residue-specific assignments, secondary chemical-shift analysis, and automated CYANA modeling to define the solution-state secondary-structure organization and conformational preferences of tirzepatide. The ¹H–¹⁵N HSQC spectrum resolves 27 backbone amide correlations, revealing two persistent α-helical segments (F6–I12 and I27–S33) within a dynamic yet non-random amphipathic ensemble. Aib residues impose a strong helical bias and reduce local flexibility, while the lipidated Lys²⁰ pre-organizes hydrophobic clustering without perturbing backbone detectability. The density of long-range and medium-range NOEs, combined with characteristic Δδ(Cα–Cβ) patterns, establishes a partially folded topology stabilized by aromatic packing and Aib-driven conformational restriction. Collectively, these data demonstrate that tirzepatide exists not as an extended random coil but as a pre-organized, segmentally ordered peptide whose amphiphilic design encodes its structural preferences in solution. This work provides a molecular framework for understanding incretin dual-agonist peptides and sets generalizable design rules for engineering next-generation Aib-rich and lipid-modified therapeutics scaffolds.