Polyproline II (PPII) helices play vital roles in biochemical recognition events and structures like collagen and form part of the conformational landscapes of intrinsically disordered proteins (IDPs). Nevertheless, this structure is generally hard to detect and quantify. Here, we report the first thorough NMR characterization of a PPII helical bundle protein, the Hypogastrura harveyi "snow flea" antifreeze protein (sfAFP). J-couplings and nuclear Overhauser enhancement spectroscopy confirm a natively folded structure consisting of six PPII helices. NMR spectral analyses reveal quite distinct Hα2 versus Hα3 chemical shifts for 28 Gly residues as well as 13Cα, 15N, and 1HN conformational chemical shifts (Δδ) unique to PPII helical bundles. The 15N Δδ and 1HN Δδ values and small negative 1HN temperature coefficients evince hydrogen-bond formation. 1H-15N relaxation measurements reveal that the backbone structure is generally highly rigid on ps-ns time scales. NMR relaxation parameters and biophysical characterization reveal that sfAFP is chiefly a dimer. For it, a structural model featuring the packing of long, flat hydrophobic faces at the dimer interface is advanced. The conformational stability, measured by amide H/D exchange to be 6.24 ± 0.2 kcal·mol-1, is elevated. These are extraordinary findings considering the great entropic cost of fixing Gly residues and, together with the remarkable upfield chemical shifts of 28 Gly 1Hα, evidence significant stabilizing contributions from CαHα ||| O═C hydrogen bonds. These stabilizing interactions are corroborated by density functional theory calculations and natural bonding orbital analysis. The singular conformational chemical shifts, J-couplings, high hNOE ratios, small negative temperature coefficients, and slowed H/D exchange constitute a unique set of fingerprints to identify PPII helical bundles, which may be formed by hundreds of Gly-rich motifs detected in sequence databases. These results should aid the quantification of PPII helices in IDPs, the development of improved antifreeze proteins, and the incorporation of PPII helices into novel designed proteins.