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NMR spectroscopy is the most important technique for understanding the structure of peptides and proteins in solution, providing information at the single-residue and single-atom level. However, written instruction in the interpretation of NMR spectra of peptides and proteins is generally focused on advanced techniques and highly complex spectra, with a lack of simple spectra and guides available for beginning students. In order to address this instructional limitation, we have generated a dataset of 1H NMR spectra of a series of simple peptides that include all canonical amino acids. Peptides examined include Ac-X(S/pS)-NH2, Ac-X(T/pT)-NH2, and Ac-XPPGY-NH2, where X = all encoded amino acids, pS = phosphorylated Ser, and pT = phosphorylated Thr. The characterization of each peptide includes a 1-D spectrum and a TOCSY spectrum, with both the raw and processed available. The spectra can be used for instructional applications including analysis of regions of the spectra (e.g. amide HN, aromatic, Hα, and aliphatic regions); identification of spin systems and residue assignment via TOCSY spectra; analysis of conformational features including amide HN chemical shift dispersion and changes due to hydrogen bonding or post-translational modifications; the 3JαN coupling constant that reports on the φ torsion angle and on order versus disorder at a given residue; conformational preferences at Hα via chemical shift index analysis; understanding of diastereotopic hydrogens; dynamic processes, including hydrogen exchange; and identification of proline cis-trans isomerism. In addition, for a limited number of peptides, NOESY spectra are included to allow sequential resonance assignment and for assignment of trans versus cis proline conformations. Spectra from closely related peptides allow the analysis of the relative effects of single amino acid changes. The paper is written to be directly accessible to students as a tutorial guide. In addition, the data can be used by instructors for problem sets and exams. 

Abstract Proline residues within proteins lack a traditional hydrogen bond donor. However, the hydrogens of the proline ring are all sterically accessible, with polarized C−H bonds at Hα and Hδ that exhibit greater partial positive character and can be utilized as alternative sites for molecular recognition. C−H/O interactions, between proline C−H bonds and oxygen lone pairs, have been previously identified as modes of recognition within protein structures and for higher‐order assembly of protein structures. In order to better understand intermolecular recognition of proline residues, a series of proline derivatives was synthesized, including 4R‐hydroxyproline nitrobenzoate methyl ester, acylated on the proline nitrogen with bromoacetyl and glycolyl groups, and Boc‐4S‐(4‐iodophenyl)hydroxyproline methyl amide. All three derivatives exhibited multiple close intermolecular C−H/O interactions in the crystallographic state, with H⋅⋅⋅O distances as close as 2.3 Å. These observed distances are well below the 2.72 Å sum of the van der Waals radii of H and O, and suggest that these interactions are particularly favorable. In order to generalize these results, we further analyzed the role of C−H/O interactions in all previously crystallized derivatives of these amino acids, and found that all 26 structures exhibited close intermolecular C−H/O interactions. Finally, we analyzed all proline residues in the Cambridge Structural Database of small‐molecule crystal structures. We found that the majority of these structures exhibited intermolecular C−H/O interactions at proline C−H bonds, suggesting that C−H/O interactions are an inherent and important mode for recognition of and higher‐order assembly at proline residues. Due to steric accessibility and multiple polarized C−H bonds, proline residues are uniquely positioned as sites for binding and recognition via C−H/O interactions.