Retention of the cis proline conformation in tripeptide fragments of bovine pancreatic ribonuclease A containing a non-natural proline analogue, 5,5-dimethylproline

Attention is focused on L-5,5-dimethylproline (dmP) as a substitute to lock L-proline (Pro) in a cis conformation in peptides and proteins, to prevent cis/trans isomerization when a protein with cis X-Pro peptide groups unfol ds. Procedures have been developed to obtain optically pure L-dmP and to in corporate this sterically hindered residue as tbe central one in tripeptide s that are suitable for fragment coupling to prepare synthetic proteins. Ba sed on the sequences of residues 92-94 (Tyr-Pro-Asn:YPN) and 113-115 (Asn-P ro-Tyr: NPY) in bovine pancreatic ribonuclease A (RNase A), in which the X- Pro peptide groups are in the cis conformation, the tripeptides Ac-Tyr-dmP- Asn (YdmPN) and Ac-Asn-dmP-Tyr (NdmPY) were synthesized, and their structur es were determined by 2D H-1 nuclear magnetic resonance (NMR) spectroscopy. YdmPN was found to exist solely in the cis conformation between 6 and 60 d egrees C, whereas NdmPY was found to have some trans component that increas ed from about 10% to about 21% as the temperature increased over the range between 6 and 80 degrees C. Both YdmPN and cis-NdmPY adopt a type VI revers e turn, as does proline. The NMR structures of YdmPN and cis-NdmPY are comp arable with the X-ray structures of the corresponding portions of RNase A, and the NMR structure of trans-NdmPY is compatible with the X-ray structure of the isolated tripeptide, Ac-NPY. These results demonstrate that L-dmP i s a promising substitute for proline in a variety of protein problems to co nstrain the X-Pro peptide group to the cis conformation.