Retention of the Cis Proline Conformation in Tripeptide Fragments of Bovine Pancreatic Ribonuclease A Containing a Non-natural Proline Analogue, 5,5-Dimethylproline

Abstract

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 unfolds. Procedures have been developed to obtain optically pure L-dmP and to incorporate this sterically hindered residue as the central one in tripeptides that are suitable for fragment coupling to prepare synthetic proteins. Based on the sequences of residues 92-94 (Tyr-Pro-Asn:YPN) and 113-115 (Asn-Pro-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 structures were determined by 2D 1 H nuclear magnetic resonance (NMR) spectroscopy. YdmPN was found to exist solely in the cis conformation between 6 and 60 °C, whereas NdmPY was found to have some trans component that increased from about 10% to about 21% as the temperature increased over the range between 6 and 80 °C. Both YdmPN and cis-NdmPY adopt a type VI reverse turn, as does proline. The NMR structures of YdmPN and cis-NdmPY are comparable 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 is a promising substitute for proline in a variety of protein problems to constrain the X-Pro peptide group to the cis conformation.

Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A

“…The nonnative proline isomers that also diminished the initial collapse may exert their effect in a similar manner. NMR investigations showed that a peptide fragment of RNase A forms a turn with a cis-Pro-93 but not with a trans-Pro-93 (20,47). Thus, the entropy of the polypeptide chain is higher in U eq than in U vf .…”

“…However, in contrast to the folded protein, an X-Pro peptide group is in equilibrium between the cis and trans forms in peptides and denatured proteins. To obtain a better estimation of the expected initial values of U vf in double-jump kinetic experiments, we explored a tripeptide, WdmPN, which contains a proline analog, dimethyl proline (dmP) (20). This analog overwhelmingly forms a cis X-Pro peptide group in peptides with the same backbone and side-chain conformations as regular cis prolines do, and this makes it a good model peptide for a cis Trp-Pro peptide group (20).…”

Time-Resolved Small-Angle X-ray Scattering Study of the Folding Dynamics of Barnase

“…43 Results of NMR investigations indicated that a peptide fragment of RNase A forms a turn with Pro93 in a cis form but not in a trans form. 44,45 Consequently, it is reasonable that the fraction of sample containing the cis proline remains in an expanded state even under the solution condition that favors folding.…”

Single Proline Residues can Dictate the Oxidative Folding Pathways of Cysteine-rich Peptides

“…35 Indeed by replacing Pro114 of RNase A with 5,5-dimethyl-L-proline, which is known to form almost exclusively cis prolyl isomers, 37 both the protein stability and the rate of refolding were significantly enhanced. 38 Similarly, 4-fluoroproline residues are known to markedly affect the stability of cis and trans peptide bonds.…”