Characterisation of the Isolated Che Y C‐terminal Fragment (79–129)

Abstract

To gain insight into how the three‐dimensional structure, stability and folding of the protein Che Y are related to one another, we have performed a conformational analysis of a long fragment of this protein, encompassing its C‐terminal 51 residues (79–129). This fragment consists of residues in the β‐strands 4 and 5 and α‐helices 4 and 5 of native Che Y. The study has been performed by two‐dimensional NMR and far‐ultraviolet circular dichroism in aqueous solution and in 30% (by vol.) trifluoroethanol/water at 273 K and 298 K. We observe little structure for this fragment in aqueous solution which could be due to low helical populations in the regions corresponding to helices 4 and 5. Within the limits of the residual helical structure experimentally detected, helix 4 appears to extend beyond the N‐terminus observed in the native structure by over four residues belonging to the preceding loop. In 30% trifluoroethanol the helical content of both helices increase and helix 4 extends further to include the preceding β‐strand 4. None of the long‐range NOES present in native Che Y are observed under the explored experimental conditions. The conformational shifts of the Hα protons within the α‐helices of fragment 79–129 are identical to those of shorter synthetic peptides corresponding to the isolated a‐helices. Thus, the fragment 79–129 appears to behave as an open chain with low local helical populations. The very low intrinsic ability for structure formation displayed by this region of Che Y at pH 2.5 suggests that in the folded protein this region could be mainly stabilised by interactions with the N‐terminal Che Y region. This is in accordance with the contact map of Che Y, which shows that the strongest non‐local contacts of C‐terminal residues are with residues of the N‐terminal region, while those within the C‐terminal region are very weak. More importantly, the relationship appears to be possibly extended to the folding properties of the protein, since the C‐terminal region is not structurally formed in the folding transition state of Che Y but in the final steps of the folding.