Characterisation of the isolated Che Y C-terminal fragment (79-129) - Exploring the structure/stability/folding relationships of the alpha/beta parallel protein Che Y

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 beta-strands 4 and 5 and alpha-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 beta-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 H alpha protons within the a-helices of fragment 79-129 are identical to those of shorter synthetic peptides corresponding to the isolated alpha-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.