CHARACTERIZATION 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 an d folding of the protein Che Y are related to one another, we have per formed a conformational analysis of a long fragment of this protein, e ncompassing its C-terminal 51 residues (79-129). This fragment consist s 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 st ructure for this fragment in aqueous solution which could be due to lo w helical populations in the regions corresponding to helices 4 and 5. Within the limits of the residual helical structure experimentally de tected, helix 4 appears to extend beyond the N-terminus observed in th e native structure by over four residues belonging to the preceding lo op. In 30% trifluoroethanol the helical content of both helices increa se 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 unde r the explored experimental conditions. The conformational shifts of t he H alpha protons within the a-helices of fragment 79-129 are identic al to those of shorter synthetic peptides corresponding to the isolate d alpha-helices. Thus, the fragment 79-129 appears to behave as an ope n chain with low local helical populations. The very low intrinsic abi lity 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 sta bilised by interactions with the N-terminal Che Y region. This is in a ccordance with the contact map of Che Y, which shows that the stronges t non-local contacts of C-terminal residues are with residues of the N -terminal region, while those within the C-terminal region are very we ak. 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 b ut in the final steps of the folding.