Characterisation of the isolated Che Y C-terminal fragment (79-129)--Exploring the structure/stability/folding relationship 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 alpha-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.
Bruix
M
M
Instituto de Estructura de la Materia, CSIC, Madrid, Spain.
Muñoz
V
V
Campos-Olivas
R
R
Del Bosque
J R
JR
Serrano
L
L
Rico
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D002633:Chemotaxis; D002942:Circular Dichroism; D004926:Escherichia coli; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010446:Peptide Fragments