STRUCTURAL CHARACTERIZATION BY NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY OF A GENETICALLY-ENGINEERED HIGH-AFFINITY CALMODULIN-BINDING PEPTIDEDERIVED FROM BORDETELLA-PERTUSSIS ADENYLATE-CYCLASE

This paper reports the solution conformation of a peptide (P-196-267) derived from the calmodulin-binding domain of Bordetella pertussis ade nylate cyclase, P-196-267 corresponding to the protein fragment situat ed between amino acid residues 196-267 was overproduced by a recombina nt Escherichia coli strain, Its affinity for calmodulin is only one or der of magnitude lower (K-d = 2.4 nM) than that of the whole bacterial enzyme (K-d = 0.2 nM). The proton resonances of the NMR spectra of P- 196-267 were assigned using homonuclear two-dimensional techniques (do uble-quantum-filtered J-correlated spectroscopy, total correlation spe ctroscopy, and nuclear Overhauser enhancement spectroscopy) and a stan dard assignment procedure, Analysis of the nuclear Overhauser effect c onnectivities and the secondary shift distribution of C alpha protons along the sequence allowed us to identify the elements of regular seco ndary structure, The peptide is flexible in solution, being in equilib rium between random coil and helical structures, Two segments of 11 am ino acids (situated between V-215 and A(225)) and 15 amino acids (situ ated between L(293) and A(247)) populate in a significant proportion t he helix conformational state. The two helices can be considerably sta bilized in a mixed solvent, trifluoroethanol/water (30/70), suggesting that the corresponding fragment in the intact protein assumes a simil ar secondary conformation, No elements of tertiary structure organizat ion were detected by the present experiments. The conformational prope rties of the isolated calmodulin target fragment are discussed in rela tion with the available NMR and X-ray data on various peptides complex ed to calmodulin. (C) 1995 Academic Press, Inc.