Sequence-specific 1H NMR assignments and secondary structure of porcine motilin.
The solution structure of the 22-residue peptide hormone motilin has been studied by circular dichroism and two-dimensional 1H nuclear magnetic resonance spectroscopy. Circular dichroism spectra indicate the presence of alpha-helical secondary structure in aqueous solution, and the secondary structure can be stabilized with hexafluoro-2-propanol. Sequence-specific assignments of the proton NMR spectrum of porcine motilin in 30% hexafluoro-2-propanol have been made by using two-dimensional NMR techniques. All backbone proton resonances (NH and alpha CH) and most of the side-chain resonances have been assigned by using double-quantum-filtered COSY, RELAYED-COSY, and NOESY experiments. Simulations of NOESY cross-peak intensities as a function of mixing time indicate that spin diffusion has a relatively small effect in peptides the size of motilin, thereby allowing the use of long mixing times to confidently make assignments and delineate secondary structure. Sequential alpha CH-NH and NH-NH NOESY connectivities were observed over a significant portion of the length of the peptide. A number of medium-range NOESY cross-peaks indicate that the peptide is folded into alpha-helix from Glu9 to Lys20, which agrees favorably with the 50% helical content determined from CD measurements. The intensities of selected NOESY cross-peaks relative to corresponding diagonal peaks were used to estimate a rotational correlation time of approximately 2.5 ns for the peptide, indicating that the peptide exists as a monomer in solution under the conditions used here.
Khan
N
N
Department of Medical Biochemistry, School of Medicine, Southern Illinois University, Carbondale 62901.
Graslund
A
A
Ehrenberg
A
A
Shriver
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009037:Motilin; D011487:Protein Conformation; D013552:Swine
D009037:Motilin; D006859:Hydrogen