Structure and dynamic properties of the single disulfide-deficient alpha-amylase inhibitor [C45A/C73A]tendamistat: an NMR study.
Covalent linkages such as disulfide bonds are important for the stabilization of proteins. In the present NMR study we compare the structure and the dynamics of the single disulfide-deficient variant C45A/C73A of the alpha-amylase inhibitor tendamistat and the wild-type protein, which contains two disulfide bonds (C11-C27 and C45-C73). Complete proton assignment was achieved by standard homonuclear 2D techniques for the variant. Chemical shift differences, intra-strand NOE effects and protected amide proton were used to compare the connectivity of the secondary structure elements of variant and wild-type. Dynamic properties of the wild-type protein were studied by 13C(alpha) heteronuclear NOE experiments with carbon in natural abundance. 15N isotope labeling was necessary to obtain the relaxation parameters of the variant, because of sample degradation. The 15N resonance assignment was achieved by a 15N 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A mutation has no influence upon the beta-barrel structure of tendamistat beside very local changes at the mutation site. The relaxation data revealed only subtle differences between variant and wild-type on a subnanosecond time scale. Only the N-terminus and G62 in the connecting loop between the anti-parallel beta-sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat.
Institut für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany.
J
J
Balbach
S
S
Seip
H
H
Kessler
M
M
Scharf
N
N
Kashani-Poor
J W
JW
Engels
D004220:Disulfides / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D004220:Disulfides; D004791:Enzyme Inhibitors; D010455:Peptides; D011994:Recombinant Proteins; D000516:alpha-Amylases; C030526:tendamistate