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 stabil ization of proteins. In the present NMR study are compare the structur e and the dynamics of the single disulfide-deficient variant C45A/C73A of the cw-amylase inhibitor tendamistat and the wild-type protein, wh ich contains two disulfide bonds (C11-C27 and C45-C73), Complete proto n assignment was achieved by standard homonuclear 2D techniques for th e variant. Chemical shift differences, intra-strand NOE effects and pr otected amide proton were used to compare the connectivity of the seco ndary structure elements of variant and wild-type. Dynamic properties of the wild-type protein were studied by C-13(alpha) heteronuclear NOE experiments with carbon in natural abundance. N-15 isotope labeling w as necessary to obtain the relaxation parameters of the variant, becau se of sample degradation. The N-15 resonance assignment was achieved b y a N-15 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A m utation has no influence upon the beta-barrel structure of tendamistat beside very local changes at the mutation site. The relaxation data r evealed only subtle differences between variant and wild-type on a sub nanosecond time scale. Only the N-terminus and G62 in the connecting l oop between the antiparallel beta-sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and t he secretion efficiency of tendamistat. (C) 1998 Wiley-Liss, Inc.