Structure and dynamic properties of the single disulfide-deficient α-amylase inhibitor [C45A/C73A]tendamistat: An NMR study
✍ Scribed by Jochen Balbach; Stephan Seip; Horst Kessler; Matthias Scharf; Noushin Kashani-Poor; Joachim W. Engels
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 176 KB
- Volume
- 33
- Category
- Article
- ISSN
- 0887-3585
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✦ Synopsis
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 ␣-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 13 C ␣ heteronuclear NOE experiments with carbon in natural abundance. 15 N isotope labeling was necessary to obtain the relaxation parameters of the variant, because of sample degradation. The 15 N resonance assignment was achieved by a 15 N 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A mutation has no influence upon the -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 antiparallel -sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat.