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Structure and dynamic properties of the single disulfide‐deficient α‐amylase inhibitor [C45A/C73A]tendamistat: An NMR study
Author(s) -
Balbach Jochen,
Seip Stephan,
Kessler Horst,
Scharf Matthias,
KashaniPoor Noushin,
Engels Joachim W.
Publication year - 1998
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(19981101)33:2<285::aid-prot11>3.0.co;2-g
Subject(s) - homonuclear molecule , heteronuclear molecule , chemistry , crystallography , molecular dynamics , covalent bond , relaxation (psychology) , resonance (particle physics) , chemical shift , protein secondary structure , protein dynamics , stereochemistry , nuclear magnetic resonance spectroscopy , computational chemistry , biochemistry , molecule , organic chemistry , biology , neuroscience , physics , particle physics
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 α‐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 α 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 β‐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 β‐sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat. Proteins 33:285–294, 1998. © 1998 Wiley‐Liss, Inc.