Oxidative Folding of a Cystine Knot Protein: the Amaranthus α-amylase Inhibitor

Čemažar, Maša (2003). Oxidative Folding of a Cystine Knot Protein: the Amaranthus α-amylase Inhibitor. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f744


The work described in this thesis is concerned with the study of the oxidative folding pathway of a cystine knot protein, Amaranthus a-amylase inhibitor (AAI). The data presented in the thesis are obtained with a combination of different biochemical and biophysical techniques, with an emphasis on NMR spectroscopy.
Chapter 1 gives a short introduction to the structural and functional superfamilies of the protein in question. Additionally, it outlines the protein folding problem, with an emphasis on oxidative folding, and stresses the importance of studying such pathways in small proteins. Chapter 2 covers both the basis of experimental techniques and the details of experimental methods used in the study. The remaining four chapters (3, 4, 5 and 6) are results and discussion chapters. Each one deals with a specific aspect of the study of the oxidative folding pathway of Amaranthus a-amylase inhibitor.
Chapter 3 presents the results from an improved solid phase peptide synthesis approach, which increased the overall yield of peptide synthesis and recovered a greater amount of AAI after the refolding step. In Chapter 4 the intermediate species that appear on the oxidative folding pathway are identified and characterised in terms of their disulfide content. An illustration of the structural features of the main folding intermediate (MFI) with NMR, photo-CID NP and computer modelling techniques is given in Chapter 5. In Chapter 6 the kinetic process of the oxidative folding of Amaranthus a-amylase inhibitor is studied at the level of a single disulfide species and at the level of a single amino acid residue.
The novelty of this thesis is two-fold. First, from the point of view of biological interest, this is the first time that oxidative folding intermediates with non-native disulfide bridges between adjacent cysteine residues are observed. Second, from the point of view of development of biophysical methods, the first application of time-resolved NMR and photo-CIDNP spectroscopy to a complete oxidative folding process is described.

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