Characterization of diphtheria toxin high-order oligomerization and energetics
Proteins, because of their great size and complexity, have a diversity of stable structures greater than most other classes of molecules and derive functional features from changes between those stable states. We determine and analyze the structures of different forms of the protein diphtheria toxin...
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| Format: | Dissertation |
| Language: | English |
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ProQuest Dissertations & Theses
01-01-2001
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| Online Access: | Get full text |
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| Summary: | Proteins, because of their great size and complexity, have a diversity of stable structures greater than most other classes of molecules and derive functional features from changes between those stable states. We determine and analyze the structures of different forms of the protein diphtheria toxin (DT) and the mechanism of changes between those forms to deduce the biological function of the molecule. In Chapter 1, the structures of the DT monomer and dimer are compared with biological information about the toxin's pathology to yield functional models. We present a model for the intoxication pathway of the whole monomer as it enters a human cell, as well as a mechanism by which the catalytic domain of the protein destroys cellular translation factors through NAD-ribosylation. In Chapter 2, we examine 3D domain swapping, in which a domain of a protein breaks its non-covalent bonds with the protein core and its place is taken by the identical domain of another molecule, creating a dimer or an oligomer. To explore the molecular basis and biological role of this conformational change, domain-swapped oligomers of diphtheria toxin were produced by freezing and thawing under a variety conditions, including in various salts and buffers, and at various temperatures. DT oligomers were also produced by exposure to acidic buffers, and were found by electron microscopy to adopt both linear and cyclized forms in a wide distribution of sizes. We present a mechanism for the formation of high-order DT oligomers by acidification that takes into account domain swapping and hydrophobic interactions. In Chapter 3, we have extended the resolution of the crystal structure of monomeric diphtheria toxin (DT) to 1.55 Å, revealing sufficient detail of its interdomain surfaces for energetic analysis of the formation of the open monomer and the domain-swapped dimer. This new structure reveals a large solvent network between the C and R domains. We propose that the formation of the open monomer is driven by the increase of entropy upon the breakup of this network rather than by the breaking of salt bridges between the C and the R domains as has been previously suggested. |
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| ISBN: | 9780493485676 0493485678 |