Characterization of high-order diphtheria toxin oligomers.
In 3D domain swapping, a domain of a protein breaks its noncovalent bonds with the protein core and its place is taken by the identical domain of another molecule, creating a strongly bound dimer or higher order oligomer. For some proteins, including diphtheria toxin, 3D domain swapping may affect protein function. To explore the molecular basis of 3D domain swapping in a well-characterized protein system, 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. Reaction yields were followed by high-performance size-exclusion chromatography. The traditional low pH pulse produced by freeze-thawing in mixed sodium phosphate buffer induces the oligomerization of DT, but the addition of alkali chloride salts was found to increase the yield in the order of Li(+) > Na(+) > K(+). Unexpectedly, oligomers also formed when DT was frozen and thawed in the presence of 1 M LiCl alone. Slower freezing and thawing of the mixture led to the production of more and larger oligomers. 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. Upon the basis of these results, the model for the production of DT oligomers by freezing and thawing was expanded to include a salt-mediated pathway. We present a mechanism for the formation of high-order DT oligomers by acidification that takes into account domain swapping and hydrophobic interactions.
Steere
B
B
UCLA-DOE Laboratory of Structural Biology and Molecular Medicine & Department of Chemistry and Biochemistry, University of California, Los Angeles, P.O. Box 951570, Los Angeles, California 90095-1570, USA.
Eisenberg
D
D
D002021:Buffers; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004167:Diphtheria Toxin / Q000737:chemistry*; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D018021:Lithium Chloride; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008958:Models, Molecular; D010710:Phosphates; D017434:Protein Structure, Tertiary; D012492:Salts; D012996:Solutions; D013499:Surface Properties
D002021:Buffers; D004167:Diphtheria Toxin; D046911:Macromolecular Substances; D010710:Phosphates; D012492:Salts; D012996:Solutions; D018021:Lithium Chloride; C018279:sodium phosphate