Structural analysis of GroE chaperonin complexes using chemical cross-linking.
In this chapter, we have shown how chemical cross-linking with a bifunctional reagent, GA, can be used to investigate the structure of large oligomeric complexes such as GroEL14GroES7 and GroEL14(GroES7)2. Cross-linking, followed by denaturing electrophoresis, confirmed the number and arrangement of GroEL and GroES subunits within each individual oligomer, which was previously known from EM analysis. Furthermore, cross-linking permitted a close examination of the effect of regulatory factors, such as nucleotides and free divalent cations, on the molecular structure of GroEL14, GroEL14GroES7, and GroEL14GroES7. Finally, cross-linking analysis permitted characterization and quantitation of various chaperonin heterooligomeric complexes, GroEL14, GroEL14GroES7, and GroEL14GroES7 in solution, under conditions that also supported protein folding and ATP hydrolysis. It was shown that GA does not induce the artifactual association or the dissociation of GroES7 from the chaperonin. On the contrary, chemical cross-linking is an obligatory procedure when the subsequent analysis is carried out using methods that can displace the equilibrium.
Azem
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A
Department of Botany, Alexander Silbermann Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
Weiss
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C
Goloubinoff
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P
D000255:Adenosine Triphosphate / Q000378:metabolism; D018835:Chaperonin 10 / Q000737:chemistry*; D018834:Chaperonin 60 / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D005976:Glutaral / Q000378:metabolism; D008854:Microscopy, Electron; D008958:Models, Molecular; D011487:Protein Conformation
D018835:Chaperonin 10; D018834:Chaperonin 60; D003432:Cross-Linking Reagents; D000255:Adenosine Triphosphate; D005976:Glutaral