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J. Biol. Chem., Vol. 276, Issue 2, 957-964, January 12, 2001
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From the We have studied the effect of macromolecular
crowding reagents, such as polysaccharides and bovine serum albumin, on
the refolding of tetradecameric GroEL from urea-denatured protein
monomers. The results show that productive refolding and assembly
strongly depends on the presence of nucleotides (ATP or ADP) and
background macromolecules. Nucleotides are required to generate an
assembly-competent monomeric conformation, suggesting that proper
folding of the equatorial domain of the protein subunits into a
native-like structure is essential for productive assembly. Crowding
modulates GroEL oligomerization in two different ways. First, it
increases the tendency of refolded, monomeric GroEL to undergo
self-association at equilibrium. Second, crowding can modify the
relative rates of the two competing self-association reactions, namely,
productive assembly into a native tetradecameric structure and
unproductive aggregation. This kinetic effect is most likely exerted by
modifications of the diffusion coefficient of the refolded monomers,
which in turn determine the conformational properties of the
interacting subunits. If they are allowed to become assembly-competent
before self-association, productive oligomerization occurs; otherwise, unproductive aggregation takes place. Our data demonstrate that the
spontaneous refolding and assembly of homo-oligomeric proteins, such as
GroEL, can occur efficiently (70%) under crowding conditions similar
to those expected in vivo.
Excluded Volume Effects on the Refolding and Assembly of an
Oligomeric Protein
GroEL, A CASE STUDY*
§,¶,
**,,, and
Unidad de Biofísica (Consejo
Superior de Investigaciones Científicas-Universidad del
País Vasco (CSIC-UPV)) y Departamento de Bioquímica y
Biología Molecular, Universidad del País Vasco, Aptdo.
644, 48080 Bilbao, Spain and Centro Nacional de
Biotecnología, CSIC, Universidad Autónoma de Madrid,
28049 Madrid, Spain
*
This work was supported by grants from the University of the
Basque Country (GO3/98), Basque Government (EX-98-28 and
PI98-30), and Comisión Interministerial de Ciencia y
Tecnología (PB97-1225).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
34-94-6012624; Fax: 34-94-4648500; E-mail: gbpmuvia@lg.ehu.es.
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