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FOR THE RECORD

Multiple equilibria of the Escherichia coli chaperonin GroES revealed by mass spectrometry

¹ Department of Chemistry, ² Department of Entomology, and ³ Department of Physics and Astronomy, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada

(RECEIVED October 21, 2004; FINAL REVISION December 21, 2004; ACCEPTED January 7, 2005)

Nanospray time-of-flight mass spectrometry has been used to study the assembly of the heptamer of the Escherichia coli cochaperonin protein GroES, a system previously described as a monomer–heptamer equilibrium. In addition to the monomers and heptamers, we have found measurable amounts of dimers and hexamers, the presence of which suggests the following mechanism for heptamer assembly: 2 Monomers Dimer; 3 Dimers Hexamer; Hexamer + Monomer Heptamer. Equilibrium constants for each of these steps, and an overall constant for the Monomer Heptamer equilibrium, have been estimated from the data. These constants imply a standard free-energy change, G⁰, of about 9 kcal/mol for each contact surface formed between GroES subunits, except for the addition of the last subunit, where G⁰ = 6 kcal/mol. This lower value probably reflects the loss of entropy when the heptamer ring is formed. These experiments illustrate the advantages of electrospray mass spectrometry as a method of measuring all components of a multiple equilibrium system.

Keywords: electrospray ionization; time-of-flight mass spectrometry; E. coli chaperone protein; GroES equilibrium

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041164305.

Reprint requests to: Lynda J. Donald, Department of Chemistry, 507 Parker Building, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada; e-mail: ldonald{at}cc.umanitoba.ca; fax: (204) 474-7608.

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