Multiple equilibria of the Escherichia coli chaperonin GroES revealed by mass spectrometry |
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Authors: | Donald Lynda J Stokell David J Holliday Neil J Ens Werner Standing Kenneth G Duckworth Harry W |
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Affiliation: | Department of Chemistry, 507 Parker Building, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada. ldonald@cc.umanitoba.ca |
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Abstract: | 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, DeltaG(0), of about 9 kcal/mol for each contact surface formed between GroES subunits, except for the addition of the last subunit, where DeltaG(0) = 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. |
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Keywords: | electrospray ionization time-of-flight mass spectrometry E. coli chaperone protein GroES equilibrium |
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