DYNAMICS OF THE GROEL PROTEIN COMPLEX - EFFECTS OF NUCLEOTIDES AND FOLDING MUTANTS

Chaperonins are a ubiquitous class of ring-shaped oligomeric protein c omplexes that are of crucial importance for protein folding in vivo. A nalysis of the underlying functional principles had relied mainly on m odel proteins the (un)folding of which is dominated by irreversible si de-reactions. We used maltose-binding protein (MBP) as a substrate pro tein for GroEL, since the refolding of this protein is completely reve rsible and thus allows a detailed analysis of the molecular parameters that determine the interaction of GroEL with non-native protein. We s how that MBP folding intermediates are effectively trapped by GroEL in a diffusion-controlled reaction. This complex is stabilized via unspe cific hydrophobic interactions. Stabilization energies for wild-type M BP increasing linearly with ionic strength from 50 kJ/mol to 60 kJ/mol . Depending on the intrinsic folding rate and the hydrophobicity of th e substrate protein, the interaction of GroEL with MBP folding interme diates leads to a dramatically decreased apparent refolding rate of MB P (wild-type) or a complete suppression of folding (MBP folding mutant Y283D). On the basis of our data, a quantitative kinetic model of the GroEL-mediated folding cycle is proposed, which allows simulation of the partial reactions of the binding and release cycles under all cond itions tested. In the presence of ATP and non-hydrolysable analogues, MBP is effectively released from GroEL, since the overall dissociation constant is reduced by three orders of magnitude. Interestingly, bind ing of nucleotide does not change the off rate by more than a factor o f 3. However the on-rate is decreased by at least two orders of magnit ude. Therefore, the rebinding reaction is prevented and folding occurs in solution. (C) 1996 Academic Press Limited