Biochemistry 1993, 32, 2554-2563
2554
Binding and Hydrolysis of Nucleotides in the Chaperonin Catalytic Cycle: Implications for the Mechanism of Assisted Protein Folding? Graham S. Jackson,'J Rosemary A. Staniforth,* David J. Halsall,* Tony Atkinson,# J. John Holbrook,* Anthony R. Clarke,* and Steven G. Burston* Molecular Recognition Centre and Department of Biochemistry, University of Bristol School of Medical Sciences, and Microbial Technology Laboratory, Public Health Laboratory Service, University Walk, Bristol BS8 1 TD, U.K., Centre for Applied Microbiology and Research, Salisbury, Wiltshire SP4 OJG,U.K. Received August 24, 1992; Revised Manuscript Received December 15, 1992
Cpn60 was labeled with pyrene maleimide in order to follow structural rearrangements in the protein triggered by the binding of nucleotides and cpnl0. The conjugate binds ATP, AMP-PNP, and ADP(Pi) with pyrene fluorescence enhancements of 60%, 60% and 1576, respectively. In each case, binding is cooperative with half-saturation (Kip) occurring a t 10 pM, 290 pM, and 2500 p M and Hill constants (nH)of 4 3 , and 3, respectively. Inclusion of the co-protein, cpnl0, tightens the binding of ATP, AMP-PNP, and ADP(Pi) to give K 1 p values of 6 pM, 100 pM,and