Nonlinear free energy relationships in Arc repressor unfolding imply the existence of unstable, native-like folding intermediates.
Under strongly denaturing conditions, the logarithm of the rate constant for dissociation/unfolding of the wild-type Arc dimer varies in a nonlinear fashion with denaturant concentration. To assess the unfolding/dissociation behavior under conditions favoring the native structure, we mixed Arc variants labeled with fluorescence acceptor or donor groups and used energy transfer to monitor the increase in heterodimer with time. Under the conditions of this experiment, the rate at which the heterodimer concentration approaches its equilibrium value is determined by rate of dissociation and unfolding of the protein. Using this method and traditional denaturant-jump experiments, rate constants for unfolding/dissociation were determined over a wide range of stabilizing and destabilizing conditions. In each case examined, plots of log(ku) versus denaturant showed significant curvature under strongly denaturing conditions, even though other kinetic experiments indicates that the unfolding/dissociation reactions remain largely two-state. This curvature can be explained most readily by a series of unstable intermediates in the unfolding pathway, with denaturant-induced changes in the kinetic step that is rate-limiting. Alternatively, curvature might result from Hammond behavior in which the structure of the transition state becomes more native-like as the stability of native Arc decreases with increasing denaturant.
Jonsson
T
T
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, USA.
Waldburger
C D
CD
Sauer
R T
RT
D000595:Amino Acid Sequence; D017100:Bacteriophage P22 / Q000737:chemistry* / Q000235:genetics; D004735:Energy Transfer; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011489:Protein Denaturation; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins
D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins