Rapid unfolding of a domain populates an aggregation-prone intermediate that can be recognized by GroEL.
Some amino acid substitutions in phage P22 coat protein cause a temperature-sensitive folding (tsf) phenotype. In vivo, these tsf amino acid substitutions cause coat protein to aggregate and form intracellular inclusion bodies when folded at high temperatures, but at low temperatures the proteins fold properly. Here the effects of tsf amino acid substitutions on folding and unfolding kinetics and the stability of coat protein in vitro have been investigated to determine how the substitutions change the ability of coat protein to fold properly. The equilibrium unfolding transitions of the tsf variants were best fit to a three-state model, N if I if U, where all species concerned were monomeric, a result confirmed by velocity sedimentation analytical ultracentrifugation. The primary effect of the tsf amino acid substitutions on the equilibrium unfolding pathway was to decrease the stability (DeltaG) and the solvent accessibility (m-value) of the N if I transition. The kinetics of folding and unfolding of the tsf coat proteins were investigated using tryptophan fluorescence and circular dichroism (CD) at 222 nm. The tsf amino acid substitutions increased the rate of unfolding by 8-14-fold, with little effect on the rate of folding, when monitored by tryptophan fluorescence. In contrast, when folding or unfolding reactions were monitored by CD, the reactions were too fast to be observed. The tsf coat proteins are natural substrates for the molecular chaperones, GroEL/S. When native tsf coat protein monomers were incubated with GroEL, they bound efficiently, indicating that a folding intermediate was significantly populated even without denaturant. Thus, the tsf coat proteins aggregate in vivo because of an increased propensity to populate this unfolding intermediate.
Doyle
Shannon M
SM
Department of Molecular and Cell Biology, University of Connecticut, 91 N. Eagleville Road, Storrs, CT 06269-3125, USA.
Anderson
Eric
E
Zhu
Dan
D
Braswell
Emory H
EH
Teschke
Carolyn M
CM
D019943:Amino Acid Substitution; D017100:Bacteriophage P22 / Q000737:chemistry*; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism*; D018834:Chaperonin 60 / Q000378:metabolism*; D006801:Humans; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D036022:Capsid Proteins; D018834:Chaperonin 60; D014364:Tryptophan; D014508:Urea