Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Biological Sciences

First Advisor

Sue G. Bartlett


Although chaperonin-assisted protein folding has been studied in vitro by a number of investigators, the features of an unfolded or partially folded polypeptide that are recognized and bound by chaperonins are not known. I addressed this question using the precursor of the small subunit (pS) of ribulose-1,5-bisphosphate carboxylase as a model substrate for GroEL, the bacterial chaperonin. The precursor protein was expressed in E. coli as a C-terminal fusion to protein A. Protein A-pS (and any associated cellular proteins) was isolated by affinity chromatography. GroEL could be eluted from the fusion protein by ATP and either GroES or casein, consistent with results of in vitro folding assays. Using deletions from the C-terminus of pS I defined the smallest truncation of pS, PAxpS90T, that binds GroEL with high avidity. A series of site-specific mutations targeting the C-terminal 15-20 amino acids of PAxpS90T was constructed and analyzed for the ability to bind GroEL. Two of these mutations bound significantly less GroEL than PAxpS90T, suggesting that this region is required for avid GroEL binding. I demonstrated a physical interaction between GroEL and this region of pS with a novel assay that utilizes the protection of tyrosine residues from iodination upon formation of specific protein-protein complexes. Finally, I further showed that at least half of the transit sequence of pS is also required for avid binding to GroEL. The association constants for the interaction of GroEL with PAxpS, PAxpS90T, or its mutated derivatives, were determined and fell within the range $3.7\times10\sp7$ to $2.7\times 10\sp6$ M$\sp{-1}$. Analysis of the affinity constants for PAxpS90T mutants allowed us to define a possible recognition motif for GroEL's interaction with pS. This motif includes the recognition of both hydrophobic and positively charged amino acids. The motif need not be helical, but structural rigidity may be a requirement for recognition (and binding) by GroEL.