CHARACTERIZATION OF THE SLOW FOLDING REACTIONS OF TRP APOREPRESSOR FROM ESCHERICHIA-COLI BY MUTATIONAL ANALYSIS OF PROLINES AND CATALYSIS BY A PEPTIDYL-PROLYL ISOMERASE

Escherichia coil trp aporepressor (TR) is a highly helical, dimeric pr otein whose folding has been shown to involve three phases whose relax ation times range from 200 ms to 50 s at 25 degrees C and pH 7.6 [Gitt elman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. Al l three phases are urea and protein concentration independent below 3 M urea, suggesting that cis/trans proline isomerization might limit th e folding of TR under these conditions. This hypothesis was tested by measuring the sensitivity of the folding reaction to site-directed mut agenesis and to cyclophilin, a peptidyl-prolyl isomerase. Each of the four proline residues in TR was replaced singly as well as simultaneou sly, and the effects on the folding mechanism were assessed. All of th ese mutants, including the version lacking prolines (des-Pro TR), reta in three slow, denaturant-independent folding phases similar to those observed for wild-type TR. However, the pattern of catalysis of the tw o slower folding phases in wild-type and mutant TRs by cyclophilin sho ws that cis/trans isomerization of the Thr44/Pro45 peptide bond can li mit folding in proteins containing Pro45. The observation of three ure a-independent, slow folding phases in des-Pro TR demonstrates that pro line isomerization is not solely responsible for this complex folding behavior. Other types of isomerization or conformational rearrangement reactions appear to Limit the folding of this dimeric protein under s trongly folding conditions.