Protein fragments as probes in the study of protein folding mechanisms: differential effects of dihydrofolate reductase fragments on the refolding of the intact protein.
We describe an approach for investigating the protein folding process, using protein fragments as inhibitory probes of the refolding protein. The refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3), reversibly unfolded in 7 M urea, was monitored by the reappearance of enzyme activity after diluting the unfolded enzyme into low urea concentrations (less than or equal to 2 M) in the presence of substrates. Of eight protein fragments produced by limited proteolysis of the 159-residue enzyme, three isolated peptides--Ser-49/Glu-90, Ile-91/Glu-154, and Gln-102/Glu-154--were evaluated for their effects on the recovery of the refolding protein's enzymatic activity. By this criterion, 13 microM peptide Gln-102/Glu-154 inhibits the refolding of 0.015 microM enzyme by approximately 80%, while the related peptide, Ile-91/Glu-154, and peptide Ser-49/Glu-90 at the same concentration inhibit the recoverable activity of the refolding enzyme by less than or equal to 20%. None of these three peptides has any significant effect on the activity of the folded enzyme. Our results indicate that peptides may inhibit refolding differentially and that these effects may be extremely sensitive to fragment sequence and composition. We suggest that peptide specificity in the inhibition of protein folding may be exploited as a structural probe of protein folding mechanisms.
Hall
J G
JG
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110.
Frieden
C
C
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D011487:Protein Conformation; D012697:Serine Endopeptidases; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014508:Urea
D010446:Peptide Fragments; D009249:NADP; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase