Refolding of denatured thioredoxin observed by size-exclusion chromatography.
Molecular sieve chromatography can resolve interactive systems into populations having different effective hydrodynamic volumes. In this report, the advantages of such resolution to protein folding are illustrated by using moderate pressure to decrease analysis time and lowered temperature to slow down the kinetics of conformational change. A 300-mm Bio-Sil TSK-125 size-exclusion column was equilibrated with a series of different concentrations of guanidine hydrochloride at 2 degrees C in 50 mM phosphate buffer, pH 7.0. Samples of native Escherichia coli thioredoxin, denatured thioredoxin, or thioredoxin equilibrated with the column solvent were injected, and the effluent was monitored at 220 nm. Injection of equilibrated protein samples defined three denaturant concentration zones identical with those observed by spectral measurements: the native base-line zone where only compact protein is observed in the effluent profile; the transition zone in which both compact and denatured forms are observed in slow exchange; and the denatured base-line zone in which only denatured protein is observed. Unfolding was observed by injection of native protein into columns having isocratic denaturant concentrations in the transition and denatured base-line zones. Effluent profiles indicated a dynamic conversion of compact to denatured protein with a time constant which appeared to decrease markedly with increasing denaturant concentration. Refolding was observed by injection of denatured protein into columns having isocratic concentrations in the transition and native base-line zones. As the denaturant concentration was decreased, the effluent profiles evidenced a persistent slow conversion of denatured to compact protein which was suddenly accelerated about midway in the native base-line zone.(ABSTRACT TRUNCATED AT 250 WORDS)
Shalongo
W
W
Ledger
R
R
Jagannadham
M V
MV
Stellwagen
E
E
D001426:Bacterial Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D013879:Thioredoxins / Q000378:metabolism*
D001426:Bacterial Proteins; D006146:Guanidines; D013879:Thioredoxins; D019791:Guanidine