Efficacy of macromolecular crowding in forcing proteins to fold.
The intrinsically unstructured protein, reduced and carboxyamidated RNase T1 (TCAM) was used to determine the degree to which macromolecular crowding agents increase the equilibrium constant for folding. TCAM is not catalytically active in an aqueous assay system alone, but becomes catalytically active on addition of 400 mg/ml dextran 70. The activity observed accounts for approximately 16% of the total available TCAM in solution. We interpret this result to mean that 16% of the TCAM becomes folded protein in the presence of the 400 mg/ml dextran 70, and this translates into an approximately five-fold increase in the equilibrium constant for folding. Sarcosine-induced folding of TCAM was performed in the presence of 0, 100, 200 and 300 mg/ml dextran 70, and apparent deltaG(o)(N-D) values determined from the linear extrapolation method provide an estimated 22% folded TCAM formed in the limit of zero sarcosine concentration and in presence of 400 mg/ml dextran 70. The increase in TCAM folding equilibrium constant using this method of determination is approximately 7.5-fold. Overall, the results indicate that macromolecular crowding agents are only modestly effective in promoting folding of this intrinsically unstructured protein.
Qu
Youxing
Y
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 5.154 MRB, Galveston, TX 77555-1052, USA.
Bolen
D W
DW
D002384:Catalysis; D002942:Circular Dichroism; D004573:Electrolytes; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry*; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet
D004573:Electrolytes; D012996:Solutions; D006163:Ribonuclease T1