Successful control of aggregation and folding rates during refolding of denatured lysozyme by adding N-methylimidazolium cations with various N'-substituents.
The present study aimed to obtain more effective refolding agents and to understand the influence of their chemical structures on their function as refolding agents. To achieve these aims, we investigated the effects of a large variety of N'-substituted N-methylimidazolium chlorides on the oxidative refolding of lysozyme in a high throughput manner. Among the molecules examined, N-methylimidazolium cations with a short N'-alkyl chain, such as an N'-ethyl or N'-butyl chain, significantly enhanced the refolding yield compared to conventional refolding additives such as arginine hydrochloride and Triton X-100. Detailed kinetic analyses revealed that the effective cations selectively decreased the aggregation rate constant (kA) without any large decreases in the folding rate constant (kN). However, when the hydrophobicity of the N'-substituent of the cations was increased, the desirable properties of the short N'-alkyl chain-type cations for protein refolding were diminished. Furthermore, increases in the N'-alkyl chain length to an N'-octyl or N'-dodecyl chain drastically decreased the kA values, thereby increasing the ratio of kN to kA, despite the very small kN values and resulting in enhanced refolding yields. Thus, by tuning the chemical structure of the N'-substituents of N-methylimidazolium chloride, five effective refolding agents (N'-ethyl-, N'-propyl-, N'-butyl-, N'-pentyl- and N'-isobutyl-N-methylimidazolium chlorides) were successfully obtained, and the kinetic parameters of folding and aggregation during the refolding process could be controlled using three different modes.
Yamaguchi
Satoshi
S
Department of Bioengineering, Graduate School of Engineering, Center for NanoBio Integration (CNBI), The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Yamamoto
Etsushi
E
Tsukiji
Shinya
S
Nagamune
Teruyuki
T
D000818:Animals; D002412:Cations; D002645:Chickens; D004531:Eggs; D007093:Imidazoles / Q000737:chemistry*; D007202:Indicators and Reagents; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology; D009113:Muramidase / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012996:Solutions
D002412:Cations; D007093:Imidazoles; D007202:Indicators and Reagents; D012996:Solutions; D009113:Muramidase