Role of non-native aromatic and hydrophobic interactions in the folding of hen egg white lysozyme.
The folding kinetics have been determined for hen egg white lysozyme and two mutants in which Trp-62 and Trp-108 have been individually replaced by tyrosine (Tyr-62-lysozyme and Tyr-108-lysozyme, respectively). An earlier study of wild-type lysozyme [Denton, M. E., Rothwarf, D. M., & Scheraga, H. A. (1994) Biochemistry 33, 11225-11236] had indicated that two transient intermediates were formed during the early stages of refolding. Both intermediates were characterized by substantial quenching of tryptophan fluorescence which suggested that, during the refolding process, Trp-62 and/or Trp-108 was involved in a non-native tertiary interaction(s). Both Tyr-108- and Tyr-62-lysozyme fold significantly faster than wild-type lysozyme (7- and 13-fold, respectively). These results indicate that the rate-limiting step in the folding of lysozyme arises not from any inherent slowness in the formation of the native structure but rather as a consequence of the formation of a highly stable intermediate which contains significant non-native structure which must be disrupted prior to, or in concert with, subsequent folding. The data suggest that aromatic and hydrophobic interactions play a pivotal role in the formation of the non-native intermediate. The general role that non-native interactions play in the folding process is discussed.
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
D M
DM
Rothwarf
H A
HA
Scheraga
D000818:Animals; D002645:Chickens; D004527:Egg Proteins / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014364:Tryptophan / Q000378:metabolism
D004527:Egg Proteins; D006146:Guanidines; D014364:Tryptophan; D009113:Muramidase; D019791:Guanidine