Compact dimension of denatured states of staphylococcal nuclease.
Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.
Chow
C-Y
CY
Institute of Physics, Academia Sinica, Nankang, Taipei 11529, Taiwan.
Wu
Ming-Chya
MC
Fang
Huey-Jen
HJ
Hu
Chin-Kun
CK
Chen
Hueih-Min
HM
Tsong
Tian-Yow
TY
D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D012474:Salmon; D014364:Tryptophan / Q000737:chemistry
D050505:Mutant Proteins; D014364:Tryptophan; D004228:Dithionitrobenzoic Acid; D008836:Micrococcal Nuclease; D019791:Guanidine; D003545:Cysteine