T-jump infrared study of the folding mechanism of coiled-coil GCN4-p1.
Partially folded intermediates have been frequently observed in equilibrium and kinetic protein folding studies. However, folding intermediates that exist at the native side of the rate-limiting step are rather difficult to study because they often evade detection by conventional folding kinetic methods. Here, we demonstrated that a laser-induced temperature-jump method can potentially be used to identify the existence of such post-transition or hidden intermediates. Specifically, we studied two cross-linked variants of GCN4-p1 coiled-coil. The GCN4 leucine zipper has been studied extensively and most of these studies have regarded it as a two-state folder. Our static circular dichroism and infrared data also indicate that the thermal unfolding of these two monomeric coiled-coils can be adequately described by an apparent two-state model. However, their temperature-jump-induced relaxation kinetics exhibit non-monoexponential behavior, dependent upon sequence and temperature. Taken together, our results support a folding mechanism wherein at least one folding intermediate populates behind the main rate-limiting step.
Wang
Ting
T
Department of Chemistry and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Lau
Wai Leung
WL
DeGrado
William F
WF
Gai
Feng
F
D050976:Basic-Leucine Zipper Transcription Factors; D003198:Computer Simulation; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry*; D013055:Spectrophotometry, Infrared / Q000379:methods*; D013997:Time Factors; D014157:Transcription Factors / Q000032:analysis* / Q000737:chemistry*
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors