Development of nonpolar surfaces in the folding of Escherichia coli dihydrofolate reductase detected by 1-anilinonaphthalene-8-sulfonate binding.
The development of nonpolar surfaces during the folding of Escherichia coli dihydrofolate reductase (DHFR) was studied by monitoring the time-dependent fluorescence of 1-anilinonaphthalene-8-sulfonate (ANS) included in the refolding solution. Stopped-flow refolding experiments demonstrated a rapid increase in fluorescence intensity within the dead time of mixing (5 ms), indicating that the earliest detectable folding intermediate contains hydrophobic surfaces which are capable of binding ANS. A further increase in fluorescence intensity over the next 300 ms coincides with the formation of a set of four intermediates which are known to contain a specific tertiary contact [Kuwajima, K., Garvey, E. P., Finn, B. E., Matthews, C. R., & Sugai, S. (1991) Biochemistry 30, 7693-7703]. Experiments performed in the presence of polar fluorescence quenching agents indicate that the binding sites for ANS in the burst phase species are more exposed to solvent than those in the subsequent set of intermediates. When considered along with the above study of the formation of secondary structure by stopped-flow circular dichroism, these results imply that DHFR initially forms a molten globule intermediate. Subdomains containing specific tertiary structure and more solvent-excluded ANS binding sites then form before ultimately being converted to native or native-like conformations during the rate-limiting steps in the folding of DHFR. The occurrence of similar kinetic phases observed by ANS binding during the folding of a number of other proteins suggests that this may be a common scheme for protein folding reactions.
Jones
B E
BE
Department of Chemistry, Pennsylvania State University, University Park 16802.
Jennings
P A
PA
Pierre
R A
RA
Matthews
C R
CR
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D004926:Escherichia coli; D066298:In Vitro Techniques; D007700:Kinetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000648:ultrastructure; D014508:Urea / Q000737:chemistry
D000178:Acrylamides; D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D012997:Solvents; D020106:Acrylamide; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase