Local and global dynamics during the folding of Escherichia coli dihydrofolate reductase by time-resolved fluorescence spectroscopy.
Time-resolved fluorescence techniques were utilized to monitor the kinetic refolding reaction of Escherichia coli dihydrofolate reductase (DHFR). Measurements of emission and anisotropy decay lifetimes of both the five intrinsic tryptophan residues and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) during the folding reaction were used to characterize the compactness and development of tertiary structure in transient intermediates formed during the folding of DHFR. Experiments monitoring bound ANS show that a rapidly-formed intermediate (< 20 ms) has a rotational time of approximately 10 ns and, therefore, a compactness similar to that for the native conformation. All of the tryptophan residues in this burst phase species rotate as freely as in the unfolded state. In the set of four intermediates which then appear over the next few hundred milliseconds, the apparent rotational time measured by ANS fluorescence increases to a maximum rotational time of approximately 20 ns. An increase in the average tryptophan lifetime for these intermediates suggests these side chains become excluded from solvent and associated dynamic quenching mechanisms. As the folding reaction proceeds to a set of four native conformers the bound ANS rotational time then decreases to approach that for the native protein, 10.5 ns, and the average tryptophan rotational time increases to the same value. During these rate-limiting, final steps in folding, the static quenching effects which reflect the formation of specific tertiary contacts involving tryptophans also appear.
Jones
B E
BE
Department of Chemistry, Pennsylvania State University, University Park 16802.
Beechem
J M
JM
Matthews
C R
CR
D000225:Adenine / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004926:Escherichia coli / Q000201:enzymology*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D008958:Models, Molecular; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase; D000225:Adenine