Design and characterization of a multisite fluorescence energy-transfer system for protein folding studies: a steady-state and time-resolved study of yeast phosphoglycerate kinase.
A multisite distance-based fluorescence resonance energy-transfer assay system was developed for the study of protein folding reactions. Single- and double-cysteine substitution mutagenesis was utilized to place sulfhydryl residues throughout the tertiary structure of the bidomain enzyme yeast phosphoglycerate kinase (PGK). These reactive cysteines were covalently modified with extrinsic donor [5-[[2-(2-iodoacetamido)ethyl]amino]-1-naphthalenesulfonic acid] and acceptor (5-iodoacetamidofluorescein) fluorescent labels. A detailed experimental strategy was followed, which revealed that, when these relatively large extrinsic fluorescent labels are covalently attached to properly selected solvent-exposed residues, they do not affect the intrinsic stability of the protein. The PGK crystal structure was combined with molecular dynamics simulations of the dyes built into the protein and time-resolved anisotropy experiments, in order to estimate a more realistic orientation factor, <kappa2>*, for each donor/acceptor pair. Time-resolved and steady-state fluorescence energy-transfer experiments revealed that this distance assay, spanning six different donor-acceptor distances, is linear and accurate (to within 10-20%) over the range of 30-70 A. This distance assay system for PGK allows for the measurement of long-range changes in intra- and interdomain spatial organization during protein folding reactions. The approach which we have developed can be applied to any protein system in which unique one- and two-site cysteine residues can be engineered into a protein. In the following paper [Lillo, M. P., et al. (1997) Biochemistry 36, 11273-11281], these multisite energy-transfer pairs are utilized for stopped-flow unfolding studies.
Lillo
M P
MP
Division of Biology, Physical Biochemistry Section, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Beechem
J M
JM
Szpikowska
B K
BK
Sherman
M A
MA
Mas
M T
MT
D018360:Crystallography, X-Ray; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D009282:Naphthalenesulfonates / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D010735:Phosphoglycerate Kinase