Slowing Down Downhill Folding: A Three-Probe Study

The mutant Tyr22Trp/Glu33Tyr/Gly46Ala/Gly48Ala of λ repressor fragment λ6−85 was previously assigned as an incipient downhill folder. We slow down its folding in a cryogenic water-ethylene-glycol solvent (−18 to −28°C). The refolding kinetics are probed by small-angle x-ray scattering, circular dich...

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Bibliographic Details
Published in:Biophysical journal 2009-07, Vol.97 (1), p.295-302
Main Authors: Kim, Seung Joong, Matsumura, Yoshitaka, Dumont, Charles, Kihara, Hiroshi, Gruebele, Martin
Format: Article
Language:English
Subjects:
BASIC BIOLOGICAL SCIENCES
Biophysics
Circular Dichroism
CRYOGENICS
DICHROISM
EXTRAPOLATION
FLUORESCENCE
GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
Guanidine - chemistry
KINETICS
Models, Molecular
MORPHOLOGY
MUTANTS
Mutation
NMR
Nuclear magnetic resonance
PHASE STUDIES
PROBES
Protein
Protein Folding
Protein Structure, Secondary
Repressor Proteins - chemistry
Repressor Proteins - genetics
Repressor Proteins - metabolism
Scattering, Radiation
SMALL ANGLE SCATTERING
SOLVENTS
Temperature
Time Factors
TRYPTOPHAN
Viral Regulatory and Accessory Proteins - chemistry
Viral Regulatory and Accessory Proteins - genetics
Viral Regulatory and Accessory Proteins - metabolism
X-Rays
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Summary:The mutant Tyr22Trp/Glu33Tyr/Gly46Ala/Gly48Ala of λ repressor fragment λ6−85 was previously assigned as an incipient downhill folder. We slow down its folding in a cryogenic water-ethylene-glycol solvent (−18 to −28°C). The refolding kinetics are probed by small-angle x-ray scattering, circular dichroism, and fluorescence to measure the radius of gyration, the average secondary structure content, and the native packing around the single tryptophan residue. The main resolved kinetic phase of the mutant is probe independent and faster than the main phase observed for the pseudo-wild-type. Excess helical structure formed early on by the mutant may reduce the formation of turns and prevent the formation of compact misfolded states, speeding up the overall folding process. Extrapolation of our main cryogenic folding phase and previous T-jump measurements to 37°C yields nearly the same refolding rate as extrapolated by Oas and co-workers from NMR line-shape data. Taken together, all the data consistently indicate a folding speed limit of ∼4.5 μs for this fast folder.
ISSN:0006-3495
1542-0086
DOI:10.1016/j.bpj.2009.04.041