The energy landscape of a fast-folding protein mapped by Ala-->Gly substitutions.
A moderately stable protein with typical folding kinetics unfolds and refolds many times during its cellular lifetime. In monomeric lambda repressor this process is extremely rapid, with an average folded state lifetime of only 30 milliseconds. A thermostable variant of this protein (G46A/G48A) unfolds with the wild-type rate, but it folds in approximately 20 microseconds making it the fastest-folding protein yet observed. The effects of alanine to glycine substitutions on the folding and unfolding rate constants of the G46A/G48A variant, measured by dynamic NMR spectroscopy, indicate that the transition state is an ensemble comprised of a disperse range of conformations. This structural diversity in the transition state is consistent with the idea that folding chains are directed towards the native state by a smooth funnel-like conformational energy landscape. The kinetic data for the folding of monomeric lambda repressor can be understood by merging the new energy landscape view of folding with traditional models. This hybrid model incorporates the conformational diversity of denatured and transition state ensembles, a transition state activation energy, and the importance of intrinsic helical stabilities.
Burton
R E
RE
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Huang
G S
GS
Daugherty
M A
MA
Calderone
T L
TL
Oas
T G
TG
D000409:Alanine / Q000235:genetics; D004268:DNA-Binding Proteins; D005998:Glycine / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D016296:Mutagenesis; D017510:Protein Folding; D012097:Repressor Proteins / Q000235:genetics; D013816:Thermodynamics; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D000409:Alanine; D005998:Glycine