Folding of the alphaII-spectrin SH3 domain under physiological salt conditions.
The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of alphaII-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the alphaII-spectrin SH3 domain.
Petzold
Katja
K
Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Ohman
Anders
A
Backman
Lars
L
D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013049:Spectrin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D018909:src Homology Domains
D011994:Recombinant Proteins; D013049:Spectrin