Folding kinetics of the SH3 domain of PI3 kinase by real-time NMR combined with optical spectroscopy.
The refolding kinetics of the chemically denatured SH3 domain of phosphatidylinositol 3'-kinase (PI3-SH3) have been monitored by real-time one-dimensional 1H NMR coupled with a variety of other biophysical techniques. These experiments indicate that the refolding kinetics of PI3-SH3 are biphasic. The slow phase (27 (+/- 8)% amplitude) is due to a population of substantially unfolded molecules with an incorrectly configured cis proline residue. The fast phase (73 (+/- 8)% amplitude) corresponds to the folding of protein molecules with proline residues in a trans configuration in the unfolded state. NMR experiments indicate that the first species populated after the initiation of folding exhibit poor chemical shift dispersion and have spectra very similar to that of the denatured protein in 8 M guanidine hydrochloride. Linear combinations of the first spectrum and of the spectrum of the native protein accurately reconstruct all of the spectra acquired during refolding. Consistent with this, native side-chain and backbone H alpha atom packing (NMR), secondary structure (far-UV circular dichroism), burial of aromatic residues (near-UV circular dichroism), intrinsic fluorescence and peptide binding activity are all recovered with effectively identical kinetics. Equilibrium unfolding and folding/unfolding kinetics yield, within experimental error, identical values for the free energy of unfolding (delta Gu-H2O = 3.38 kcal mol-1) and for the slope of the free energy of unfolding versus denaturant concentration (meq = 2.33 kcal mol-1 M-1). Together, these data provide strong evidence that PI3-SH3 folds without significant population of kinetic well-structured intermediates. That PI3-SH3 folds slowly (time constant 2.8 seconds in H2O at 20 degrees C) indicates that slow refolding is not always a consequence of kinetic traps but may be observed even when a protein appears to fold via a simple, two-state mechanism.
Guijarro
J I
JI
New Chemistry Laboratory, Oxford Centre for Molecular Sciences, University of Oxford, UK.
Morton
C J
CJ
Plaxco
K W
KW
Campbell
I D
ID
Dobson
C M
CM
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D018909:src Homology Domains
D009842:Oligopeptides; D011994:Recombinant Proteins; D019869:Phosphatidylinositol 3-Kinases; D019791:Guanidine