Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1.
Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as an intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions followed by dynamic fluorescence were markedly different. Specifically, fluorescence anisotropy data showed that a relaxation spectrum of tryptophan contained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentrations of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. The fast nanosecond motion had an average time of 0.8 ns and showed no dependence on the formation of native structure. Overall, dynamic fluorescence revealed two preliminary stages in GB1 folding, which are equated with the formation of local structure in the beta(3)-strand hairpin and the initial collapse. Both stages exist without alpha-helix formation, i. e., before the appearance of any ordered secondary structure detectable by circular dichroism. Another stage in GB1 folding might exist at very low ( approximately 1 M) GdnHCl concentrations.
Tcherkasskaya
O
O
Laboratory of Experimental and Computational Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. tcherkasskaya@nih.gov
Knutson
J R
JR
Bowley
S A
SA
Frank
M K
MK
Gronenborn
A M
AM
D000942:Antigens, Bacterial / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017452:Receptors, IgG / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013291:Streptococcus / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D017452:Receptors, IgG; D014364:Tryptophan; D019791:Guanidine