Statistical description of the denatured structure of a single protein, staphylococcal nuclease, by FRET analysis

Abstract

Structural characterization of fully unfolded proteins is essential for understanding not only protein-folding mechanisms, but also the structures of intrinsically disordered proteins. Because an unfolded protein can assume all possible conformations, statistical descriptions of its structure are most appropriate. For this purpose, we applied Förster resonance energy transfer (FRET) analysis to fully unfolded staphylococcal nuclease. Artificial amino acids labeled with a FRET donor or acceptor were introduced by an amber codon and a four-base codon respectively. Eight double-labeled proteins were prepared, purified, and subjected to FRET analysis in 6 M urea. The observed behavior could be explained by a power law, R = αN^0.44, where R, and N are the distance and the number of residues between donor and acceptor, and α is a coefficient. The index was smaller than the value expected for an excluded-volume random coil, 0.588, indicating that the fully unfolded proteins were more compact than polypeptides in good solvent. The FRET efficiency in the native state did not necessarily correlate to the distance obtained from crystal structure, suggesting that other factors such as the orientation factor made a substantial contribution to FRET.

Keywords: Denatured structure; FRET; Protein; Random coil; Staphylococcal nuclease.

Fig. 1

Designed DNA sequence for the acceptor site at the fifth position and the donor site at the 33rd position (33_D5_A). The four-base codon and amber codon are demarcated by a solid box and a broken box respectively. The single underline represents the expected stop codon when the four-base codon is read as an ordinary three-base codon and a frameshift occurs. The double underline represents the 6xHis-tag. The stop codon, indicated by the wavy underline, is TGA

Fig. 2

a Gel images of the purification process of 33_D5_A. Lanes are as follows: 1 and 2, cell-free translation solution; 3 and 4, supernatant of Ni-beads; 5 and 6, cleaning solution of Ni-beads; 7, elution of Ni-beads; 8, final elution by gel filtration chromatography; 9, fluorescent marker. Top, fluorescence image of 515 nm emission at 460 nm excitation; bottom, fluorescence image of 605 nm emission at 460 nm excitation. Images were taken on an LAS4000 (GE Healthcare). b Gel images of eight purified samples. Top, fluorescence image of 520 nm emission at 488 nm excitation; center, fluorescence image of 580 nm emission at 532 nm excitation. Images were taken on an FMBIO-III (Hitachi Software Engineering). Bottom, western blot analysis of double-labeled His-tagged SNase. Proteins were visualized with an anti–His-tag antibody

Fig. 3

Normalized fluorescence spectra of double-labeled SNase in the native state (black solid line) and in the presence of 6 M urea (red broken line). a 33_D5_A, b 48_D5_A, c 70_D5_A, d 97_D5_A, e 123_D5_A, f 134_D5_A, g 143_D5_A, and h 146_D5_A

Fig. 4

Comparison of the distance estimated by FRET with the inter-Cα distance obtained from the coordinates in 2SNS (Cotton et al. 1979). Note that the coordinates of the 143rd and 146th position are ambiguous. Although the coordinates of the fifth position are also ambiguous, we used as a reference point because this residue is common for all double-labeled proteins

Fig. 5

FRET efficiency of the fully unfolded state in 6 M urea, plotted as a function of inter-residue number. The solid line is the result of fitting, described in the text