Mapping Multiple Distances in a Multidomain Protein for the Identification of Folding Intermediates

Abstract

The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation.

Figure 1

(Left) A 3D structure of yPGK (PDB: 1QPG) with the N-terminal domain (residues 1–185) highlighted in red, the C-terminal domain (residues 205–415) highlighted in blue, and the hinge region (residues 186–204) highlighted in green. Black spheres indicate positions at which natural amino acids are replaced by cysteines. In total, three different interdye distances are depicted: interdomain distance Q135C-S290C, intradomain distance in the N-terminal domain S1C-Q135C, and intradomain distance within the C-domain T202C-D256C. (Right) yPGK structures of the three mutants are shown with AVs (red and blue volumes) localized at respective dye attachment positions for the three mutants. For each mutant, the C_α-distances of the mutated cysteines, the distance obtained from AV calculations, and the interdye distances obtained experimentally from the position of the smFRET peak are given. To see this figure in color, go online.

Figure 2

(Upper graphs) 3D structures of yPGK are shown with individual dye attachment positions for all three intermolecular FRET-pairs. Amino acids and related structural elements localized between the attachment positions in the amino acid sequence are highlighted in red. For the intradomain mutants, the enclosed structure elements (N1 mutant: 134 residues; C1 mutant: 54 residues) include N1 mutant with 4 β-sheets and 2 α-helices and C1 mutant with 2 β-sheets and 2 α-helices. (Lower panels) Transfer efficiency histograms for each measurement at a specific GndHCl concentration are shown. The solid lines over the histograms represent fits with one or two Gaussian peaks. To see this figure in color, go online.

Figure 3

Properties of two further intradomain distances within the C-domain. The interdye distance of the C2 mutant is given by a measured R_DA = 57 Å, whereas that of the C3 mutant yields a measured R_DA = 31 Å. Structure elements enclosed between the dye attachment positions are highlighted in red (C2 mutant: 88 residues; C3 mutant: 34 residues) and includes C2 mutant with 4 β-sheets and 3 α-helices and C3 mutant with 2 β-sheets and 1 α-helix. To see this figure in color, go online.