Structural investigation of disordered stress proteins. Comparison of full-length dehydrins with isolated peptides of their conserved segments

Abstract

Dehydrins constitute a class of intrinsically disordered proteins that are expressed under conditions of water-related stress. Characteristic of the dehydrins are some highly conserved stretches of seven to 17 residues that are repetitively scattered in their sequences, the K-, S-, Y-, and Lys-rich segments. In this study, we investigate the putative role of these segments in promoting structure. The analysis is based on comparative analysis of four full-length dehydrins from Arabidopsis (Arabidopsis thaliana; Cor47, Lti29, Lti30, and Rab18) and isolated peptide mimics of the K-, Y-, and Lys-rich segments. In physiological buffer, the circular dichroism spectra of the full-length dehydrins reveal overall disordered structures with a variable content of poly-Pro helices, a type of elongated secondary structure relying on bridging water molecules. Similar disordered structures are observed for the isolated peptides of the conserved segments. Interestingly, neither the full-length dehydrins nor their conserved segments are able to adopt specific structure in response to altered temperature, one of the factors that regulate their expression in vivo. There is also no structural response to the addition of metal ions, increased protein concentration, or the protein-stabilizing salt Na(2)SO(4). Taken together, these observations indicate that the dehydrins are not in equilibrium with high-energy folded structures. The result suggests that the dehydrins are highly evolved proteins, selected to maintain high configurational flexibility and to resist unspecific collapse and aggregation. The role of the conserved segments is thus not to promote tertiary structure, but to exert their biological function more locally upon interaction with specific biological targets, for example, by acting as beads on a string for specific recognition, interaction with membranes, or intermolecular scaffolding. In this perspective, it is notable that the Lys-rich segment in Cor47 and Lti29 shows sequence similarity with the animal chaperone HSP90.

Figure 1.

Amino acid sequences of Cor47 (SK₃), Lti29 (SK₃), Lti30 (K₆), and Rab18 (Y₂SK₂). The K-segment is highlighted in green, the Y-segment in red, the S-segment in yellow, and the Lys-rich segment (ChP-1) in purple.

Figure 2.

CD spectra of the full-length dehydrins (top) and their conserved segments (bottom) at 4°C.

Figure 3.

The CD signal at 222 nm versus protein concentration shows linear relations indicating that the dehydrins and their conserved segments have low propensities to aggregate. The K-segment, ▪; the Y-segment, ○; Lti30, X; Lti29, □; ChP-1, ▾; and Cor47, •.

Figure 4.

CD difference spectra showing the degree of structural induction at 90% TFE at 4°C. The full-length dehydrins (top) and their conserved segments (bottom).

Figure 5.

The increase in helical content upon titration with TFE at 4°C, as derived from CD data by the spectral analysis software CDPro. The full-length dehydrins (top) and their conserved segments (bottom).

Figure 6.

Changes in the CD signal at 222 nm upon titration of the full-length dehydrins with GdmCl.

Figure 7.

Structural response to increased temperature in pure buffer and in the presence of TFE. Top, CD spectra of Lti29; spectra are reading from the top at 220 nm Lti29 at 4°C, Lti29 at 25°C after having been heated to 85°C, Lti29 at 25°C, and Lti29 at 85°C. Bottom, Combined effect of elevated temperature and TFE addition on the K-segment.

Figure 8.

Temperature scans at 222 nm indicating the gradual loss of PII helices. The full-length dehydrins and a destabilized mutant of SOD1 (top) and the conserved segments (bottom).

Figure 9.

Plot of the CD signals at 222 nm versus 200 nm for a set of disordered proteins (○; adapted from Uversky, 2002). The dataset indicates two clusters, one representing intrinsic coils (top left) and the other premolten globules (right). Data for the full-length dehydrins and their conserved segments at 4°C, 25°C, and 85°C are shown as joined plots progressing from top left to bottom right. At elevated temperatures, both the dehydrins and the peptide segments shift consistently toward the premolten globule cluster. Cor47, •; Lti29, ▪; Lti30, X; Rab18, ♦; K-segment, +; ChP-1, ▵; and ChP-2, .

Figure 10.

α-Helical propensity per amino acid of Cor47, Lti29, Lti30, and Rab18 as predicted from their sequence composition by AGADIR. The maxima in α-helical propensity match overall the positions of the conserved segments.