The Effect of Positive Charge Distribution on the Cryoprotective Activity of Dehydrins

Abstract

Dehydrins are intrinsically disordered proteins expressed ubiquitously throughout the plant kingdom in response to desiccation. Dehydrins have been found to have a cryoprotective effect on lactate dehydrogenase (LDH) in vitro, which is in large part influenced by their hydrodynamic radius rather than the order of the amino acids within the sequence (alternatively, this may be a sequence specific effect). However, it seems that a different mechanism may underpin the cryoprotection that they confer to the cold-labile yeast frataxin homolog-1 (Yfh1). Circular dichroism spectroscopy (CD) was used to assess the degree of helicity of Yfh1 at 1 °C, both alone and in the presence of several dehydrin constructs. Three constructs were compared to the wild type: YSK₂-K→R (lysine residues substituted with arginine), YSK₂-Neutral (locally neutralized charge), and YSK₂-SpaceK (evenly distributed positive charge). The results show that sequence rearrangements and minor substitutions have little impact on the ability of the dehydrin to preserve LDH activity. However, when the positive charge of the dehydrin is locally neutralized or evenly distributed, the dehydrin becomes less efficient at promoting structure in Yfh1 at low temperatures. This suggests that a stabilizing, charge-based interaction occurs between dehydrins and Yfh1. Dehydrins are intrinsically disordered proteins, expressed by certain organisms to improve desiccation tolerance. These proteins are thought to serve many cellular roles, such as the stabilization of membranes, DNA, and proteins. However, the molecular mechanisms underlying the function of dehydrins are not well understood. Here, we examine the importance of positive charges in dehydrin sequences by making substitutions and comparing their effects in the cryoprotection of two different proteins.

Keywords: charge; circular dichroism; cryoprotection; dehydrins; intrinsically disordered proteins; lactate dehydrogenase; sequence order; yeast frataxin homolog 1.

Figure 1

Sequences of wild-type YSK₂ and the three constructs. (A) Multiple sequence alignment of YSK₂, YSK₂K→R, YSK₂-SpaceK, and YSK₂-Neutral. The alignment was performed using ClustalW and the image was created using ESPript. Conserved regions are shown bounded by a blue box. White letters on a red background show residues conserved among all sequences, while red letters on a white background show residues that are similar. (B) Distribution of lysine and negatively charged residues in the YSK₂-SpaceK and YSK₂-Neutral constructs. All negatively charged residues are shown in red and all lysine residues are shown in blue.

Figure 2

Comparison of the disorder of the wild-type YSK₂ and the constructs. CD spectra of 10 μM protein in 10 mM Tris, pH 7.4, were collected at 25 °C. Symbols are shown as an inset. Each spectrum is an average of three replicates with six accumulations each. Error bars represent the standard deviation.

Figure 3

Cryoprotection of LDH by the YSK₂ constructs. The LDH assay was performed as described by [31]. The legend is shown as a figure inset. Lines were fitted using the equation described in Section 2, where the percent LDH preservation is relative to untreated LDH in the absence of additives. Red line, YSK₂-K→R; solid black line, YSK₂-Neutral; YSK₂-SpaceK, yellow line; YSK₂, dashed line with diamond symbols; BSA, dashed line with square symbols.

Figure 4

Spectra of Yfh1 at 25 and 1 °C. CD spectra of 10 μM Yfh1 at 25 °C and 1 °C in 10 mM Tris, pH 7.4. The spectra shown here are an average of eight replicates with error bars representing standard deviation.

Figure 5

Yfh1 spectra in the presence of YSK₂ constructs. CD spectra of 10 μM Yfh1 at 1 °C in the presence of 5 (red), 7.5 (yellow), 10 (blue), 15 (green), and 20 μM (purple). (A) Yfh1 spectra in the presence of YSK₂, (B) Yfh1 spectra in the presence of YSK₂-K→R, (C) Yfh1 spectra in the presence of YSK₂-SpaceK and (D) Yfh1 spectra in the presence of YSK₂-Neutral. The panels also contain the spectra of 10 μM Yfh1 alone at 1 °C (black circles) and at 25 °C (black × symbols). Each spectrum is an average of three replicates. All samples were in 10 mM Tris, pH 7.4, buffer.

Figure 6

The Relationship between Yfh1 helicity and construct concentration. The CD spectrum of 10 μM Yfh1 was determined at 1 °C in 10 mM Tris, pH 7.4, in the presence and absence of the various dehydrin constructs. The equation described in Section 2 was used to find the percent α-helicity of Yfh1. Three replicates were used to create this image and the standard deviation is represented by the error bars. The dashed line indicates the helicity of Yfh1 alone at 25 °C, while the dotted line indicates the helicity of Yfh1 alone at 1 °C. YSK₂, light blue line; YSK₂-SpaceK, red line; YSK₂-Neutral, green line; YSK₂-K→R, yellow line.

Figure 7

The effect of PEG 10,000 on Yfh1 Structure. CD spectra of 10 μM Yfh1 in the presence of various concentrations of PEG 10,000 at 1 °C in 10 mM Tris, pH 7.4.

Figure 8

¹⁵N-HSQC spectra of YSK₂ in the presence and absence of Yfh1. ¹⁵N-HSQC spectra overlap of 0.5 mM ¹⁵N-labelled YSK₂ alone (red) and in the presence of 0.5 mM Yfh1 (blue). All samples are in pH 6.0 phosphate buffer. Data were collected at 300 K.