Cryoprotective mechanism of a small intrinsically disordered dehydrin protein

Abstract

Dehydration proteins (Dehydrins) are expressed during dehydration stress in plants and are thought to protect plant proteins and membranes from the loss of water during drought and at cold temperatures. Several different dehydrins have been shown to protect lactate dehydrogenase (LDH) from damage from being frozen and thawed. We show here that a 48 residue K 2 dehydrin from Vitis riparia protects LDH more effectively than bovine serum albumin, a protein with known cryoprotective function. Light scattering and 8-anilino-1-naphthalene sulfonate fluorescence experiments show that dehydrins prevent aggregation and unfolding of the enzyme. The cryoprotective effects of LDH are reduced by the addition of salt, suggesting that the positively charged K-segments are attracted to a negatively charged surface but this does not result in binding. Overall K 2 is an intrinsically disordered protein; nuclear magnetic resonance relaxation experiments indicate that the two-terminal, Lys-rich K-segments show a weak propensity for ahelicity and are flexible, and that the central, polar rich phi-segment has no secondary structure preference and is highly flexible. We propose that the phi-segments in dehydrins are important for maintaining the disordered structure so that the protein can act as a molecular shield to prevent partially denatured proteins from interacting with one another, whereas the K-segments may help to localize the dehydrin near the enzyme surface.

The Importance of Size and Disorder in the Cryoprotective Effects of Dehydrins

“…Dehydrins are thought to be involved in dehydration protection, since their transcription and translation are increased during dehydration, and a correlation exists between drought tolerance and the amount of dehydrin present. In vitro, dehydrins have been shown to protect enzymes from freeze-thaw damage (Lin and Thomashow, 1992;Kazuoka and Oeda, 1994;Momma et al, 2003;Goyal et al, 2005;Hughes and Graether, 2011) and heat denaturation (Kovacs et al, 2008), interact with and protect membranes from cold and dehydrative stresses (Rahman et al, 2010;, and bind water (Tompa et al, 2006), ions (Alsheikh et al, 2003), and nucleic acids (Hara et al, 2009). Dehydrins have also been suggested to prevent the growth of ice crystals by functioning in a manner similar to antifreeze proteins (AFPs; Wisniewski et al, 1999;Simpson et al, 2005).…”

“…The only clear role shown to date for the K-segments is their ability to bind anionic membranes (Koag et al, 2009). It has been suggested that the K-segment may be an amphipathic helix, although NMR studies showed that the segment is only very weakly helical in solution (Hughes and Graether, 2011). Some dehydrins have two additional conserved amino acid motifs, known as the Y-segment and the S-segment.…”

“…Overall, dehydrin sequences are highly hydrophilic and generally lack Cys or Trp residues, although one dehydrin has been discovered to have Cys residues (Peng et al, 2008;Reyes et al, 2008). Not surprisingly, the high polar content results in dehydrins being intrinsically disordered proteins (IDPs; Hughes and Graether, 2011), and as such, they do not have a single, well-defined protein structure (Lisse et al, 1996;Tompa, 2002;Uversky, 2002a). IDPs are generally highly flexible and have minimal secondary structure, although some undergo a disorder-to-order transition after binding a ligand (Tompa, 2002).…”

“…We have previously characterized the Vitis riparia K 2 dehydrin Livernois et al, 2009;Hughes and Graether, 2011). This protein appears to be a splice variant of YSK 2 mRNA whose transcription is up-regulated after cold stress (Xiao and Nassuth, 2006).…”

CRISPR-induced null alleles show that Frost protects Drosophila melanogaster reproduction after cold exposure

“…We determined whether FST can protect enzyme activity during freezing using a lactate dehydrogenase (LDH) cryoprotection assay as performed previously (Hughes and Graether, 2011). The samples were immersed five times in liquid nitrogen for 30 s and thawed by immersion in a circulating water bath at 4°C for 5 min.…”