Temperature-Induced Extended Helix/Random Coil Transitions in a Group 1 Late Embryogenesis-Abundant Protein from Soybean

Abstract

Group 1 late embryogenesis-abundant (LEA) proteins are a subset of hydrophilins that are postulated to play important roles in protecting plant macromolecules from damage during freezing, desiccation, or osmotic stress. To better understand the putative functional roles of group 1 LEA proteins, we analyzed the structure of a group 1 LEA protein from soybean (Glycine max). Differential scanning calorimetry of the purified, recombinant protein demonstrated that the protein assumed a largely unstructured state in solution. In the presence of trifluoroethanol (50% [w/v]), the protein acquired a 30% ␣-helical content, indicating that the polypeptide is highly restricted to adopt ␣-helical structures. In the presence of sodium dodecyl sulfate (1% [w/v]), 8% of the polypeptide chain adopted an ␣-helical structure. However, incubation with phospholipids showed no effect on the protein structure. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein existed in equilibrium between two conformational states. Ultraviolet absorption spectroscopy studies also showed that the protein became more hydrated upon heating. Furthermore, circular dichroism spectral measurements indicated that a minimum of 14% of amino acid residues existed in a solvent-exposed, left-handed extended helical or poly (l-proline)-type (PII) conformation at 20°C with the remainder of the protein being unstructured. The content of PII-like structure increased as temperature was lowered. We hypothesize that by favoring the adoption of PII structure, instead of the formation of ␣-helical or ␤-sheet structures, group 1 LEA proteins retain a high content of surface area available for interaction with the solvent. This feature could constitute the basis of a potential role of LEA proteins in preventing freezing, desiccation, or osmotic stress damage.Late embryogenesis-abundant (LEA) proteins accumulate to high concentrations in plant embryos during the latter stages of seed development before desiccation (Baker et al., 1988;Dure et al., 1989;Hughes and Galau, 1989). LEA proteins also accumulate in vegetative tissues exposed to exogenous abscisic acid, as well as dehydration, osmotic, and lowtemperature stress (Chandler and Robertson, 1994;Ingram and Bartels, 1996; Bray, 1997; Close, 1996 Close, , 1997Thomashow, 1998; Nylander et al., 2001). More than seven different groups of LEA proteins have been described and categorized by virtue of similarities in their deduced amino acid sequences (Baker et al., 1988;Dure et al., 1989). The majority of LEA proteins are highly hydrophilic and display a preponderance (e.g. Ala, Gly, Glu, and Thr) or lack (e.g. Trp and Cys) of certain amino acid residues (Dure, 1993a(Dure, , 1993b(Dure, , 1997. Thus, LEA proteins are part of a larger, evolutionarily conserved group of hydrophilic proteins termed "hydrophilins" involved in various adaptive responses to hyperosmotic conditions (Garay-Arroyo et al., 2000).Various functions have been proposed for different groups of LEA proteins ranging from wa...

Conformation of a Group 2 Late Embryogenesis Abundant Protein from Soybean. Evidence of Poly (l-Proline)-type II Structure

“…Otherwise, we should expect a decrease in ␣-helical content as the temperature increases and a negative CD band above 200 nm in the difference spectrum. Interestingly, these two features observed in the difference spectrum are present in the CD spectra of peptides rich in poly (l-Pro)-type II (PII) structures (Woody, 1992;Park et al, 1997;Fox et al, 1999;Bienkiewicz et al, 2000;Soulages et al, 2002). This similarity suggested that the spectral changes observed in rGmDHN1 could be due to a temperature-induced extended helix/unordered transition.…”

“…Additional support for this conformation arises from the coordinates of the isodichroic point observed in Figure 4, A and B (at 208 nm and Ϫ1.85 m Ϫ1 cm Ϫ1 ). These coordinates are coincident with the coordinates observed in model polypeptides and proteins that undergo PII/unordered transitions (Tiffany and Krimm, 1968;Tiffany, 1975;Woody, 1992;Siligardi and Drake, 1995;Park et al, 1997;Fox et al, 1999;Bienkiewicz et al, 2000;Soulages et al, 2002). Park et al (1997) have investigated a series of host/ guest peptides and suggested limiting values of ellipticity for the PII and the unordered structures of ϩ9,580 and Ϫ5,560°cm 2 dmol Ϫ1 at 220 nm.…”

“…The absorption spectrum of a tyrosyl residue is sensitive to the polarity of its environment, and therefore the intensity and shape of the absorption band can be used to determine the degree of hydration of Tyr. The spectral features affected by the hydration are conveniently monitored by the second derivative spectra rather than the zero order spectra (Ragone et al, 1984;Demchenko, 1986;Soulages and Bendavid, 1998;Soulages et al, 2002). The second derivative spectra of rGmDHN1 as a function of temperature are shown in the Figure 2B.…”

Genetic approaches towards overcoming water deficit in plants - special emphasis on LEAs

“…The charged amino acid motifs in LEA3 may aid in sequestering ions that accumulate under water deficit conditions (Dure, 1993). LEA proteins exhibit increase in folding when water activity is decreased using trifluoroethanol, high salt, SDS or even under dehydration conditions (Lisse et al, 1996;Ismail et al, 1999;Soulages et al, 2002). These LEA proteins are natively unfolded or intrinsically disordered but nevertheless this does not mean lack of function as increased folding is known to occur on binding to biological targets.…”

“…NMR spectroscopy of a recombinant carrot (Daucus carota) group 1 protein, EMB-1, expressed in Escherichia coli revealed no defined secondary or tertiary structure of the protein in solution (Eom et al, 1996). UV absorption and CD spectroscopy studies disclosed the unstructured protein (rGmD-19), with 6 % to 14 % of the protein occupying a left-handed extended helical or poly (l-Pro)-type (PII) conformation instead of a α-helical or β-sheet structure (Soulages et al, 2002). Circular dichroism (CD) spectroscopy and hydrodynamic modeling of the wheat group 1 LEA protein, Em divulged 70 % random coil or nonregular and a flexible secondary structure (McCubbin et al, 1985).…”