Some proteins are highly expressed during late stage(s) of seed development and are referred to as LEA (late embryogenesis abundant) proteins. These proteins are found in a wide range of plant species and are suggested to involve in desiccation tolerance based on their accumulation and physicochemical properties. [1] LEA-type proteins fall into a number of families with diverse structures and functions, that differ in the arrangement and number of conserved motifs. These proteins are also hydrophilic in nature, and are transcriptionally regulated in response to ABA. [2] Prediction of secondary structures suggests that these proteins exist as largely unfolded molecules in their native state although a few members do exist as dimers or tetramers. [3] The precise function of LEA-type proteins is largely unknown. However, their considerable synthesis during the late stage of embryogenesis, induction by stress and other biophysical characteristics, such as hydrophilicity, random coils and repeating motifs permit prediction of some of their possible functions. LEA-type proteins are reported to act as water-binding molecules, in ion sequestration and in macromolecule and membrane stabilization. [2,4]

LEA proteins are ubiquitous among photosynthetic organisms and have been reported in mono- and dicot plants as well as in nematodes, yeast, bacteria and cyanobacteria. [5] These proteins are encoded by multigene families with different number of conserved residues motifs arranged in tandem as reported in cotton, maize, barley, Arabidopsis, mung bean, soybean etc. [ 6] Our earlier work showed the occurrence of early-methionine (Em)-labeled proteins in the mung bean (Vigna radiata) axes, referred as EMV proteins, the first ever report of such proteins in the Fabaceae family. [7] The cDNAs encoding these proteins were isolated, characterized and found to show certain level of similarity with other Em/ LEA proteins.

The results of an in silico analyses of these proteins based on their deduced amino acid sequences prove that these belong to group 1 LEA protein with possible DNA/RNA binding function. Such a property may facilitate hydrogen bonding of these proteins with essentially any macromolecule or membrane thus protecting the internal structures of the cell from being damaged due to altered physiological conditions.

The occurrence of a 20 residues long repeat in group 1 LEA proteins (Table 1) identified a 20-mer motif ‘GGQTRKQQLGSEGYHEMGRK’ at positions 44-63 and 64-83 in EMV1 and at position 44-63 in EMV2, characteristic in plants and other organisms indicating that these proteins belong to group1 LEA family based on the revised classification system proposed by Wise [17]. This remarkable conservation points to an important role of LEA proteins in stress adaptation.

Table 1 Characteristics of group1 LEA proteins from Vigna radiata and other sources

Figure 1 Hydropathy analysis of predicted proteins EMV1 and EMV2 based on Kyte and Doolittle values, using a seven-residue window. Those values below zero are negative and obviously hydrophilic. Areas corresponding to highly conserved blocks are highlighted (more ...)

The structure prediction program and intrinsic disorder prediction suggest that EMV proteins maybe largely or entirely unstructured in solution. Firstly, the consensus identified for EMV proteins by structure prediction programs revealed predominantly random coil structure with two small regions of β sheets and five distinct helical blocks (Figure 2). Secondly, the GLOBPLOT analysis (Fig. 3) revealed intrinsic disorders in EMV1 (residue positions 20-29, 40-46, 49-66 and 80-91) and EMV2 (residues 20-28, 40-46, 60-71 and 93-97). Secondary structures were also observed as hydrophobic clusters and corresponded with the 1D and 3D representations (Figure 4). Low hydrophobic levels of proteins with relatively high overall charge are associated with a lack of compactness in proteins under physiological conditions resulting in a natively unfolded structure [18]

Figure 2 Secondary structure analysis of the predicted EMV proteins performed with PELE program available on the SDSC Biology Workbench (http://workbench.sdsc.edu [13]). Seven different structure predictions are shown, with the most likely structural (more ...)

Figure 3 GLOBPLOT analysis of EMV proteins indicating the regions of intrinsic disorders and conserved LEA_5 domain along the length of sequence.

Figure 4 Correspondence between 1D sequence, 2D HCA plot and 3D organization of secondary structures of EMV proteins.

Figure 5 Predicted structure of the EMV proteins. A) Backbone stereo view of EMV proteins (EMV 1- residues 1-112; EMV2- residues 1-99). B) Ribbon view of the representative EMV proteins structure (closest to average). Numbers represents the order of helices, (more ...)

Homology models validated by PROCHECK essentially satisfy the stereo-chemical parameters with well-refined structures at similar resolutions. [20] The distribution of residues in the most favored regions of the Ramachandran plot for EMV1 and EMV2 is 87.6 % and 93 %, respectively (Table 2). Thus, EMV1 is ‘fairly good’ while that of EMV2 is a ‘good’ hypothetical protein model. The homology model of mung bean LEA proteins, thus generated in this study, could aid in determining the mechanistic function of this important class of proteins.

Table 2 Ramachandran plot statistics of EMV proteins

Vigna radiata EMV proteins are classified under group 1 LEA proteins based on their extreme hydrophilicity and predominantly random-coiled arrangement of the residues along with the adoption of helical conformation as revealed by ab initio secondary structure predictions. Function assignments of these two LEA proteins suggest that they are involved in DNA/RNA binding action like other group 1 LEA proteins. Such a property may facilitate hydrogen bonding of these EMV proteins with essentially any macromolecule or membrane thus protecting the internal structures of the cell from being damaged due to altered physiological conditions. EMV proteins with the consistent spatial arrangements hence point to the possibility that they have a functional role in the plant's response to dehydration.

S.R is grateful to CSIR (Council of Scientific and Industrial Research) New Delhi, India for the Research Fellowship. Authors thank Dr. L. Arul for critical reading of the manuscript. We acknowledge the crews of NCBI, EBI, MRC Lab-UK and SIB for making computational biology data/tools publicly available.