LRR Conservation Mapping to Predict Functional Sites within Protein Leucine-Rich Repeat Domains

Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains

Growth Response, Biochemical Composition and Fatty Acid Profiles of Four Antarctic Microalgae Subjected To UV Radiation Stress

The effects of ultra-violet radiation (UVR) stress on the growth, biochemical composition and fatty acid profiles of four Antarctic microalgae from the University of Malaya Algae Culture Collection (UMACC) were investigated. The microalgae studied were Chlamydomonas UMACC 229, Navicula UMACC 231, Chlorella UMACC 237 and Klebsormidium UMACC 227 which were isolated from samples collected from Casey Station, Antarctica. Three experiments were conducted. In the first experiment, the cultures were exposed to to days of high UVB (515 µWcm⁻²) + Photosynthetically Active Radiation (PAR), UVA (845 µWcm⁻²) + PAR, and PAR alone. In the second experiment, the cultures were illuminated for to days with low UVB (117 µWcm⁻²) + PAR and PAR alone. In the third experiment, the cultures were subjected to short-term (48 hours) exposure of low UVB (117 µWcm⁻²) + PAR and PAR alone. There was no marked difference amongst the four Antarctic microalgae in terms of their growth response to UVB. All the species tested did not grow well after exposure to low (117 µWcm⁻²) or high (515 µWcm⁻²) UVB for 10 days. In contrast, exposure of UVA at 845 µWcm-2 for to days did not affect the growth of the microalgae. Growth of the cultures exposed to UVA for 48 h was only slightly affected compared to those exposed to PAR alone. The effects of UVR on lipid, carbohydrate and protein content did not show consistent trends. However, exposure to UVB resulted in marked decrease of the percentage of polyunsaturated fatty acids (PUFA) in the four microalgae. Thus, fatty acid profile is a distinct biomarker for UVR stress