Salt-affected soil is a prominent challenge in agriculture. Nowadays, more than 800 million hectares of land (about 6% of the world’s total land area) are induced with high salt concentrations, and thus, are unsuitable for growing typically salt-sensitive crop plants. The ongoing salinization of arable land exacerbates this limitation. To address this issue, the development of salinity-tolerant crop plants has gained considerable interest, with a protein identified by Prof. Mark Tester's group, named "SALTY2," offering promising potential. SALTY2 is overexpressed in response to NaCl treatment on Salicornia plants conferring salinity tolerance, and following the function of the SALTY2 protein from Salicornia and analogous proteins in Arabidopsis, yeast and in vitro, a universal mechanism in evolution is suggested. During my thesis, we analyzed the biophysical properties of SALTY2, and based on spectroscopic methods we confirmed it is an intrinsic disordered protein (IDP), which is consistent with previous studies claiming that IDPs play a vital role in stress response pathways. We have identified and characterized the loss-of-function "RG/RGG" to "KG/KGG" type mutation and a deltaSTM1 N-terminal mutation, and investigated the interaction of SALTY2 and other cellular components, including short fragment RNA, and 80S ribosome. Together with state-of-the-art high-resolution NMR and Cryo-EM methods we validated the direct interaction of SALTY2 with plant ribosomes, and 25nts random RNA, and determined the 3D structure of ribosome with the potential binding site of the SALTY2 protein. Combining biophysical, structural and functional analyses of the wild-type and loss-of-function mutants of SALTY2, we proposed a potential mechanism by which the IDP protein SALTY2 confers salinity tolerance in plants. These findings offer a deeper understanding of the molecular basis of salinity tolerance in plants via IDPs and contribute to the ongoing efforts to develop salinity-tolerant crop plants.

abstract: Spider dragline silk is an outstanding biopolymer with a strength that exceeds steel by weight and a toughness greater than high-performance fibers like Kevlar. For this reason, structural and dynamic studies on the spider silk are of great importance for developing future biomaterials. The spider dragline silk comprises two silk proteins, Major ampullate Spidroin 1 and 2 (MaSp1 and 2), which are synthesized and stored in the major ampullate (MA) gland of spiders. The initial state of the silk proteins within Black Widow MA glands was probed with solution-state NMR spectroscopy. The conformation dependent chemical shifts information indicates that the silk proteins are unstructured and in random coil conformation. 15N relaxation parameters, T1, T2 and 15N-{1H} steady-state NOE were measured to probe the backbone dynamics for MA silk proteins. These measurements indicate fast sub-nanosecond timescale backbone dynamics for the repetitive core of spider MA proteins indicating that the silk proteins are unfolded, highly flexible random coils in the MA gland. The translational diffusion coefficients of the spider silk proteins within the MA gland were measured using 1H diffusion NMR at 1H sites from different amino acids. A phenomenon was observed where the measured diffusion coefficients decrease with an increase in the diffusion delay used. The mean displacement along the external magnetic field was found to be 0.35 μm and independent of the diffusion delay. The results indicate that the diffusion of silk protein was restricted due to intermolecular cross-linking with only segmental diffusion observable. To understand how a spider converts the unfolded protein spinning dope into a highly structured and oriented in the super fiber,the effect of acidification on spider silk assembly was investigated on native spidroins from the major ampullate (MA) gland fluid excised from Latrodectus hesperus (Black Widow) spiders. The in vitro spider silk assembly kinetics were monitored as a function of pH with a 13C solid-state Magic Angle Spinning (MAS) NMR approach. The results confirm the importance of acidic pH in the spider silk self-assembly process with observation of a sigmoidal nucleation-elongation kinetic profile. The rates of nucleation and elongation and the percentage of β-sheet structure in the grown fibers depend on pH. The secondary structure of the major ampullate silk from Peucetia viridians (Green Lynx) spiders was characterized by X-ray diffraction (XRD) and solid-state NMR spectroscopy. From XRD measurement, β-sheet nano-crystallites were observed that are highly oriented along the fiber axis with an orientational order of 0.980. Compare to the crystalline region, the amorphous region was found to be partially oriented with an orientational order of 0.887. Further, two dimensional 13C-13C through-space and through-bond solid-state NMR experiments provide structural analysis for the repetitive amino acid motifs in the silk proteins. The nano-crystallites are mainly alanine-rich β-sheet structures. The total percentage of crystalline region is determined to be 40.0±1.2 %. 18±1 % of alanine, 60±2 % glycine and 54±2 % serine are determined to be incorporated into helical conformations while 82±1 % of alanine, 40±3 % glycine and 46±2 % serine are in the β-sheet conformation. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015