LATE EMBRYOGENESIS ABUNDANT PROTEIN Chaperonin Capacity: Unraveling Protective Mechanisms Potentiating a Stable Cellular Milieu During Anhydrobiosis.

Society takes advantage of the wide availability of refrigeration to maintain food, medical supplies (vaccines, plasma), chemicals, and whole cells in a useful state. The cold-chain (CC; as it has become known) ensures that valuable, fragile biological materials (FBM) are stored under conditions that maximize FBM utility. The absolute requirement for uninterrupted CC maintenance to safeguard FBM function underlies the vulnerability of the CC to electrical disruption. Power outage frequently occurs during natural disasters and armed conflict while remote areas remain without access to electricity entirely. Drying FBM while maintaining its serviceability (usually defined as activity or even viability upon rehydration) offers numerous advantages relative to the CC which is expensive, difficult to extend to regions without it, subject to disruption, centrally distributed, and conspicuous by its nature (heavy, bulky, noisy refrigerators/generators and equipment heat signatures at a central locale). The focus of my research is to understand how certain organisms can, at some stage of their life cycle, withstand almost complete loss of water without dying. While in the desiccated state these organisms display extreme resistance to abiotic perturbation and aging. Generally, the attributes cells acquire leading to anhydrobiosis (life with little water) include the accumulation of specific, non-reducing oligosaccharides (trehalose, and sucrose:raffinose admixtures) and some form of intrinsically disordered protein (in plants and some animals, LATE EMBRYOGENESIS ABUNDANT PROTEINs, LEAPs). These macromolecules are thought to: 1) replace water around otherwise desiccation sensitive cellular constituents; 2) vitrify the cellular interior, embedding cellular contents in a glass that severely restricts movement and represses metabolism and; 3) shield sensitive cellular components from reactive oxygen species and denaturation/aggregation. Our current scientific objective is a step-wise investigation of if and how the hallmark anhydrobiotic molecules, oligosaccharides and a LEAP (SEED MATURATION PROTEIN1 (SMP1)), perform in synergy to enhance the stability of a specific fragile client protein of SMP1, the CANCER SUSCEPTIBILITY CANDIDATE3 (CASC3) to salt, freezing, and desiccation. Assessments of the affinity of non-reducing sugars, SMP1, and mixtures of these protective molecules for CASC3 in various solutions, after a variety of stresses, will use the Nanotemper Dianthus instrument, the subject of this request. The Dianthus can also assess the aggregation of CASC3 following stress and the capacity of SMP1 to dissipate these aggregates over time. Protective oligosaccharide recovery (assessed by HPLC) from CASC3, SMP1 mixtures after stress will unveil if the protective non-reducing sugars can quantitatively release CASC3/SMP1 post-stress, allowing the rapid depletion of the sugars to normal levels. An undergraduate researcher will be recruited from the University of Kentucky Medical and Agricultural Biotechnology program to assist in this project. They will help forward our rudimentary understanding of how two groups of molecules, polyhydric, non-reducing sugars and intrinsically disordered, hydrophilic LEAPs may protect labile macromolecules from desiccation-induced damage. The relative effectiveness of the protective sugar trehalose versus 3:1 sucrose:raffinose in defined concentrations in affording protection to CASC3 will be ascertained. The synergy in the protective influence of specific sugars and SMP1, in defined ratios and amounts, will be revealed.