Exploring Cold-Adapted Eye Lens Proteins and Discovery of an Antimicrobial Protein from a Carnivorous Plant

This thesis explores the eye lens proteins from the Antarctic toothfish Dissostichus mawsoni and how they have evolved to the subfreezing temperatures of their environment. This includes showing that toothfish γS1- and γS2-crystallins are less stable than their homologous human counterparts and working toward solving their structures using solution-state NMR. By making structural and biophysical comparisons, inferences can be made about how these crystallins have become cold adapted. Another unique adaptation of D. mawsoni is the ability to completely resist cold cataract, a liquid-liquid phase separation of the proteins. By studying γM-crystallins from D. mawsoni that are susceptible to phase separation and performing site specific mutagenesis it was discovered the temperature of phase separation could be controlled by simply swapping between lysine and arginine residues. These results hint at hydration effects and salt bridges as being a major factor influencing the crystallin’s propensity to self associate into a separate liquid phase. To further characterize these proteins their functional role of providing refractive power was measured. The results of these measurements demonstrated that all the crystallins tested had a refractive index much higher than would be predicted by their amino acid composition. This suggests that protein conformation has a large impact on protein refractivity and the assumed models used to predict protein refractivity must be approached much more carefully.Also described is the D1-PSI discovered in the genome of the carnivorous plant Drosera capensis. As a saposin-like protein, it has demonstrated the ability to interact with membranes in a way that can exhibit anti-microbial growth. Results show that this PSI is able to disrupt membranes, but seems to lack bias for which lipid head groups it interacts with in the context of a stable lipoprotein complex. To better understand what is structurally happening to the D1-PSI while interacting with a membrane, solid-state NMR experiments are ongoing. Such information can inform the mechanism by which D1-PSI is capable of disrupting membranes