Isolation of Cyanide Hydratase Mutants from Gloeocerospora Sorghi at alkaline pH

Cyanide is both a useful and dangerous chemical compound that serves as a crucial component in multiple industrial processes, including metal mining. The leaching process that utilizes cyanide ions to help separate target metals and increase mining yield is an industrial standard for chemical leaching. However, this method of ore extraction results in toxic cyanide waste that requires dangerous, costly, and potentially environmentally damaging remediation systems to degrade. As cyanide is a naturally occurring substance, several organisms contain enzymes capable of oxidizing cyanide into less toxic compounds. Despite the effectiveness of these proteins, they lack stability and functionality at the alkaline pH levels industrial cyanide is stored at. This project attempts to optimize the screening and mutagenesis methods in hopes of a isolating an alkaline tolerant mutant of cyanide hydratase, an enzyme originally found in the fungus Gloeocercospora sorghi. This approach incorporates random mutagenesis of the target fungal gene using error-prone polymerase chain reaction and an in vivo picric acid assay that tests the activity of the mutant enzymes at target conditions. Experimentation was used to determine the ideal conditions for a screening method by testing the activity of the wild-type positive control at different reaction conditions. The final, optimized screening conditions for the high throughput assay combined a 50 μL aliquot of cell culture grown overnight in a 96 well plate with a 50 μL of 0.1 M CAPS buffered to pH 10.5. As screening continues, these conditions can be used to identify a viable, alkaline tolerant mutant. If such a mutant is identified, the molecule would be a strong bioremediation candidate for the metal mining industry and could lead to more efficient and environmentally friendly degradation of cyanide waste

ELUCIDATING GENERAL EFFECTS OF BILAYER HYDRATION AND MECHANICAL FORCES ON β-BARREL MEMBRANE PROTEIN ENERGETICS

A quantitative model describing how molecular driving forces balance to shape structure and function in biological membranes is immensely important for both understanding cellular vitality and curing diseases. To accomplish this goal, general principles that report on the unique physical chemistry of protein and lipid interactions must be determined. The lipid bilayer is a chemically diverse and asymmetric structure, and this complexity gives rise to a multitude of factors that influence the energy landscape of integral membrane proteins. In this work, we investigate how depth-dependent changes in local water concentration and the transduction of mechanical forces via the bilayer alter structure and thermodynamics of β-barrel membrane proteins. We determined that backbone hydrogen bonds located in the bilayer interface and those found in soluble portions contribute equally to the folding free energy of the protein. This was achieved using a combination of experimental and theoretical methods, and our findings show that backbone hydrogen bond stabilities in OmpW are similar in magnitude to those found in water soluble proteins. In addition, we developed a theoretical framework describing how transmembrane β-barrel proteins respond to bilayer tension. We then applied this framework to study a total of eight outer membrane proteins from Gram negative bacteria. Our studies revealed that β-barrel membrane proteins are the most rigid component of the cell envelope and strengthen the mechanical properties of the outer membrane in a concentration dependent fashion. This stiffness is observed in all outer membrane proteins investigated, regardless of size, species of origin, or function. Finally, we observe that bilayer tension can remodel β-barrel shape to make these proteins more circular. Protein-protein interactions reduce this reshaping and suggest a general function for complex formation in outer membrane proteins. These studies help to clarify the role of the membrane in influencing protein energetics and motivate future research on the interplay between lipid and proteins in the native environment

ELUCIDATING GENERAL EFFECTS OF BILAYER HYDRATION AND MECHANICAL FORCES ON β-BARREL MEMBRANE PROTEIN ENERGETICS

A quantitative model describing how molecular driving forces balance to shape structure and function in biological membranes is immensely important for both understanding cellular vitality and curing diseases. To accomplish this goal, general principles that report on the unique physical chemistry of protein and lipid interactions must be determined. The lipid bilayer is a chemically diverse and asymmetric structure, and this complexity gives rise to a multitude of factors that influence the energy landscape of integral membrane proteins. In this work, we investigate how depth-dependent changes in local water concentration and the transduction of mechanical forces via the bilayer alter structure and thermodynamics of β-barrel membrane proteins. We determined that backbone hydrogen bonds located in the bilayer interface and those found in soluble portions contribute equally to the folding free energy of the protein. This was achieved using a combination of experimental and theoretical methods, and our findings show that backbone hydrogen bond stabilities in OmpW are similar in magnitude to those found in water soluble proteins. In addition, we developed a theoretical framework describing how transmembrane β-barrel proteins respond to bilayer tension. We then applied this framework to study a total of eight outer membrane proteins from Gram negative bacteria. Our studies revealed that β-barrel membrane proteins are the most rigid component of the cell envelope and strengthen the mechanical properties of the outer membrane in a concentration dependent fashion. This stiffness is observed in all outer membrane proteins investigated, regardless of size, species of origin, or function. Finally, we observe that bilayer tension can remodel β-barrel shape to make these proteins more circular. Protein-protein interactions reduce this reshaping and suggest a general function for complex formation in outer membrane proteins. These studies help to clarify the role of the membrane in influencing protein energetics and motivate future research on the interplay between lipid and proteins in the native environment