Current Projects in the Vaden Research Group
Understanding the structures and stabilities of proteins in the presence of ionic liquids (Funded by NSF DMR-1904707)
Ionic liquids are liquid salts that can help stabilize or destabilize proteins for in vitro biochemical technology. The right combination of cations and anions can be used to destabilize unwanted proteins and stabilize desired proteins for a specific application. Our research focuses on understanding what molecular forces and ionic liquids properties are needed to design specific-protein stabilizing ionic liquid biomaterials.
Our current example of this protein-stabilization is directed to the azurin-nitrite reductase complex which is used in bacterial denitrification:
Crystal structure of three proteins (Cu(I/II) in green) for bioremediation development. A: Pseudoazurin-NiR complex (PDB id: 5B1J); B: Azurin (PDB id: 4MFH); C: NiR (PDB id: 1MZY); D: Laccase (PDB id: 1GYC).
Denitrification is the process of reducing soil/water nitrates into atmospheric nitrogen. Excess nitrates from fertilizers and industry can cause atmospheric problems due to generation of NOx pollutants and the greenhouse-gas N2O, which are denitrification intermediates. A key step in bacterial denitrification is the NO2- to NO reduction at the nitrite reductase (NiR)/azurin redox protein system. Azurin, bound to NiR, transfers the electrons via the Cu (I/II) metallocenter. Utilizing this protein system for engineered denitrification has potential for industrial-scale environmental remediation, especially in the removal of nitrates from contaminated water. If it can be selectively stabilized and enhanced in non-physiological environments, NiR/azurin may be exploited for environmentally-friendly industrial and environmental chemistry. Together, the complex can be isolated and utilized for environmental remediation. Separately, azurin can be targeted for electrochemistry and the NiR enzyme can catalyze nitrogen-based chemistry. By altering the protein structures and copper metallocenters the copper reduction voltages can be tuned to modulate protein activities. Hence, it should be possible to selectively enhance NiR and azurin by using solution additives to stabilize or destabilize their structures.
Experimentally, we can express and purify proteins (eg, azurin and nitrite reductase) and then use biophysical chemistry techniques to quantify how they are affected by different ionic liquids. These techniques include IR, UV, and Fluorescence spectroscopy versus temperature to watch the protein unfold as a function of temperature, and mass spectrometry coupled with HDX reactions to quantify the tertiary structure (open and loose versus tight and closed).
Here below is a recent poster that summarizes some technical results for the protein azurin:
Assessing the lipid membrane permeabilities of ionic liquid drug-delivery formulations (Funded by Rowan University SEED Grant)
