Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Jose Castellon - California State University Los Angeles
Co-Author(s): Audrey Kishishita and Josh Lug, CSULA, Los Angeles, CA
Antifreeze proteins (AFPs) are highly known for the ability to bind and inhibit ice crystals, thus allowing species such as fish and insects to survive sub-zero temperatures. The relative flat protein surface contributes to the adsorption of the protein onto the crystal surface. It has been also reported by our laboratory that AFPs can control the crystallization of non-ice like compounds. We have demonstrated that AFPs can inhibit the formation of calcium carbonate crystals. It is speculated that charged residues (e.g., aspartate, glutamate, and arginine) on the proteins’ surfaces play an important role in this effect by forming ionic interactions with calcium or carbonate ions on the crystal’s surface. In this study, two AFP mutants are genetically modified in order to alter the charged residues on the proteins’ surfaces for an AFP from Tenebrio molitor (TmAFP). One of the mutants is engineered to contain a ladder of about equally dispersed aspartate residues. The other mutant is designed as a control by removing all negatively charged residues. The mutant with a ladder of about equally dispersed aspartate residues are expected to have higher affinity for the calcium site on the calcium carbonate crystalline surfaces and thus inhibit crystal growth more effectively. The effects of the mutants on calcium carbonate crystallization are investigated and compared to those of the wild-type AFPs using pH, absorbance, and zeta potential assays. The results shed light on the molecular mechanism of this novel function of AFPs. The achieved structural information will be useful for designing next-generation of antiscalants and anticaking agents.
Funder Acknowledgement(s): This material is based upon work supported by the National Science Foundation under Grant #HRD-1463889.
Faculty Advisor: Xin Wen, email@example.com
Role: Designed mutants using molecular graphics programs (Molsoft, pyMol). Designed primers for mutations. Performed mutagenesis, PCR, miniprep, and transformation of mutants onto competent cells. Performed cell growth and extracted protein through various biochemical techniques, including; cation-exchange chromatography, dialysis, SDS-PAGE gel electrophoresis, and buffer preparation. Conducted analysis using different techniques such as absorbance reading, pH detection, zeta potential detection, and electronegativity detection. Prepared supersaturated solutions of sodium carbonate and calcium chloride to produce calcium carbonate crystals for analysis.