Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Velia Garcia - Fisk University
Co-Author(s): Sashari Pinnace, Saffron Little, and Steven Damo, Fisk University, Nashville, TN
In proteins, structure is determined by the primary amino acid sequence and it is this structure that dictates protein function in the cell. Staphylococcus aureus has two manganese (Mn) dependent superoxide dismutases (Sods), SodA and SodM. These Sods are ~80% similar in amino acid sequence, which suggests these two proteins should have essentially the same function. Since it is rare for an organism to express two of the same protein, we hypothesized that these Sods must have different functions in the cell. SodM and SodA were expressed in E. coli using the pBG100 plasmid to drive heterologous expression of the Sod protein, which possessed a cleavable N-terminal histidine tag. We determined the activity of SodA and SodM using a colorimetric assay based on Sod’s ability to inhibit a competing superoxide reaction with WST-1. SodA was found to be 7 times more active than SodM. SodA required 37.74 ± 1.88 μg for 50% WST-1 product inhibition while SodM required 269.52 ± 7.95 μg. This difference in enzymatic activity is inconsistent with what we know about how proteins work. As a step toward revealing the structural basis of this difference in enzymatic activity of SodA and SodM, we used x-ray crystallography to determine the structure of SodM. SodM crystals diffracted to 2.0 Å in space group P43212. The phase was solved using molecular replacement, and the asymmetric unit contains two molecules. The refined model has R=18% and Rfree=22%. Future research will include determining the structure of SodA using x-ray crystallography and comparing the structures of SodA and SodM to better understand the structural basis for their differences in activity.Not Submitted
Funder Acknowledgement(s): We gratefully acknowledge the National Science Foundation for funding these studies. Velia Garcia is supported by the TSLAMP Bridges to the Doctorate program, NSF HRD1500320. This work is also supported in part by a NSF/HBCUP RIA HRD1400969 awarded to Steven Damo.
Faculty Advisor: Steven Damo, email@example.com