Isolation and Characterization of yeast Sod1 & Ccs1 Taylor Sells, Stefanie Boyd Sneed, Li Liu and Duane D. Winkler Department of Biological Sciences - University of Texas at Dallas Background Approach HI-FI Binding Analysis Superoxide dismutase (Sod1) is a 32-kDa homo-dimeric metallo- enzyme that purges the cell of superoxide radicals. The mature holo-enzyme requires insertion of a catalytic copper ion, a stabilizing zinc ion, and oxidation of an intra-subunit disulfide bond. At least two of these post-translational modifications are completed by a helper protein termed the copper chaperone for Sod1 (Ccs1), though the mechanistic details of this transaction are unknown1. Mutant forms of the Sod1 molecule have been implicated in an inherited form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS)2. Pathogenic Sod1 variants are aggregation prone causing motor-neuron dysfunction leading to paralysis and death by respiratory failure. However, the mode of pathogenesis remains elusive as numerous seemingly unrelated Sod1 mutants cause an virtually indistinguishable disease. Cloning – The yeast sod1 gene was cloned into the pAG8H vector using restriction sites NarI and SalI (diagram below): Expression – BL21 DE3 pLysS cells were transformed with the cloned vector. Cells were then grown to an optical density of 0.6 and induced for 4 hours with 1mM IPTG. Purification – Recombinant protein was then purified using an AKTA-FPLC with a HisTrap HP Column. The His6-tag is then cleaved with TEV protease and separated. Source Q anion exchange followed to produce a pure protein sample. HI-FI Assays – Sod1 is fluorescently labeled at C57 and used in quantitative binding assays to determine, for the first time, dissociation constants for the Sod1•Ccs1 complex. Sod1 Labeling – The Sod1 molecule (cysteine 57) is labeled with an Alexa-546 dye via a maleimide linkage for the purpose of low-concentration binding studies. Quantitative Binding Assays – In 384-well clear bottom plates, labeled Sod1 is kept constant and titrated with increasing Ccs1. ySod His8 T7 promoter TEV cleavage Nar1 Sal1 HindIII M 25 20 15 1 2 3 4 The labeled Sod1 protein (lanes 3-4) is well behaved and runs similar to the unmodified Sod1 protein (lane 2). (Top panel: coomassie, Bottom panel Alexa 546) The change in fluorescence upon binding is measured on a fluoro-imager (bottom panel). The data points are then fit and a dissociation constant (KD) for the complex can be determined (right), which has never been done before now. KD = 114 nM Abstract Results Recent studies have shown that it is likely the immature (unmodified) forms of Sod1 that are noxious to motor neurons3. Higher order aggregate structures are formed by an over population of immature Sod1 molecules in the cell. This makes understanding mechanistic details of Ccs1-mediated Sod1 maturation that more important. Previously in this lab, the crystal structure of an immature form of Sod1 (green) bound in a hetero- dimeric conformation to the Ccs1 molecule (cyan/blue/red) was determined at high resolution. The structure suggests a model for Ccs1 action where the copper delivery domain (red) of Ccs1 reaches into the “open” active site of Sod1 to deliver the copper ion and oxidize the intra-subunit disulfide bond. Here, we amplified the sod1 gene from yeast cDNA, cloned, expressed, and purified the Sod1 construct. The isolated protein was then used to quantify interactions during Ccs1-mediated Sod1 maturation using a newly established fluorescence-based binding assay system termed HI-FI (High-throughput Interactions by Fluorescence Intensity). Cloning – Insertion of the ysod1 gene was confirmed via dual restriction digest using NarI and SalI (right, lane 3), followed by DNA sequencing. (ysod1 gene ≈ 450 bp) Expression – IPTG induced over-expression of the ysod1 gene was monitored at 1hr time points via Western blot utilizing an anti-His6 antibody. Ponceau-S stained transfer (lanes 1-6) and blot (7-10) are shown to the right. Purification – The 3-step purification proceeds as follows: (1) His-Trap HP chromatography, (2) TEV protease cleavage, rerun over the His-Trap HP column, and (3) Source Q anion exchange chromatography, resulting in ≥ 99% purity (below). (Ccs1 is purified in a similar manner using the same 3-steps) 1 2 3 Conclusions and Future Work Conclusions The ysod1 gene has successfully been cloned, expressed, and the Sod1 protein has been purified to homogeneity The Sod1 protein has been successfully labeled with Alexa-546 at a single cysteine residue (C57) Quantitative ySod1•yCcs1 binding data indicates extremely tight binding between immature Sod1 and Ccs1 Future Work Continue binding studies with increasingly modified Sod1 Crystallize Sod1 in complex with metallated Ccs1 1 2 3 4 5 6 - 7 8 9 10 Acknowledgements Thank you to the members of the Winkler lab who helped me in my research, in particular Dr. Li Liu and Stefanie Boyd Sneed. This project would not be possible without the generosity of the Undergraduate Research Program at UTD. His-Trap HP TEV-cleavage Source Q M 75 50 37 25 20 15 References Winkler, D.D., et al. (2009) Journal of Biological Chemistry 48 (15), 3436-47 Rosen, D. R., Siddique, T., Patterson, D., et al. 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