Changes in biophysical characteristics of PFN1 due to mutation causing amyotrophic lateral sclerosis

Abstract

Single amino acid mutations in profilin 1 (PFN1) have been found to cause amyotrophic lateral sclerosis (ALS). Recently, we developed a mouse model for ALS using a PFN1 mutation (glycine 118 to valine, G118V), and we are now interested in understanding how PFN1 becomes toxically lethal with only one amino acid substitution. Therefore, we studied mutation-related changes in the PFN1 protein and hypothesized that such changes significantly disturb its structure. Initially, we expressed and studied the purified PFN1^WT and PFN1^G118V proteins from bacterial culture. We found that the PFN1^G118V protein has a different mean residue ellipticity, as measured by far-UV circular dichroism, accompanied by a spectral shift. The intrinsic fluorescence of PFN1^G118V showed a small fluctuation in maximum fluorescence absorption and intensity. Moreover, we examined the time course of PFN1 aggregation using SDS-PAGE, western blotting, and MALDI-TOF/TOF and found that compared with PFN1^WT, PFN1^G118V had an increased tendency to form aggregates. Dynamic light scattering data confirmed this, showing a larger size distribution for PFN1^G118V. Our data explain why PFN1^G118V tends to aggregate, a phenotype that may be the basis for its neurotoxicity.

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