# Correlation of calpain sensitivity, Bradford assay instability, and electrophoretic mobility in phosphomimetic mutants of GlyT2 N-terminus

## TL;DR

This study investigates how phosphorylation-like mutations affect the structure of the GlyT2 transporter's N-terminus using biochemical assays.

## Contribution

The study introduces a correlation between calpain sensitivity, Bradford instability, and electrophoretic mobility shifts in phosphomimetic GlyT2 mutants.

## Key findings

- Phosphomimetic mutations in GlyT2's N-terminus alter calpain cleavage patterns and SDS gel mobility.
- Bradford instability and SDS mobility shifts are correlated in phosphomimetic GlyT2 mutants.
- Neutral mutations do not significantly affect GlyT2N properties, unlike phosphomimetic ones.

## Abstract

The glycine transporter GlyT2 plays an important role in glycine-inhibitory neurotransmission of the hindbrain and spinal cord. Its special feature is the extended N-terminus, which contains a large number of potentially phosphorylated serine and threonine residues. Due to its unstructured nature, it is difficult to address the changes introduced by potential phosphorylation. Here, we used relatively simple methods such as calpain sensitivity, Bradford instability, and SDS electrophoretic mobility shift to investigate the effect of multiple phosphomimetic mutations versus neutral mutations on GlyT2N properties. The replacement of several serines or threonines with neutral alanines did not have a significant effect on the studied GlyT2N properties. Replacement of the same residues with phosphomimetic aspartate resulted in significant alterations in calpain cleavage patterns, Bradford instability, and SDS gel protein mobility. Interestingly, a correlation between the relative intensity of the measured effects was observed, indicating that they all reflect similar structural changes introduced by potential phosphorylation in vivo. Results indicate that a potential single or multiple phosphorylation significantly alters the proteomic properties of the glycine transporter GlyT2 N-terminus. Assays can be helpful in the first screening of structurally significant and possibly phosphorylated residues in the N-terminus of GlyT2.

•Blockade of major GlyT2 N calpain cleavage sites reveals multiple calpain sensitivity.•GlyT2 N-terminal phosphomimetic mutations cause a SDS gel mobility shift.•GlyT2 N-terminal SDS gel mobility shift is correlated with its Bradford instability.

Blockade of major GlyT2 N calpain cleavage sites reveals multiple calpain sensitivity.

GlyT2 N-terminal phosphomimetic mutations cause a SDS gel mobility shift.

GlyT2 N-terminal SDS gel mobility shift is correlated with its Bradford instability.