Česká společnost pro hmotnostní spektrometrii

(CEEPC/IPM/CMSC 2022 - ThP-41)
Insights into the transport dynamics of prokaryotic chloride/proton antiporter

Jasmína Portašiková ^1,2 *, Lukáš Fojtík ^1,2, Petr Novák ¹, Petr Man ¹

1. MBÚ AV ČR - BioCeV
2. PřF UK

Abstrakt

Chloride channel family includes transmembrane channels and transporters, which are involved in many cellular processes and their mutation can cause serious illnesses. To treat these diseases, a description of their transport mechanism is needed. Chloride transporter CLC-ec1 from E. coli is often used as a model protein to study the protein family of chloride channels. The protein functions as an antiporter of a single proton for two chloride anions. During transport of ions, the protein converts between inward and outward-facing conformations. Outward-facing state is induced by protons, when Glu residues which are involved in ion transport are protonated. This state can be mimicked by a QQQ mutant, in which three key Glu residues are mutated to Gln residues [1]. So far, CLC-ec1 transport has been studied by X-ray crystallography that provided detailed but static images. Here we used hydrogen/deuterium exchange mass spectrometry to extend the recent findings and provide more detailed insight into the transport dynamics of this protein. Full-length wild type and QQQ CLC-ec1 were overexpressed in bacteria, isolated via detergent solubilization and purified by affinity chromatography and gel filtration. HDX-MS based digestion was carefully optimized to provide full sequence coverage with reasonably sized peptides. Next, HDX-MS experiment was conducted at different conditions. First, we compared WT and QQQ proteins at pH 7.4, which pointed to differences between the two forms underlying their different conformations. Next, to address the role of protonation, both proteins were followed at four pH values spanning pH range 4.4-7.4. These data highlight the stepwise protonation and the associated structural changes across the ion transport path.

* Korespondující autor: portasij@natur.cuni.cz

Literatura

1. Chavan T. et al: Elife 9, e53479 (2020).

Poděkování:

Financial support from CSF project 22-27695S and CIISB (LM2018127) is gratefully acknowledged.