ASSEMBLY AND DISASSEMBLY OF THE TETRAMERIC POTASSIUM CHANNEL KCSA

PhD student project 9
(Early Stage Researcher 9, ESR9)

ASSEMBLY AND DISASSEMBLY OF THE TETRAMERIC POTASSIUM CHANNEL KCSA

Supervision: Antoinette Killian

Early Stage Researcher: Jonas Dörr

   

Aim:
Understanding how lipids modulate the folding, self-assembly and stability of the tetrameric ion channel KscA.

Methodology:
ESR9 will set up an in vitro transcription/translation assay with lipid acceptor vesicles to establish how lipids influence folding and assembly of the tetrameric channel KcsA and relevant protein mutants (collaboration Braakman). A new assay will be developed for general purification of membrane protein complexes without the use of detergent, from which also other ESRs in the network will profit (collaboration UPE). This procedure will allow biochemical and biophysical characterization of native protein/lipid complexes and will facilitate reconstitution into lipid vesicles of well-defined composition. ESR9 will use solid-state NMR approaches to characterize native and reconstituted complexes of KcsA (mutants) with lipids. The project will also involve crystallisation trials (collaboration Gros) and functional assays in native and reconstituted systems (collaboration Thiel).

Collaborators:
Braakman, Gros; Thiel (TU Darmstadt); UPE

Key publications:

  1. Raja M, Spelbrink REJ, De Kruijff B and Killian JA (2007) Phosphatidic acid plays a special role in stabilizing and folding of the tetrameric potassium channel KcsA. FEBS Lett. 581, 5715-5722.
  2. Nyholm, TKM, Özdirekcan S and Killian JA (2007) How protein transmembrane segments sense the lipid environment. Review. Biochemistry 46, 1457-1465.
  3. Spelbrink REJ. Kolkman A, Slijper M, Killian JA and De Kruijff B (2005) Detection and identification of stable oligomeric protein complexes in E. coli inner membranes. A proteomics approach. J. Biol. Chem. 280, 28742-28748.