Computational investigation of function of membrane proteins : Amt/Rh Ammonium transporters and SecY translocon

Baday, Sefer. Computational investigation of function of membrane proteins : Amt/Rh Ammonium transporters and SecY translocon. 2014, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_10962

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In this thesis, we studied the function of the Amt/Rh family of proteins and of the SecY/Sec61 translocons using computational methods. The Amt/Rh proteins mediate transport of ammonium across the lipid bilayer. SecY and Sec61 translocons facilitate the insertion of membrane proteins or translocation of secreted proteins in prokaryotes and eukaryotes, respectively. We investigated on the molecular details of ammonium transport in E.Coli AmtB and human RhCG proteins, and the effect of the hydrophobicity of the SecY translocon pore in membrane protein insertion.
Functional studies have revealed that Amt proteins transport the charged form of ammonium (NH4+) while Rh proteins transport neutral ammonia (NH3). However, permeation mechanisms at a molecular level have not been understood clearly. Here, we present molecular details of ammonium transport in AmtB and RhCG proteins. Our calculations show that ammonium ion binds and deprotonates at the hydrophobic pore of AmtB. Then, ammonia diffuses down the hydrophobic pore while the excess proton is transported with the help of a highly conserved histidine dyad (H168 and H318). Ammonia gets re-protonated when it reaches the bottom of the pore and leaves the channel as ammonium. To recruit a new ammonium substrate the protonation states of the histidine dyad has to be reset. This is achieved through water molecules forming a single-file chain in the pore. Thus, hydration of the pore plays an important role in the transport mechanism in AmtB protein. Our simulations of RhCG protein have revealed that the pore of RhCG protein is not hydrated. Lack of hydration in the pore suggests that the excess proton cannot be transported across the hydrophobic pore as it is proposed for AmtB. We show that ammonium binds and deprotonates at a histidine residue (H185) lining the hydrophobic pore of RhCG. After deprotonation, ammonia diffuses down the pore. Then, the excess proton is circulated back to the extracellular site through a network of hydrogen bonds connecting H185 to D177. In conclusion, our calculations suggest that RhCG protein transports neutral ammonia while AmtB transports charged ammonium.
Experimental findings showed that mutation of the pore-ring residues of Sec61 translocon changed the hydrophobicity threshold for membrane integration. Our free energy calculations suggested that mutation of the pore-ring residues influences the stability of peptides in the pore, thus affecting the probability of membrane integration. In addition, insertion experiments of oligo alanine peptides, which contain a cluster of three leucines at various positions, revealed an asymmetry in the membrane integration profile. In particular, a significant drop in membrane integration was observed when the three-leucine cluster aligns with the pore-ring residues. We simulated the wild-type SecY and its pore-ring mutants with the oligo-alanine peptides initially placed into the pores. Analysis of these simulations suggested that hydration of the leucine side-chains drops dramatically when the three-leucine cluster is aligned with the pore-ring residues. The reduced hydration of the leucine residues stabilizes the peptide in the translocon pore and favors its translocation.
Advisors:Bernèche, Simon
Committee Members:Schwede, Torsten
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Computational Biophysics (Bernèche)
UniBasel Contributors:Baday, Sefer and Bernèche, Simon and Schwede, Torsten
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:10962
Thesis status:Complete
Number of Pages:166 S.
Identification Number:
edoc DOI:
Last Modified:22 Apr 2018 04:31
Deposited On:28 Oct 2014 15:12

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