Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane

Abstract

In silico algorithms have been the common approach for transmembrane (TM) protein topology prediction. However, computational tools may produce questionable results and experimental validation has proven difficult. Although biochemical strategies are available to determine the C-terminal orientation of TM proteins, experimental strategies to determine the N-terminal orientation are still limited but needed because the N-terminal end is essential for membrane targeting. Here, we describe a new and easy method to effectively determine the N-terminal orientation of the target TM proteins in Escherichia coli plasma membrane environment. D94N, the mutant of bacteriorhodopsin from Haloarcula marismortui, can be a fusion partner to increase the production of the target TM proteins if their N-termini are in cytoplasm (Nin orientation). To create a suitable linker for orientating the target TM proteins with the periplasmic N-termini (Nout orientation) correctly, we designed a three-TM-helix linker fused at the C-terminus of D94N fusion partner (termed D94N-3TM) and found that D94N-3TM can specifically improve the production of the Nout target TM proteins. In conclusion, D94N and D94N-3TM fusion partners can be applied to determine the N-terminal end of the target TM proteins oriented either Nin or Nout by evaluating the net expression of the fusion proteins.

The de novo design of a biocompatible and functional integral membrane protein using minimal sequence complexity

“…Although it is well-attested that fusion proteins can support the expression of synthetic transmembrane segments, it is less clear that this extends to standalone de novo sequences and examples are relatively scarce. The available evidence is that redesigned and de novo proteins comprised of a limited selection of hydrophobic amino acids (mainly L, I and A) can generally be expressed by the cell 11 , 22 , 23 , 40 and localized to the plasma membrane 11 , 22 , 23 . Goparaju and colleagues 40 used a fusion protein strategy to deliberately express a minimal de novo protein into inclusion bodies, so the intrinsic localization of that sequence is unknown.…”

“…A logical extension of this approach is to develop new minimal systems that are geared towards a deeper understanding of oligomeric and multipass transmembrane proteins. It has already been shown that bottom-up designs with very low sequence complexity can be tolerated by the cell 11 , 22 , 23 , but it is not yet clear whether these simplistic proteins can bridge the gap between being both resident in cellular membranes and compatible with the in vitro biophysical methods necessary for protein characterization. A reasonable design target for such a protein would be a tetrahelical bundle, which is a common motif in natural membrane proteins and can support various functions 2 – 4 .…”

Unraveling Gardnerella vaginalis Surface Proteins Using Cell Shaving Proteomics

“…Both BAQ32849 and BAQ33018 were localized in the cytoplasm with PSORT and in the cell membrane with Gpos-mPLoc (more details in Supplementary Table S2 ). In the literature, it is common to find discrepancies between the in silico -predicted topology and the experimental data ( Rodriguez-Ortega et al, 2006 ; Lee et al, 2015 ). We only found two discrepancies when comparing the characteristic motifs of surface-exposed proteins predicted using bioinformatics tools with the mapping of the MS identified peptides on the primary sequence of the proteins (Supplementary Table S1 and Supplementary Data).…”