Abstract
Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an invivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design.
Highlights
The central role of integral membrane proteins (IMPs) in many biological functions motivates structural and biophysical studies that require large amounts of purified protein, often at considerable costs in terms of both materials and labor
The employment of TatC was well suited for this study as it is reasonably sized, non-essential, and found broadly throughout bacteria; TatC homologs previously have been observed to exhibit widely varying expression levels in E. coli (Ramasamy et al, 2013), suggesting the importance of sequence-level details in the expression of this IMP
Previous expression trials of TatC homologs identified that AaTatC is readily produced at high levels in E. coli, which enabled the solution of its structure (Ramasamy et al, 2013; Rollauer et al, 2012)
Summary
The central role of integral membrane proteins (IMPs) in many biological functions motivates structural and biophysical studies that require large amounts of purified protein, often at considerable costs in terms of both materials and labor. Related IMP homologs can vary dramatically in the amount of protein available after expression (Lewinson et al, 2008), which raises a fundamental question: what differentiates the expression of IMP homologs? Following the co-translational or posttranslational insertion of nascent protein sequences into the translocon channel, hydrophobic segments pass through the lateral gate of SecY into the membrane to form TMDs. Following the co-translational or posttranslational insertion of nascent protein sequences into the translocon channel, hydrophobic segments pass through the lateral gate of SecY into the membrane to form TMDs Factors such as TMD hydrophobicity (Harley et al, 1998; Hessa et al, 2005) and loop charge (Heijne, 1986; Goder and Spiess, 2003) have been shown to affect the efficiency of TMD integration and topogenesis. TMD hydrophobicity is directly related to the probability with which TMDs partition into the lipid bilayer, while positively charged residues in the loop alter TMD orientation by preferentially occupying the cytosol (Goder and Spiess, 2003; Hessa et al, 2005; Heijne, 1986)
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