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Regulation of multispanning membrane protein topology via post-translational annealing.

Van Lehn RC, Zhang B, Miller TF - Elife (2015)

Bottom Line: This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies.The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins.The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States.

ABSTRACT
The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

No MeSH data available.


Comparison of the average position of the slowest-flipping loop in the EOT ensemble to the average position of that same loop in the ensemble of fully integrated configurations.For the K3, L85R, nEmrE, and nT28R1 mutants, which have two slow-flipping loops, the axes of this plot are defined so as to account for the positions of both loops, as described in the Alternative definition of the slowest-flipping loop position for mutants with two slow-flipping loops section of the ‘Materials and methods’. The black dashed line indicates perfect correlation.DOI:http://dx.doi.org/10.7554/eLife.08697.018
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fig7s1: Comparison of the average position of the slowest-flipping loop in the EOT ensemble to the average position of that same loop in the ensemble of fully integrated configurations.For the K3, L85R, nEmrE, and nT28R1 mutants, which have two slow-flipping loops, the axes of this plot are defined so as to account for the positions of both loops, as described in the Alternative definition of the slowest-flipping loop position for mutants with two slow-flipping loops section of the ‘Materials and methods’. The black dashed line indicates perfect correlation.DOI:http://dx.doi.org/10.7554/eLife.08697.018

Mentions: In Figure 7A, the K3 and L85R mutants deviate most significantly from the plotted correlation between the EOT ensemble and the final topology; as seen in Figure 6, these two mutants exhibit a pair of slow loop-flipping frequencies rather than a single, well-separated slowest loop-flipping frequency. For a more detailed analysis of these special cases that involve a pair of slow loop-flipping frequencies, we direct the reader to the Alternative definition of the slowest-flipping loop position for mutants with two slow-flipping loops section of the ‘Materials and methods’ and the corresponding results in Figure 7—figure supplement 1. However, we emphasize that the close agreement between the results in Figure 7A and Figure 7—figure supplement 1 indicate that our conclusions regarding the strong correlation between the EOT ensemble and the final topology are robust with respect to the details of the definition of the slowest-flipping loop.


Regulation of multispanning membrane protein topology via post-translational annealing.

Van Lehn RC, Zhang B, Miller TF - Elife (2015)

Comparison of the average position of the slowest-flipping loop in the EOT ensemble to the average position of that same loop in the ensemble of fully integrated configurations.For the K3, L85R, nEmrE, and nT28R1 mutants, which have two slow-flipping loops, the axes of this plot are defined so as to account for the positions of both loops, as described in the Alternative definition of the slowest-flipping loop position for mutants with two slow-flipping loops section of the ‘Materials and methods’. The black dashed line indicates perfect correlation.DOI:http://dx.doi.org/10.7554/eLife.08697.018
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4635508&req=5

fig7s1: Comparison of the average position of the slowest-flipping loop in the EOT ensemble to the average position of that same loop in the ensemble of fully integrated configurations.For the K3, L85R, nEmrE, and nT28R1 mutants, which have two slow-flipping loops, the axes of this plot are defined so as to account for the positions of both loops, as described in the Alternative definition of the slowest-flipping loop position for mutants with two slow-flipping loops section of the ‘Materials and methods’. The black dashed line indicates perfect correlation.DOI:http://dx.doi.org/10.7554/eLife.08697.018
Mentions: In Figure 7A, the K3 and L85R mutants deviate most significantly from the plotted correlation between the EOT ensemble and the final topology; as seen in Figure 6, these two mutants exhibit a pair of slow loop-flipping frequencies rather than a single, well-separated slowest loop-flipping frequency. For a more detailed analysis of these special cases that involve a pair of slow loop-flipping frequencies, we direct the reader to the Alternative definition of the slowest-flipping loop position for mutants with two slow-flipping loops section of the ‘Materials and methods’ and the corresponding results in Figure 7—figure supplement 1. However, we emphasize that the close agreement between the results in Figure 7A and Figure 7—figure supplement 1 indicate that our conclusions regarding the strong correlation between the EOT ensemble and the final topology are robust with respect to the details of the definition of the slowest-flipping loop.

Bottom Line: This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies.The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins.The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States.

ABSTRACT
The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

No MeSH data available.