Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

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Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (ΔG app ) of "snorkeling" of charged amino acids toward the lipid-water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone hydrogen bonds (H-bonds) on ΔG app . These results help establish a quantitative basis for our understanding of membrane protein assembly in eukaryotic cells. Using a cotranslational insertion assay, we previously measured the contribution of each of the 20 natural amino acids to the membrane integration efficiency of model transmembrane segments, and derived a "biological" hydrophobicity scale that assigns an apparent free-energy of membrane integration, ΔG aaðiÞ, j app , to each amino acid of type i [aa(i) = A, C, D, E, . . ., W, Y] as a function of its position j within a transmembrane α-helix (2). To further probe the physicochemical basis for membrane insertion, we also analyzed a series of aliphatic and aromatic nonproteinogenic amino acids, using the same insertion assay (3). This approach made it possible to quantitate the "hydrophobic effect" during membrane protein insertion, giving nonpolar solvation energy parameter values of -10 cal/(mol·Å 2 ) and -7 cal/(mol·Å 2 ) for aliphatic and aromatic surface area, respectively.Here, we extend our analysis of nonproteinogenic amino acids to analogs of the positively charged amino acids Arg and Lys. We provide estimates of the influence on the apparent free energy of membrane integration (ΔG app ) of "snorkeling" of charged amino acids toward the lipid-water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone H-bonds on ΔG app .

Section: Resultsmentioning

confidence: 38%

Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids

Öjemalm

Higuchi

Lara

et al. 2016

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“…Perhaps more likely, the Bam complex might lower the free energy of membrane insertion, but the efficiency of insertion is ultimately dictated by the biophysical properties of each substrate. Indeed a recent study showed that although the Sec machinery greatly enhances the partitioning of α-helical transmembrane segments into a lipid bilayer, insertion efficiency is influenced by the nonpolar surface area of individual amino acid side chains (37). Moreover, although the generality of our results remains to be determined, it is noteworthy that the mutation of a conserved glycine residue in the β domain of a trimeric autotransporter also appears to affect its membrane insertion (38).…”

Section: Discussionmentioning

confidence: 63%

Mechanistic link between β barrel assembly and the initiation of autotransporter secretion

Pavlova

Peterson

Ieva

et al. 2013

126

Autotransporters are bacterial virulence factors that contain an N-terminal extracellular ("passenger") domain and a C-terminal β barrel ("β") domain that anchors the protein to the outer membrane. The β domain is required for passenger domain secretion, but its exact role in autotransporter biogenesis is unclear. Here we describe insights into the function of the β domain that emerged from an analysis of mutations in the Escherichia coli O157:H7 autotransporter EspP. We found that the G1066A and G1081D mutations slightly distort the structure of the β domain and delay the initiation of passenger domain translocation. Site-specific photocrosslinking experiments revealed that the mutations slow the insertion of the β domain into the outer membrane, but do not delay the binding of the β domain to the factor that mediates the insertion reaction (the Bam complex). Our results demonstrate that the β domain does not simply target the passenger domain to the outer membrane, but promotes translocation when it reaches a specific stage of assembly. Furthermore, our results provide evidence that the Bam complex catalyzes the membrane integration of β barrel proteins in a multistep process that can be perturbed by minor structural defects in client proteins.

“…Öjemalm et al (22) found that the apparent translocon-to-membrane free energy of insertion of nonproteinogenic amino acids at a particular position in the TM segment varied linearly with σ, as expected for hydrophobic-driven partitioning. However, σ was found to be less favorable than that for bulk water/hydrocarbon partitioning and to depend on position within the model TM segment (Fig.…”

mentioning

confidence: 99%

Anomalous behavior of water inside the SecY translocon

Capponi

Heyden

Bondar

et al. 2015

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The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359-E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a "primed" open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182-17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple waterfilled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications. membrane protein folding | molecular dynamics | protein-conducting channel | protein hydration | confined water T he heterotrimeric SecY translocon complex (SecYEG in bacteria, SecYEβ in archaea, Sec61αβγ in eukaryotes) is required for the cotranslational assembly of membrane proteins and the secretion of soluble proteins (1-3). The SecY subunit (Fig. 1A) has 10 transmembrane helices comprised of two fivehelix domains related by pseudo-twofold symmetry around an axis parallel to the membrane (4-8). These helices form an hourglass-shaped water-filled pore that spans the membrane. The so-called hydrophobic pore ring (HR) comprised of six hydrophobic residues forms the narrowest part of the hourglass, located near the bilayer center. Sitting just above the ring on the extracellular side is a small, distorted helix [transmembrane 2a (TM2a)], called the plug domain that is believed to impede the passage of water and solutes across the membrane. Access to the membrane from the water-filled hourglass-shaped interior of SecY is provided by the so-called lateral gate, formed by helices TM2b and TM7 (Fig. 1A). SecG is not required for function, but SecE is indispensable. Experimental (9-11) and computational studies (12-15) emphasize the importance of interactions of the gate helices with nascent-chain se...