Free-energy cost for translocon-assisted insertion of membrane proteins

Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid's contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal∕ðmol · Å 2 Þ, the solvation parameter for transfer from translocon to bilayer is 6-10 cal∕ðmol · Å 2 Þ, pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a thermodynamic partitioning model and insights into the physical properties of the translocon.flexizyme | hydrophobicity | nonproteinogenic amino acid | transmembrane helix I n eukaryotic cells, membrane proteins destined for the plasma membrane and the various compartments along the endo-and exocytic pathways are synthesized by endoplasmic reticulum (ER)-bound ribosomes and cotranslationally integrated into the ER membrane in a process mediated by the Sec61 translocon complex; the homologous SecYEG translocon mediates membrane-protein integration into the inner membrane of prokaryotes (1, 2). Subsequent to membrane integration, membrane proteins fold and oligomerize in the ER and are then moved further along the secretory pathway by vesicular transport.During the membrane-integration step, hydrophobic segments in the translocating nascent polypeptide chain exit the Sec61 translocon through a lateral gate and become embedded in the surrounding lipid bilayer (3-5). Cotranslational, transloconmediated integration of transmembrane α-helices into the ER membrane sets the stage for all subsequent folding and oligomerization events and hence represents a critical step in the maturation of membrane proteins.In previous studies, we have provided quantitative data on the propensities of the 20 natural amino acids to promote the integration of transmembrane helices into the ER membrane and have shown that they depend both on hydrophobicity and on position within the helix (3, 6). Although the partitioning of transmembrane helices between the Sec61 translocon and the lipid membrane bears strong similarities to partitioning of solutes between water and lipid membranes, translocon-to-bilayer partitioning may not be equivalent to water-to-bilayer partitioning (7). Insights into the differences between the two partitioning processes might be revealed i...

Section: Discussionmentioning

confidence: 55%

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

Öjemalm

Higuchi

Jiang

et al. 2011

“…The original implication has been that the translocon effect is relatively small and that the experimental findings are related to the energetics of the specific amino acid in the center of the membrane. This issue is important in view of attempts to point out the possibility that the experiments have not established that the controversial low ΔG app for a positively charged Arg corresponds to Arg in the center of the membrane, since we have an obvious possibility that the Arg side chain is tilted toward the membrane surface and that this explains the low ΔG app (9,10,17). We point out in this respect that one can use a more trivial suggestion, just saying that the center of the helix can move and place the central charge closer to the membrane surface.…”

Section: Introductionmentioning

confidence: 99%

“…In fact, this was implied by the attempts to correlate ΔG app with the water-membrane partitions. However, different works (8)(9)(10) implied that the corresponding equilibrium constant corresponds to the equilibrium between the TR and the membrane (see also Background). Unfortunately, none of these works has offered a clear physical rationale for why one has to consider such equilibrium without considering the transfer to water.…”

mentioning

confidence: 99%

Exploring the nature of the translocon-assisted protein insertion

Rychkova

Warshel

2012

The elucidation of the molecular nature of the translocon-assisted protein insertion is a challenging problem due to the complexity of this process. Furthermore, the limited availability of crucial structural information makes it hard to interpret the hints about the insertion mechanism provided by biochemical studies. At present, it is not practical to explore the insertion process by brute force simulation approaches due to the extremely lengthy process and very complex landscape. Thus, this work uses our previously developed coarse-grained model and explores the energetics of the membrane insertion and translocation paths. The trend in the calculated free-energy profiles is verified by evaluating the correlation between the calculated and observed effect of mutations as well as the effect of inverting the signal peptide that reflects the "positive-inside" rule. Furthermore, the effect of the tentative opening induced by the ribosome is found to reduce the kinetic barrier. Significantly, the trend of the forward and backward energy barriers provides a powerful way to analyze key energetics information. Thus, it is concluded that the insertion process is most likely a nonequilibrium process. Moreover, we provided a general formulation for the analysis of the elusive apparent membrane insertion energy, ΔG app , and conclude that this important parameter is unlikely to correspond to the freeenergy difference between the translocon and membrane. Our formulation seems to resolve the controversy about ΔG app for Arg.coarse-grain modeling | hydrophobicity scale | topology T he establishment of the correct functional topology of membrane proteins is a subject of great current interest (e.g., refs. 1-3). It is known that the protein-conducting channel named translocon (TR) plays a vital role in membrane proteins biogenesis (4). Although biochemical and structural (e.g., refs. 5 and 6) studies have provided crucial information about the insertion process, the understanding of this process is still limited. The difficulties in gaining detailed understanding are also apparent from the emerging problem in fully defining the molecular meaning of the intriguing results about the apparent free energy, ΔG app . That is, the concept of ΔG app , introduced by Hessa et al. (7) for the assessment of inserted versus secreted helical domains (Background), appeared in recent years to be more complex than previously thought. Apparently, the most logical implication of the original descriptions of ΔG app has been that it represents an equilibrium result of the partition between membrane and water. In fact, this was implied by the attempts to correlate ΔG app with the water-membrane partitions. However, different works (8-10) implied that the corresponding equilibrium constant corresponds to the equilibrium between the TR and the membrane (see also Background). Unfortunately, none of these works has offered a clear physical rationale for why one has to consider such equilibrium without considering the transfer to water. We note that eve...

“…SecG is not required for function, but SecE is indispensable. Experimental (9)(10)(11) and computational studies (12)(13)(14)(15) emphasize the importance of interactions of the gate helices with nascent-chain segments during TM helix insertion.…”

mentioning

confidence: 99%

Anomalous behavior of water inside the SecY translocon

Capponi

Heyden

Bondar

et al. 2015

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...