2015 Mar 13
Mechanisms of Integral Membrane Protein Insertion and Folding
Item in Clipboard
Mechanisms of Integral Membrane Protein Insertion and Folding
J Mol Biol
The biogenesis, folding, and structure of α-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, as well as the transport of solutes and signals across membranes. Nearly all MPs require translocons--often referred to as protein-conducting channels--for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here, we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of MP insertion and the folding of polytopic MPs in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure but also is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion.
lipid–protein interactions; membrane protein biogenesis; membrane protein folding; transmembrane helix.
Copyright © 2014 Elsevier Ltd. All rights reserved.
This schematic cartoon represents in broad terms current thinking about the insertion of multi-span proteins into membranes. Two ideas are captured in the cartoon. First, TM segments (red) emerge from the ribosome and pass into the translocon (blue). Second, the nascent segments partition into the membrane from the translocon. As a starting point for discussion, we present alternative views of the membrane protein insertion pathway in Fig. 15.
Alpha-helical MPs exist in their native state in highly thermally disordered lipid bilayers, as illustrated here for the SecYEG translocon  from
Methanococcus jannaschii. The image is from a molecular dynamics simulation of the translocon (PDB code 1RHZ) executed in a phospholipid bilayer. In this view, parallel to the membrane plane, the so-called gate helices 2b and 7 (red cylinders) were exposed by cutting away the lipid bilayer. Water molecules within the translocon are shown as van der Waals spheres in red (oxygen) and white (hydrogen). Waters surrounding the bilayer are shown as H-O-H bonds in blue-gray. Lipid acyl chains are white and the phospholipid headgroups are red. Image provided courtesy of J. Alfredo Frietes and Stephen H. White.
Summary of the various interactions that stabilize MPs stably folded in fluid lipid bilayers (blue lines are interface boundaries, red lines represent boundaries of the lipid hydrocarbon core). “Global bilayer effects” accounts for changes in the structure and stability of the lipid bilayer when perturbed by the protein , emphasizing that the bilayer itself sits in a free energy minimum determined by the tendency of the system to minimize exposure of the acyl chains (grey) to water (blue) on the one hand, and the tendency to maximize the distance between headgroups on the other . Both the bilayer and protein must adjust structurally to minimize the free energy of the protein plus bilayer system. The protein shown (red helices) is bacteriorhodopsin determined to a resolution of 1.55 Å, PDB Code 1C3W . Image modified from .
A four-step thermodynamic cycle for describing the energetics of the partitioning, folding, insertion, and association of an α-helix (red helices) in a lipid bilayer (grey). The process can follow an interfacial path, a water path, or a combination of the two. Studies of folding along the interfacial path are experimentally more tractable . The Δ
G symbols indicate standard transfer free energies. The subscript terminology indicates a specific step in the cycle. The subscript letters are defined as follows: w = water, i = interface, h = hydrocarbon core, u = unfolded, f = folded, and a = association. With these definitions, for example, the standard free energy of transfer from water to interface of an unfolded peptide would by Δ G. However, some parts of the cycle (dashed box) are generally inaccessible experimentally. Image modified from . wiu
The energetics of inserting an α-helix into lipid bilayers (grey) is dominated by the peptide bonds, as illustrated here for the glycophorin A (GpA) TM helix. Even with the helical backbone internally H-bonded, it is costly to dehydrate the H-bonded peptide bonds (Δ
G) upon insertion into the bilayer. For the helix to be stable, the favorable free energy of transfer of the sidechains (Δ bb(f) G), determined by the hydrophobic effect, must compensate for the unfavorable Δ sc G. In the case GpA, the net stability of the helix Δ bb(f) G is −12 kcal mol TM −1. The energetic cost of unfolding the polyglycine helix within the bilayer is immense: Δ G is greater than 100 kcal mol bb(u) −1! Modified from .
Equilibrium microsecond-scale simulations of the folding and membrane insertion of polyleucine sequences (here 10 leucines) reveal only two states . The simulation shown here begins with the unfolded peptide (U) in water (W) about 10 Å from the phosphatidylcholine bilayer (grey). Within a few nanoseconds (ns) it absorbs to the membrane interface and never returns to the bulk water phase. After the next 40 ns, the peptide becomes α-helical and fluctuates between being on the surface (S) and across the membrane (TM) during the ensuing several microseconds. The trajectory of the simulation is represented as a plot of the insertion depth of the peptide's center-of-mass against its helicity. The sampled time points are connected sequentially by blue lines. Modified from . This simulation reveals the importance of the membrane interface in membrane protein folding, and suggests that the interface may play a role in translocon-guided insertion of TM helices.
Helix-helix interactions of the glycophorin A (GpA) dimer (blue and orange helices) based upon the structure of GpA in SDS . A. Glycine (or other small residues) separated by three residues allow the helices to pack tightly. B. Other amino acids in the structural vicinity of the GXXXG motif can facilitate or inhibit specific binding of complementary surfaces .
High-resolution structure of a mammalian ribosome-Sec61 complex. The structure was obtained using advanced cryo-EM methods. PTC indicates the peptidyl transferase center. Image from Voorhees et al. .
Structure of the SecYE translocon from
Pyrococcus furiosus . The left panel shows a view in the plane of the membrane, the right panel shows a view from the cytoplasm. The plug domain is circled, and the residues in the hydrophobic ring are shown as van der Waals spheres, indicated by *. The arrow points into the lateral gate between TMH2b and TMH7. SecE is shown in red, SecY is color coded from N-terminus (blue) to C-terminus (orange).
Two models for translocon-mediated insertion of a N
out-C in orientated TMH in a single-span (type I) membrane protein. (a) The “In-out” model”. The TMH (in black) first moves all the way into the central translocon channel, and then exits sideways through the lateral gate. ( b) The “sliding” model. The TMH slides along the lateral gate into the membrane, with one side exposed to lipid at all times. The leading polar segment penetrates through the lateral gate and is shielded from lipid contact. SecE is shown in red, SecY is color coded from N-terminus (blue) to C-terminus (orange). On the right-hand side a schematic of the translocon (blue) interacting with a membrane protein (green) and a membrane inserting TMH (red) are shown.
Structure of the YidC translocon from
Bacillus halodurans  viewed in the plane of the membrane, looking into the hydrated cleft in the center of the protein. The left panel shows a surface representation; the right panel shows the walls of the cavity, with the rest of the protein in stick representation.
Amino acid Δ
G values for Sec61-, SecYEG-, and YidC-dependent membrane insertion of a model TMH [84, 85, 102]. The SecYEG (panel a) and YidC (panel b) data are plotted against the Sec61 data. Full lines indicate linear fits to the data (with the equations given in the panels), while the broken line in panel b is the linear fit obtained when including only non-polar and weakly polar residues (slope = 0.8) . app
Using APs as
in vivo force sensors . ( a) An AP (AP; in blue) is inserted into a membrane protein with two natural TMHs (TM1, TM2; in black) and a model TMH composed of six leucines and 13 alanines (H; in red). Depending on the length L of the tether between the H-segment and the AP, the H-segment will be in different locations relative to the translocon at the time when the ribosome reaches the last codon in the AP, as shown in the cartoon. The pulling force F( L) on the nascent chain will determine the fraction of stalled vs. full-length protein produced. The fraction full-length protein can be determined by [ 35S]-Met pulse-labeling of growing E. coli, followed by immunoprecipitation of the protein construct and analysis by SDS-PAGE, as shown on the right for a construct with L=63 residues (a control construct in which a critical proline in the AP has been mutated to alanine, preventing stalling, is also shown). ( b) Fraction full-length protein as a function of L for a set of constructs designed as shown in panel a (top).
Folding spaces during membrane protein structure formation. During nascent chain synthesis by the ribosome (brown), helical segments (shown in blue and orange) can associate already in the ribosomal vestibule (1)  or within the translocon channel (2a, green) , where polar residues can be shielded within the helix-helix interface (2b, red spheres) and hydrophobic side-chains (orange spheres) form a membrane insertion competent surface. Helices can furthermore interact during insertion into a membrane (3, grey) whereas one segment is already inserted into the membrane and drives the insertion of a less hydrophobic segment by shielding hydrophilic residues within the interaction interface . Interactions of helices within different proteins can also occur co-translationally (5) and was shown do drive membrane protein complex assembly .
An alternative view of translocon-aided insertion of multi-span membrane proteins and the secretion of soluble proteins. The idea of this alternative view is shown in a cartoon fashion in panel
a (also see Fig. 10). We suggest that initial contact of the nascent chain (green, with red TMHs) is with the membrane interface in the vicinity of the translocon (blue), and that the chain does not immediately thread into the translocon. Rather, the translocon has YidC-like behavior; it provides a pathway for polar components of membrane proteins to cross the membrane. This is not to say that it cannot form a passageway through the membrane, but we suggest that it does this only for polar polypeptide segments in membrane proteins and for secreted proteins, as shown in panel b.
All figures (15)
Spontaneous transmembrane helix insertion thermodynamically mimics translocon-guided insertion.
Nat Commun. 2014 Sep 10;5:4863. doi: 10.1038/ncomms5863.
Nat Commun. 2014.
25204588 Free PMC article.
How translocons select transmembrane helices.
Annu Rev Biophys. 2008;37:23-42. doi: 10.1146/annurev.biophys.37.032807.125904.
Annu Rev Biophys. 2008.
Cotranslational folding of membrane proteins probed by arrest-peptide-mediated force measurements.
Proc Natl Acad Sci U S A. 2013 Sep 3;110(36):14640-5. doi: 10.1073/pnas.1306787110. Epub 2013 Aug 19.
Proc Natl Acad Sci U S A. 2013.
23959879 Free PMC article.
The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding.
J Biol Chem. 2015 Nov 27;290(48):28944-52. doi: 10.1074/jbc.M115.672261. Epub 2015 Aug 7.
J Biol Chem. 2015.
26254469 Free PMC article.
Marginally hydrophobic transmembrane α-helices shaping membrane protein folding.
Protein Sci. 2015 Jul;24(7):1057-74. doi: 10.1002/pro.2698. Epub 2015 May 30.
Protein Sci. 2015.
25970811 Free PMC article.
Sec translocon has an insertase-like function in addition to polypeptide conduction through the channel.
F1000Res. 2019 Dec 20;8:F1000 Faculty Rev-2126. doi: 10.12688/f1000research.21065.1. eCollection 2019.
32025287 Free PMC article.
Membrane-Protein Unfolding Intermediates Detected with Enhanced Precision Using a Zigzag Force Ramp.
Biophys J. 2020 Feb 4;118(3):667-675. doi: 10.1016/j.bpj.2019.12.003. Epub 2019 Dec 13.
Biophys J. 2020.
Membrane receptor activation mechanisms and transmembrane peptide tools to elucidate them.
J Biol Chem. 2020 Feb 14;295(7):1792-1814. doi: 10.1074/jbc.REV119.009457. Epub 2019 Dec 25.
J Biol Chem. 2020.
31879273 Free PMC article.
A pair of non-optimal codons are necessary for the correct biosynthesis of the
Aspergillus nidulans urea transporter, UreA.
R Soc Open Sci. 2019 Nov 13;6(11):190773. doi: 10.1098/rsos.190773. eCollection 2019 Nov.
R Soc Open Sci. 2019.
31827830 Free PMC article.
Designing minimalist membrane proteins.
Biochem Soc Trans. 2019 Oct 31;47(5):1233-1245. doi: 10.1042/BST20190170.
Biochem Soc Trans. 2019.
31671181 Free PMC article.
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Cell Membrane / metabolism
Membrane Proteins / metabolism
Protein Structure, Tertiary / physiology
LinkOut - more resources
Full Text Sources Other Literature Sources Miscellaneous