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Marginally hydrophobic transmembrane helices shaping membrane protein folding Keywords 1. Introduction • Membrane protein A typical organism devotes about a quarter of its genes to produce integral membrane proteins (1,2) . Their importance is further implied by the fact that more than half of the targets in the pharmaceutical industry are membrane proteins (3,4) . I will begin this review by presenting the hydrophobic effect - fundamental to biochemistry and research on membrane protein insertion - before describing biological membranes and their dynamics. The protein machinery essential to membrane protein biogenesis has been previously reviewed and is only briefly mentioned in order to allow me to focus on alpha-helical integral membrane proteins. Most of this review is devoted to marginally hydrophobic transmembrane helices (mTMHs). These transmembrane segments are deficit in the defining character of transmembrane helices (TMHs) - hydrophobicity. Nevertheless, they are fairly common in membrane proteins. • Translocon recognition • Protein folding • Hydrophobicity • Molecular dynamics 2. The hydrophobic effect Water molecules are electric dipoles, where the electronegative oxygen has a partial negative charge and the small hydrogen atoms have a partial positive charge. Water molecules therefore have dipole interactions with each other as well as to other molecules. The dipole interaction between a hydrogen atom bonded to a electronegative nucleus (nitrogen, oxygen,fluoride and chloride in particular) and a second such nucleus is termed a hydrogen bond. The hydrogen bond is particularly strong, mainly due to the large dipole moment caused by the electronegative nuclei’s polarization of the XH bond (where X is an electronegative atom and H a hydrogen), but also due to a partial covalent character (5) . Liquid water forms a tightly hydrogen bonded network between the molecules (typically 3 hydrogenbonds/molecule (6) ), and the relative strength of these interactions is demonstrated clearly by the textbook comparison (e.g. Pauling (5) ) to hydrogen sulfide, which Preprint submitted to Elsevier February 5, 2015 is gaseous at room temperature. Hydrogen bonds are the strongest intermolecular interaction between overall neutral molecules. When liquid water forms an interface at the boundary to some other phase to which it does not bond (e.g. a vacuum or a negliglbly-interacting gas), the water molecules at this boundary can not form as many hydrogen bonds since they are not surrounded by other molecules on all sides. The orientations of the molecules at the interface are also more restricted, resulting in a decrease in entropy (7) . Molecules at the surface therefore have a higher energy than those in the bulk, resulting an effective force ”pulling” the surface molecules towards the bulk - the well-known phenomenon of surface tension. Another well-known and related fact is the observation that ”oil and water don’t mix”, known as the hydrophobic effect in chemical terminology. If a nonpolar molecule is placed in bulk water, it disrupts the hydrogen-bond network of the water, in effect creating a ’bubble’ around the non-polar molecule. The energetic cost of this may be accounted in two parts: First, the energy required by surface tension to create an interface and form a void in the water. Second, the energy gained from the weak van der Waals (vdW) interactions (Debye, London forces) that exist between the non-polar molecule placed into the void and the surrounding water. Since vdW interactions are much weaker than hydrogen bonds, the former term is guaranteed to dominate in the case of a purely non-polar molecule, and it will cost energy to place the non-polar molecule into the bulk water. The energetically favored situation is for the non-polar molecules to ”lump together” into droplets or bubbles, and ultimately a separate phase, in order to minimize the interface area with the water. The nonpolar substance is immiscible in water. The term “hydrophobic” can be misleading. While bulky hydrophobic molecules may form strong intermolecular bonds (e.g. oils have a higher boiling point than water), they do not bond to each other more strongly than they do to water in terms of the interfacial area (7) . Non-polar molecules interact with water through the Debye (induced dipole moment) and London (dispersion) interactions, while non-polar molecules bind to each other through the weaker (but longer-range) London force alone. Therefore, the driving force behind the hydrophobic effect is not that nonpolar molecules bind to each other more strongly than to water, but rather that water binds to itself more strongly than it does to non-polar molecules. Although a completely non-polar molecule is immiscible, hydrophobicity is not an either or phenomenon. The more polar and hydrogen-bonding a molecule is in relation to its size, the more the energetic cost of creating a void in the water is offset. Hence methanol is entirely miscible in water (8) , 1-pentanol is weakly soluble, while 1-decanol is normally considered insoluble. Figure 1: Structure of a phospholipid. A.The structure of phospholipids is illustrate with phosphatidylcholine. The head group consists of the glycerol backbone (in green), linked thruogh a phosphate (in cyan) to a polar or charged head group (in magneta). Here this head group is choline, but it can be replaced by for instance ethanolamine in phosphatidylethanolamine. The two remaining carbons of the glycerol are attached to two fatty acids through ester linkage. The fatty acids can be saturated or unsaturated (in yellow). The saturation status as well as the properties of the head group determine the physiochemcial properties of a phospolipid. B. Schmatic representation of lipids with different overall shapes and on how the fatty acid structure influences membrane curvature. 2.1. The hydrophobic effect and membrane lipids Biological membranes are composed of amphiphatic lipids, consisting of a hydrophilic head group and a hydrophobic acyl chain (see Figure 1 A.). Despite the head group, they are still largely immiscible in water, and therefore aggregate spontaneously. They will also arrange themselves with their head groups at the water interface, where those groups may form hydrogen and polar bonds to the surrounding bulk water. This lowers the interfacial energy sufficiently that a stable emulsion can be formed, rather than lipids aggregating into an ”oily” phase. Surface tension will lead to the formation 2 of a spherical miscelle, unless the acyl chains are sufficiently bulky for their steric repulsion to counter it. In the latter case a stable lipid bilayer membrane is formed, with oppositely-oriented layers of the amphipilic lipds. 3.1. Membrane lipids The bulk of a biological membrane consists of lipids. The lipid composition determines the physical properties of the membrane such as the surface charge, thickness, fluidity and curvature. All these characteristics must be maintained within an appropriate range and can be adjusted depending on the changes in environmental conditions (16,17) . To achieve this, different types of lipids are needed, albeit membrane lipids share the same general structure with one polar and one non-polar region. Phospholipids are the major component of all cell membranes. Glycerophospholipids consist of a glycerol backbone with two hydrophobic acyl chains attached via ester linkage to the first and second carbons. The third glycerol carbon is attached to a polar or charged head group through a phosphodiester linkage (16) . The fatty acid chains vary; in general carbon one is usually linked to a C16 or C18 saturated fatty acid whereas the second carbon usually bears a C18 or C20 unsaturated fatty acid , see Figure 1 A. 2.2. The hydrophobic effect and proteins Amino acids are polar compounds with good water solubility. On forming peptide bonds, the charged groups become the amide and keto groups of the peptide backbone, which are hydrophilic but substantially less so than the original charged groups. The aminoacid side chains may be polar, charged or hydrophobic. In addition, the folding and geometry of the protein can make the various groups inaccessible from the surrounding medium. Therefore, proteins can exhibit a wide range of hydrophobicity, both as a whole and in localized regions of the protein. Globular proteins, which exist in the cytosol, are hydrophilic in the sense that they are water-soluble, which in turn implies that their surfaces bind strongly to water relative the protein’s interfacial area. The hydrophobic effect will manifest itself as a force working to bring hydrophobic portions of the protein together, away from the bulk solution and more hydrophilic regions. Strongly hydrophobic regions are concentrated towards the interior of the protein. This, and other forms of inter- and intramolecular bonding within the protein results in a specific three-dimensional structure of the protein, determined mainly by its primary amino-acid sequence (9) . For membrane proteins, the hydrophobic effect plays a more complicated role and is discussed in more detail in later chapters. 3.1.1. Physical properties of lipids shape the characteristics of a biological membrane The lipid composition of membranes varies between different organisms, cell types and in time. In addition to the plasma membrane, eukaryotic cells contains a number of internal membranes, each with a specialized set of lipids and proteins (17) . Organisms can devote up to 5% of their genome towards lipid metabolism (17) and the number of different lipids range from several hundreds in bacteria to up to thousands in eukaryotes (18) . While the combination of different lipid head groups shape the characteristics of the membrane surface, the physical properties of the membrane are largely dependent on the head groups and various fatty acid side chains (19) . Cylindrical lipids are prone to forming bilayers and are abundant in biological membranes (1 B). However, non-bilayer lipids are also present in the membrane; Cone-shaped lipids have head groups with a cross-sectional area smaller than their acyl chains( 1 C). They promote a negative curvature in membranes whereas lipids with a inverted-cone shape tend to form micelles and promote positive curvature in membranes (20) (Figure 1 D). Ultimately, the ratio between bilayerand non-bilayer forming lipids determines the intrinsic curvature of the membrane as well as shape and integrity (14,16) . Additionally, it influences the membrane flexibility, which is important for fusion/fission events as well as for membrane protein function (10,21,22) . 3. Biological membranes Biological membranes are mainly composed of lipids and proteins. Together they form an essential barrier between living cells and their external milieu. In addition, they allow compartmentalization of intracellular organelles within eukaryotes. While membrane lipids have been seen as merely solvents for membrane proteins, the picture today is more complex; Biological membranes are dynamic structures, that vary in their lipid, protein and carbohydrate composition coevolving and functioning together (10) . Indeed, the role of lipids in membrane protein structure (11,12) , topology (13) , function (14) and in signaling (15) have become evident. 3 Membrane fluidity depends on lipid types and acyl chain composition. Saturated fatty acids have a linear acyl chain allowing it to pack tightly, whereas unsaturated fatty acids contain kinks resulting in increased fluidity. In addition, incorporation of rigid steroidic lipids, such as cholesterol, confer stability (16) . Membrane fluidity is also dependent on temperature and some organisms can adjust their lipid composition in response to changes of temperature (23) . As an example, some bacteria can maintain membrane fluidity at both higher and lower growth temperatures by increasing the number of saturated and unsaturated fatty acids, respectively (24,25) . are embedded in (see Figure ?? in Introduction). The model assumes that membrane proteins are interacting with lipids mainly due to hydrophobic forces (31) . However, more recent results have added new layers of complexity to this model. Specialized membrane domains and extramembraneous structures can limit motion and lateral diffusion of membrane components and proteinlipid interactions can be essential for protein structure and function (29,32) . Biological membranes are rich in protein - reported lipid:protein ratios vary depending on cell type but range between 18 - 80% protein by mass (reviewed in (33) . Membrane proteins can be crudely divided into two groups, the peripheral and integral membrane proteins. Peripheral membrane proteins attached to lipids or proteins in the membrane through non-covalent interactions, whereas integral membrane proteins have segments that cross the membrane. They can be further grouped into β-barrel membrane proteins with amphiphatic β-strands and (II) α-helical membrane proteins with an α-helices crossing the membrane. The vast majority of integral membrane proteins are α-helical. It has been suggested that the amino-acid sequence of transmembrane domains would reflect on the properties of the bilayers in which they reside. Indeed, the amino acid composition do seem to vary based on the membrane where a particular helix is found embedded (34) . 3.1.2. The lipid environment in biological membrane is heterogenic The structure for a 1,2-Dioleoyl-sn-glycero-3phosphocholine (DOPC) lipid bilayer has been solved using X-ray and neutron scattering data. The 30 Å thick hydrophobic core is lined with a 15 Å thick interface region on each side. Transition from the core to the interfaces is not sharp and should be viewed as a zone with gradual change in hydrophobicity (26) . The interfacial regions can also vary between the two leaflets. While the lipids are uniformly distributed on both faces of the bilayer in the endoplasmic reticulum membrane and the Golgi apparatus, the two leaflets of plasma membranes are asymmetric (17) . A common feature in many animal cells is that the extracytoplasmic leaflet contains most of the phosphatidylcholine, sphingomyelin, and glycosphingolipids, while phosphatidylserine and phosphatidylethanolamine are enriched in the cytoplasmic leaflet (27) . How the asymmetry is established and maintained is not well understood. In addition to the differences between leaflets, membranes can also exhibit lateral asymmetry. Membrane domains can be defined as short-range ordered structures (0.001 and 1.0 µm in diameter), enriched in particular lipids and proteins. This lateral heterogeneity has been related to characteristic functional properties (28) and both protein- and lipid-based mechanisms for membrane domain formations have been described (reviewed in (29) . For some time, lateral heterogeneity was assumed to be an eukaryotic feature but lipid domains were later also found in bacteria (30) . 3.2.1. Lipid-protein interactions So far the characteristics of a lipid bilayer have been discussed in relation to the physical and chemical properties of lipids. However, an interesting consideration is also how membrane proteins and lipids interact with each other. Several features of the membrane have been described to influence the function of membrane proteins: the thickness and phase of the lipid bilayer and the presence of a specific phospholipid (35) . 3.2.2. Hydrophobic mismatch An ideal transmembrane domain would span the hydrophobic core of the membrane, which would require an α-helix with 20 amino acids. However, both longer and shorter transmembrane segments exist and do not match the thickness of the lipid bilayer, a phenomenon termed - hydrophobic mismatch. If the transmembrane helix is too long, hydrophobic sequences are exposed to the aqueous environment. And if it is too short, hydrophilic amino acids have to reside within the membrane. Both situations are energetically unfavorable. To minimize the free energy of the system, long transmembrane helices may either tilt or distort through coils 3.2. The fluid mosaic model and beyond The fluid mosaic model describes how proteins and lipids are organized in biological membranes. It depicts the membrane as a two-dimensional viscous lipid matrix, within which freely diffusing membrane proteins 4 to compensate for the mismatch, thus preventing the exposure of hydrophobic side chains to the aqueous environment (36) . Another alternative is that the lipid tails around the protein extend by acyl chain ordering (37) . When the transmembrane domain is shorter than the hydrophobic core of the bilayer, the tails of the surrounding lipids may compensate by chain disordering, resulting in compression (38–40) . However, it remains unclear whether lipids in living cells can change the thickness of the membrane (41) . Nevertheless, mismatch has been implicated in the functionality of several proteins (32,42) and may also be reflected in membrane protein sorting in eukaryotes (34,43) . binding site for Phosphatidylinositol 4,5-bisphosphate (PIP2) has been identified in K+ channels and its functional importance was demonstrated in the Kv7.1 channel, where the coupling of the Kv7.1 channel voltagesensing domain and the pore domain requires PIP2 (44) . A more general feature, such as the lipid head-group size, can also be important for protein function (14) . 4. Protein machineries in the biogenesis of α-helical membrane proteins Although some α-helical membrane proteins are able to spontaneously insert into the membrane, the membrane integration in vivo is generally aided by protein machineries (51,52) . A common pathway for the transport, translocation, and insertion of α-helical membrane proteins is dependent on the Signal Recognition Particle (SRP) (53) . An overview of this pathway is shown in ?? A. The major components are the cytosolic SRP, its membrane bound signal recognition particle receptor (54) and the core of the secretase (Sec) translocon all universally conserved in the three domains of life (53) . 3.2.3. Membrane properties shaping protein function Anionic phospholipids can regulate protein activity and structure (44,45) as well as influence targeting to the membrane (46) . Interaction with anionic phospholipids can be rather nonspecific. Unstructured regions enriched in arginine and lysine residues can form electrostatic interactions with a negatively charged lipid domain in the membrane (47) . Rhodopsin function, in turn, can be related to the requirement for a relatively unstable membrane environment, brought about by lipids with small head groups and bulky acyl chains (48) . The mixing of bilayer prone and non-bilayer lipids can cause the tail region to over-pack while the head group region remains under-packed. This curvature stress results in a higher lateral pressure in the middle of the membrane, which in turn may affect membrane protein structure. A transmembrane protein may relieve this stress by adopting an hourglass shape, a feature found in a large number of membrane protein structures (49) . In addition, lipid composition of the membrane can direct the topology of at least some proteins in a non-specific manner (19) . 4.1. The SRP-dependent pathway A N-terminal signal sequence is crucial for the transport of a nascent polypeptide chain to its target membrane (53) . The signal sequence is characterized by a hydrophobic core, surrounded by positively charged amino acids at its N-terminus and polar amino acids at its C-terminus (53) . Since the hydrophobic nature of the signal sequence core is the most important feature for SRP recognition (55) , the first transmembrane domain of a membrane protein can also act as SRP substrate (54) . SRP is recruited to a ribosome-nascent chain complex (RNC) as soon as the signal sequence emerges from the ribosome exit tunnel (53) , in eukaryotes, this leads to stalling of the ribosome. Formation of the RNC-SRP complex is followed by its association with the ER (eukaryotes) or plasma membrane (prokaryotes), mediated by the GTP dependent interaction between SRP and its receptor. Once at the membrane, the RNC complex is transferred on to the translocon, followed by GTP hydrolysis in SRP and SR resulting in their dissociation (54) . These events are presented schematically in Figure 2 A. The RNC and translocon are associated with each other such, that the ribosomal exit tunnel is aligned with the translocon pore and the polypeptide synthesized by the ribosome can be fed into the translocon (56) . The translocon possesses a dual nature, allowing both a passage across the membrane and into the membrane. 3.2.4. Implications of specific lipid properties in membrane protein function Some lipids interact specifically with a protein regulating its function. Since membrane proteins are generally solubilized in detergent solutions before crystallization, lipids are probably underrepresented in crystal structures. In addition, the lipid molecules present in the crystal may represent a few tightly bound lipid molecules and do therefore not represent typical protein-lipid interactions. Regardless, examples where the structure and function of a membrane protein is linked to a specific interaction do exist (35,47) . For instance, the full activation of Protein Kinase C is dependent on a specific interaction between phosphatidylserine (50) . Likewise, a conserved 5 Transmembrane domains are recognized and shunted sideways into the membrane bilayer, while polar domains either remain in the cytoplasm or translocate to the periplasm/ER lumen. Since the translocon itself is passive, a driving force is required. In the co- translational mode, this force is provided by GTP hydrolysis during polypeptide synthesis (57) . Knowledge of the translocon as well as the properties of amino acids are both needed to understand how transmembrane regions are recognized (57) . The structure of the translocon is described below while the fifth chapter will focus on the molecular code for translocon mediated recognition of transmembrane segments. 4.2. The Sec translocon The Sec61αβγ-translocon is a heterotrimer and facilitates the translocation of secretory proteins across, and insertion of membrane proteins into the ER membrane (shown in (Figure 2 B-C.). The functional homolog in bacteria is SecYEG and in archaea SecYEβ, both which reside in the plasma membrane. Sec61α and γ share high sequence similarity with SecY respectively SecE. A crystal structure of the Methanococcus jannaschii SecYEβ revealed fascinating details about the channel structure (58) . The model for translocon function in membrane protein biogenesis is based on both structural data as well as many biochemical studies (reviewed in (59? ) ). The major component of the translocon is the αsubunit, a membrane protein with 10 transmembrane helices. When viewed from the side, it has an hourglassshape with a narrow pore composed of hydrophobic amino acids (58) , through which secretory proteins and the loops of membrane proteins cross the membrane (60) . The exact dimensions of the pore have remained controversial and the dimensions may adjust to the nascent protein chain (61) . From top view, Secα has a clamshell like shape with two halves formed by TMH1-5 and TMH6-10 (Figure 2 B). The loop between TMH5 and 6 functions as a hinge, allowing the opposite side of the protein to open up towards the lipid bilayer (58) (Figure 2 C). The lateral gate is mainly formed by TMH2 on one side and by TMH7 on the other (58) (Figure ?? D). The opening allows exposure of a translocating polypeptide to the hydrophobic environment of the lipid bilayer, with the possibility of said polypeptide to partition into the membrane (62) . The exact mechanism is unclear, but experimental and molecular dynamics simulation data suggests that the binding of a prospective transmembrane domain to the translocon could stabilize the open conformation of the lateral gate (63,64) . The membrane Figure 2: Protein machinery involved in the membrane integration of alpha-helical membrane proteins. A. A cartoon over the SRP-dependant co-translational pathway. When the N-termianl signal sequence (orange) emerges, it is recognized by the Signal Recognition Particle (SRP, in cyan). The ribosome-nascent chain comples is brought to the translocon (green) through the interaction between SRP and SRP-receptor (red). B. Top view of the translocon heterotrimer.The alpha subunit (purple and green) forms a clam-shell like structure with SecE (in cyan) associated at its back. C. The loop between the 5th and 6th TMHs forms a hinge, which allows the lateral opening of the chanel. D. The lateral gate formed by the 2nd (in green) and 7th (in purple) TMH is highlighted. barrier is maintained in part by the narrow pore ring and its interactions with a short helix on the non-cytosolic side, namely TM2a (65) , which forms a plug (Figure ?? D) blocking the passage of small molecules (58) . In eukaryotes, a lumenal protein BiP has also been implicated in preventing leakage (66) . The mammalian Secγ and the prokaryotic SecE are single-spanning membrane proteins in most species. Like Sec61α/SecY, they are essential. Studies in yeast suggest that the γ-subunit is important for translocon stability (67) and it is found associated to Sec61α on the back/hinge side in the crystal structure (58) . The third, non-essential subunit (Sec61β/SecG) is usually a singlespanning protein in archaea and eukaryotes but has two transmembrane domains in bacteria. 4.3. Associated proteins The active translocon has been observed to be a multimer of Sec heterotrimers and associated with other 6 proteins. However, the actual number of subunits and their identities have remained controversial (68–70) . Cryo-EM reconstructions of native ribosome-translocon complexes suggested a complex with two dimers of the Sec61 heterotrimer and two tetrameric transloconassociated protein complexes (TRAP) (68) , while crosslinking studies indicate that a single Sec61 heterotrimer is responsible for protein translocation (70) . Perhaps the controversy is in part due to versatility of the Sectranslocon, where the number and identify of interacting proteins is at least occasionally dependent on its substrate (71–73) . The co-translational insertion of many membrane proteins and bacteria seem to require YidC, a membrane protein with six transmembrane domains (74) . In eukaryotes, the monomeric translocation-associated membrane protein (TRAM) may fulfill a similar function (75,76) . Both TRAP and TRAM have been crosslinking to nascent peptide as they emerge from the translocon, possibly influencing integration into the bilayer (75,77) or the topology (78) . In the ER, two modifying proteins are also found in the close vicinity of the translocon namely the signal peptidase complex (SPC) (79) and Oligosaccharyl transferase (OST) (80) . The SPC cleaves signal sequences of some membrane proteins and from secreted proteins, releasing them to the exoplasm (81) . OST is a membrane protein complex in the ER membrane with its active side on the lumenal side. It recognizes a consensus sequence of Asn-X-Ser/Thr and catalyzes the N-linked glycosylation of the protein sequence; attaching a sugar moiety to the asparagine (57) in a co-translational manner (82) 5.1. Defining amino acid hydrophobicity Just as there is an energetic cost associated with introducing a non-polar molecule (or amino-acid side chain) into an aqueous environment, there is an energetic cost from introducing a polar molecule or side chain into the nonpolar membrane interior, due to the loss of polar or hydrogen bonding with water. The polar peptide back-bone opposes membrane insertion, even though the free energy cost can be reduced by secondary structure formation (85,86) . Recent experiments with nonproteinogenic amino acids have demonstrated that the hydrophobic surface area of an amino-acid side chain is directly proportional to membrane insertion (87) . In other words, the hydrophobic effect will promote partitioning of a peptide when said peptide contains a certain number of hydrophobic amino acid side chains In the cell, transmembrane helices are recognized by the translocon based on the average hydrophobicity of a stretch of amino acids. Several experimental and computational studies of artificial systems, as well as in vitro ( (85,88) ), in vivo ( (88–90) ) and statistical (91,92) methods have been used to assess amino acid hydrophobicity. The biological hydrophobicity scale, also known as the Hessa scale, defines the individual contributions of amino-acid side chains in a position specific manner (88,93) (Figure ?? a). For the work presented in this thesis, the biological hydrophobicity scale and tools for predicting the change in free energy upon insertion based on this scale were used. Correlation between the different hydrophobicity scales is in general good. Compared to other experiemental scales, polar and charged side chains are tolerated unexpectedly well in the membrane (88,93) . Concurrently, the hydrophobicity of proline varies significantly. It is hydrophobic in the GES and WimleyWhite scale (94,95) , but rather hydrophilic according to the Hessa scale. The later being understandable, given its helix-breaking nature (96) . Overall the differences between hydrophobicity scales may be attributed to the high abundance of proteins in biological membranes, compared to the uniformly hydrophobic environment composed of membrane mimics such as organic solvents (95) . 5. Properties of amino acids in membrane protein structure Topology is often used to describe α-helical membrane proteins. In this context it refers to the number and orientation of transmembrane helices as well as the location of the N- and C-termini of a protein. Membrane proteins tend to follow a predictable pattern of topological organization; anti-parallel transmembrane helices cross the membrane from one side to the other with hydrophilic loops alternating between cytoplasmic and non-cytoplasmic location. By now many properties directing the topology of a nascent polypeptide chain have been reported (reviewed in (83,84) ); Transmembrane helices are recognized through sufficiently long and hydrophobic sequences of amino acids and the orientation is determined mainly by the distribution of positively charged amino acids according to the positive-inside rule (57) . 5.2. The properties of amino acids in membrane proteins To establish the Hessa scale, a series of artificial, potential transmembrane segments were designed and presented to the translocon in microsomal membranes. The in vitro expression system allowed a quantitative assessment of membrane insertion efficiency of the 7 test segments (88) . The probability of insertion conformed to a Boltzmann distribution, suggesting that the translocon-mediated insertion is an equilibrium process (Figure ??b). Hence, the apparent free energy of insertion (∆Gapp ) can be calculated and used to express the potential for membrane insertion of a given polypeptide (57,88) . By systematically varying the amino acid composition of these test segments, the molecular code for translocon mediated membrane insertion was deciphered (88,93,97) (Figure ??a). Similar studies were carried out in the E. coli inner-membrane (90) , baby hamster kidney cells (88) and yeast (89) . Positively charged residues close to transmembrane helices are strong topogenic signals, which will be discussed later on. In contrast, acidic residues are much less potent topology determinants and show no statistical preference for loops on either side of the membrane (105,106) . However, they have been reported to influence topology under special conditions, such as when negative charges are present in high numbers (107) , in close proximity of marginally hydrophobic transmembrane helices (108) or when present within seven flanking residues from the end of a transmembrane helix (109) . 5.2.3. Aromatic amino acids are unequally distributed in transmembrane helices Tryptophan and tyrosine are enriched near the ends of the helices, often referred to as the aromatic belt (49,105,110) . They are believed to interact favorably with the lipid head groups and have been shown to anchor and stabilize tilt angles of transmembrane helices relative to the bilayer (111,112) . Phenylalanine, on the other hand, is entirely hydrophobic and thus more abundant in the central core region of transmembrane helices (49,112) . 5.2.1. Hydrophobic and helix-forming amino acids promote membrane insertion A transmembrane helix is exposed to a varying milieus in the lipid bilayer, also reflected in the statistical difference in amino acid distribution within it (49,91) . Which amino acids are present in transmembrane helices is not only determined by hydrophobicity and bulkiness but also in their abilities to form interactions with the protein itself and the environment it resides in. Isoleucine, leucine, phenylalanine and valine promote membrane integration and dominate in the central region of a transmembrane helix. Cystein, methionine and alanine have a ∆Gapp ≈ 0 kcal/mol, placing them at the threshold between those amino acids that promote membrane integration and those that do not (Figure ?? a). However, alanines are good α-helix-formers and often found in transmembrane helices (105? ) . Polar and charged residues are rare in the membrane core, but some of them show biased distribution in membrane proteins. These amino acids and their role in membrane proteins are discussed below. 5.2.4. Proline and glycine in transmembrane segments Proline is a unique amino acid as its amine nitrogen is part of a ring structure bound to two alkyl groups. Its rigid structure disrupts an α-helix (96) introducing either a kink in a transmembrane domain or promoting the formation of helical hairpins (two closely spaced TMHs with a tight turn) in sufficiently long hydrophobic segments. Proline induced kinks may be critical for the proper structural stability and/or function of membrane proteins (113,114) and the transmembrane domains of membrane proteins contain more prolines than αhelices in globular proteins (115) . Amino-acid sequences rich in prolines and glycines tend to form coils, that is, regions lacking regular secondary structure. The presence of coils allows a higher degree of structural flexibility, creating swivels and hinges (114,116) as well as reentrant regions (49,98,117) . They are especially common in channels and transporters and are often required for function (117) . Glycine is also involved in helix-helix interactions. The GxxxG motif is fundamental in helix-helix associations (118–120) . Here, the two small glycine residues are separated by one turn creating a groove on the helix surface. This groove serves as a contact surface for another helix with the same motif. The GxxxG or variations of it (G/A/S)xxxGxxxG and GxxxGxxx(G/S/T) occur in more than 10% of all known membrane protein structures (118) . 5.2.2. Charged amino-acid side chains in transmembrane helices The increased number of crystal structures have resulted in many findings of charged residues and irregular secondary structure within the hydrophobic core (98) , despite polar residues having high ∆Gapp values (88,93) . Long, aliphatic side chains allow charged amino acids to orient their side chains towards the interfacial region and to interact with lipid head groups (99,100) . This so-called snorkeling may explain why polar groups are better tolerated in biological membranes than expected (101) . Simulations suggest, that snorkeling also allows the polar side chains to create polar microenvironments for themselves by pulling water into the membrane core (102) . In addition, intramembrane salt bridges have been found in some membrane proteins, with both structural and functional importance (103,104) . 8 5.3. Positive inside rule in establishing topology detail when the positive-inside rule can assert its influence on a membrane protein. Studies on membrane proteins with known topology revealed a bias for positive charged residues in cytoplasmic loops. This uneven occurrence in amino acid distribution is generally referred to as “the positive-inside rule”, and has been shown to hold for most organisms (1,105,121) . The presence of positive charges influence both the insertion and orientation of a transmembrane helix. For instance, a single Arg or Lys residue placed downstream of a transmembrane segment in Cin orientation can lower the apparent free energy of insertion by ≈ 0.5 kcal/mol. The effect is additive and dependent on the distance from the positive charge to the transmembrane segment (122) . However, the effect may also contribute globally, as a single positive residue placed at the very C-terminus of the dual topology protein EmrE, was able to flip the topology of the entire protein (123) . The exact mechanisms behind the positive inside rule are not fully understood. Since the cellular conditions for membrane protein insertion vary within the three domains of life, the effect of positive charges may even differ between Archaea, Bacteria and Eukarya. One explanation to why the retaining effect of positive charges is more pronounced in E. coli than in microsomes (124) is the membrane potential. The electrochemical potential across the bacterial inner-membrane is stronger than that of the ER membrane. However, the positive-inside rule does apply both in the ER and in the bacterium Sulfolobus acidocaldarius, with a reversed membrane potential (125) . Likewise, if the membrane potential was responsible, negatively charged residues would be expected to have topogenic effect. This does not seem to be the case (49,105,106) . A more likely suggestion is that the anionic phospholipids prevent membrane passage of positive charges. Basically, the negative headgroup of anionic phospholipids form electrostatic interactions to positively charged residues in protein domains retaining the loop in the cytoplasm (47,126,127) . As anionic phospholipids are present in all membranes (47) , this might explain the ubiquity of the positive inside rule. Specific interactions with the translocon have also been reported to contribute to the orientation of the signal sequence (128,129) . Mutagenesis studies on yeast Sec61p identified three charged residues, which influenced the topology of some membrane proteins (128) . Further indications of the role of translocon in membrane topogenesis, came from studies where substitutions in the lateral gate altered the topology of membrane proteins (129) . It would be interesting to study in 5.4. The two stage model and topology The knowledge of properties of transmembrane domains, topology signals and folding of a membrane protein are often combined into the two-stage model. In this model, topology is established during the first step, as helices are inserted individually into the membrane. The second stage consists of interactions between the membrane embedded helices, folding into the final tertiary structure (130,131) . Although this model applies to many proteins, others show more complex behavior (98,132–135) . Figure 3: Different paths into the membrane A. sequential and B. non-sequential membrane integartion. 5.4.1. The first stage - establishing topology Topology is in general established during cotranslational membrane integration (136) . Hydrophobicity and the positive-inside rule appear to be the main determinants but other more subtle features may contribute as well (133,137–141) . 5.4.2. The second stage - membrane protein folding Unlike soluble proteins, membrane folding of membrane proteins occurs in an environment that is different in its character (the aqueous extra- and intracellular environment and the hydrophobic core of the membrane) (131) . To add to that, the orientation of transmembrane domains is considered at least relatively fixed. One might think that the loops would be important in keeping the membrane protein together but transmembrane helices are known to assemble into a functional protein without the presence of loops (142,143) This 9 suggests, that the native fold of membrane proteins is mostly stabilized by interactions between transmembrane domains. VdW - (specifically dispersion) - forces have been suggested to be major driving force for membrane protein folding (110,144) . The amino-acid residues in transmembrane regions are in general more buried than residues in soluble proteins or extramembrane regions (131,145) , resulting in higher numbers of vdW interactions even if not necessarily stronger ones. The backbones of transmembrane helices can also be involved in “non-conventional” αC-H...O hydrogen bonds, and have been suggested to contribute to helixhelix interactions (146,147) . While their influence on stability remains controversial (148,149) , they might be important for helices at close distances and be suitable for maintaining stability but also provide structural flexibility (150,151) . Occasional salt bridges may also contribute towards membrane protein structure and stability. Salt bridges are defined as electrostatic interactions between ions of opposite charge. Due to the low di-electric constant, such interactions are strong (103,104) . Further, in an oxidative environment, two cysteines can form a disulfide bond. In vivo this occurs in the ER of eukaryotes and in the periplasm of gram negative bacteria and is catalyzed by enzymes (152,153) . In both of these cases, the final topology and functional structure is achieved through later repositioning events. Concurrently, an increasing number of results suggest that topology may not be as fixed as previously thought. Membrane proteins can undergo dramatic re-organizations, including inversion of transmembrane domains and translocation of extracellular loops, in response to changed lipid composition in vitro and in vivo. Since this can occur without the involvement of a cellular machinery, the activation energy must be low for re-assembly. (13,158) . This is an important observation, not least considering dual topology membrane proteins. Dual topology membrane proteins adopt two opposite topologies (159) and a single positively charged residue at the very C-terminus can be sufficient to convert an established topology to an opposite one (123) . 6.1. Marginally hydrophobic helices A marginally hydrophobic transmembrane helix (mTMH) can be defined as a transmembrane domain unable to insert into the membrane by itself. A surprisingly large fraction (¿30 %) of transmembrane helices in multi-spanning proteins of known three-dimensional structure have a high ∆Gpred and may thus be dependent on extrinsic sequence characteristics for membrane insertion (93) . While at least some of these helices can insert in the membrane surprisingly well, those with even higher ∆Gpred above tend to require other parts of the same protein for efficient membrane integration (133–135,154,160,161) . Even though polar residues in transmembrane regions hamper membrane integration, they are more conserved than other residues in transmembrane segments (157) . This is in part explained by their functional involvement and their tendency of being buried within the structure (157,162) . Interestingly, polar residues are more common in polytopic membrane proteins with many transmembrane domains. Membrane integration of mTMHs depend on sequence features extrinsic to the hydrophobic segment itself. Some features have been identified (illustrated in Figure 5) and include charged amino-acid residues flanking the hydrophobic segment (93,122,133) , interactions between polar residues in adjacent TMHs (163–165) , neighboring helices with strong orientational preference (166) and repositioning of TMHs relative to the membrane during folding and oligomerization (98) . However, the mechanisms by which these events take place and how frequent they are is still unclear. This section is devoted to describing both the function and problems polar groups in transmembrane helices can 6. Non-sequential membrane integration of αhelical membrane proteins The text book version of a membrane protein is an α-helical bundle, where each of the hydrophobic transmembrane helices cross the membrane in more or less perpendicular orientations (154) . The topology is assumed to form sequentially and to be relatively fixed once established (See Figure 3 A). However, the increasing number of solved membrane protein structures show far more variation in the structural elements of membrane proteins (155) . Transmembrane segments can vary significantly in length (49,100,137) , adopt strongly tilted conformations (156) , have kinks, polar groups and non-helical regions in the middle of the membranes (157) as well as re-entrant regions - helices that span only a part of the membrane before looping back. Transmembrane segments are not necessarily recognized as such by the translocon, which results in nonsequential membrane insertion. As a consequence, some proteins have been reported to initially insert in an intermediate topology (See Figure 3 B). Also, the initially inserted transmembrane regions may differ from the membrane embedded regions in the final structure. 10 bring by, as well as how they can be successfully integrated into the membrane despite high energetic cost. machineries have indeed been implicated and are described below. AQP1 folding also proposes significant plasticity in membrane protein topogenesis and folding. Similar behavior has been observed for several polytopic membrane proteins (135,160,161,175) . The ability to reorient transmembrane segments may not be restricted to membrane protein folding. E. coli SecG has been reported to undergo reversible topology inversion as part of its function (176) . Even more so, at least three membrane proteins (LacY, PheP and GabP) are known to undergo dramatic reorganization upon changes in membrane lipid composition (reviewed in (13) ). The dramatic differences in the topogenesis of AQP1 and AQP4 are dependent on surprisingly few differences in their primary sequences. Two residues (Asn49 and Lys51) in AQP1 mTMH2 are responsible for its hydrophilic nature. When exchanged to the corresponding residues in AQP4 (a methionine and leucine), the transmembrane domains of AQP1 are inserted in a sequential manner (172) . A previously uncharacterized difference lies in the nature of the region just before TMH3, which contains the helical section of reentrant region 1, the loop between the re-entrant region and TMH3, along with the N-terminal part of TMH3. In AQP4, this segment is hydrophilic (∆Gpred 4 kcal/mol) while the corresponding region in AQP1 is hydrophobic (∆Gpred 0 kcal/mol). We suggest that this difference may account for the flexibility in AQP1 topogenesis since the third transmembrane helix of AQP1 may shift out of the membrane core simultaneously bringing in the preceding “R1-H3 loop”into the membrane (177) . 6.1.1. Consequences of mTMH in AQP1 folding Aquaporin 1 (AQP1) is part of the ubiquitous family of water channels with six transmembrane helices and two re-entrant loops with a central water conducting pore (167) . When AQP1 topology was studied in Xenopus oocytes, it was shown to initially insert as a four-helix intermediate before folding into its final structure at a later stage (168,169) (see Figure 4). These results were regarded with some skepticism based on contradicting results from experiments in mammalian cells (170) . However, this apparent controversy has been solved by the observation that the intermediate is less stable in mammalian cells (169) . Biogenesis of the close homolog Aquaporin 4 (AQP4), occurs more conventionally, with sequential and co-translational insertion of each transmembrane segment. The sequence features underlying the different behavior of AQP1 have attracted a lot of attention. In essence, the second transmembrane helix is not sufficiently hydrophobic to integrate into the membrane (133,171,172) , which in turn results in altered behavior of the rest of the protein (172) . For one, TMH3 will be inverted in its opposite orientation while TMH4 is unable to integrate into the membrane, due to a number of positively charged residues at its C-terminus. Transition from this intermediate to the final topology requires the 180 ◦ rotation of TMH3 as well as the translocation of the loops between helices 3-4 and 4-5 (Figure 4). An occurrence requiring the presence of the transmembrane helices four, five and six (169,171) . These observations imply interesting and novel features in membrane proteins: a dilemma regarding features required for function and correct folding as well as the unexpected flexibility in membrane protein structures. AQP1 demonstrates clearly the consequences of a mTMH in a membrane protein. The residues rendering mTMH2 unable to integrate to the membrane are functionally important (172) . At the same time, correct orientation is a prerequisite for adequate exposure of extramembranous domains on the functionally relevant side of the membrane. How does the cell solve the dilemma of keeping the unfavorable residues within the mTMH yet obtaining functional protein? After all, not only is the production of a non-functional protein costly (173,174) , it also poses a burden for cellular quality control along with protein degradation systems. Clearly there must exist a mechanism to ensure proper membrane integration - some sequence features and cellular 6.2. Cost of polar groups in the membrane The hydrocarbon core of biological membranes has been considered to be a “forbidden zone” for charged amino acids (178) . However, the membrane structure derived from X-ray and neutron scattering data depicts a rather dynamic structure. Only the very center of the membrane is entirely hydrophobic, the rest is a mixture of polar lipid head-groups and water molecules. Consequently, the membrane may partially permit the presence and passage of polar groups (26) . In regards to mTMHs and the ability of extramembraneous regions to cross the membrane it is of interest to learn how unfavorable polar groups in the membrane really are. Arginine is often thought of as the most hydrophilic of amino acids, yet it is frequent in transmembrane helices (178) . Molecular dynamics estimations suggest a barrier around 17 kcal/mol (179) and experimental scales report values ranging between 12 11 to 1.8 kcal/mol (94) . However, direct comparison of different hydrophobicity scales is not straight forward as they are often normalized in different ways (95) . According to the Hessa scale, the cost of an arginine in the middle of an hydrophobic helix is only ≈ 2.5 kcal/mol (88,180) and strongly position specific (180) . Several mechanisms seem to decrease the free energey penalty for a charged group in the membrane. Charged residues with long side chains can snorkle toward the membrane interface and polar groups can draw lipid head-groups and water deep into the core (157,179,181) . Additional, arginine (and other charged residues) may partition into the membrane as a sufficient number of hydrophobic amino acids can to overcome the cost (178) . vice versa (140,141) . Since these observation strongly rely on artificial model proteins it remains unclear whether this trend is prevalent in native proteins. In addition, several studies suggest that the first transmembrane helix may not be sufficient in determining the topology of the entire protein (183–185) . 6.3.2. Specific interactions between polar groups Specific interaction within or between helices can improve their propensity to insert into the membrane (see Figure 5c). As discussed before, polar or charge groups hamper insertion of transmembrane domains. However, helices with charged or polar residues can interact with each other before entering the lipid bilayer, effectively “hiding” the energetically unfavorable groups in interhelical hydrogen bonds (164,165) . 6.3. Sequence features implicated in the insertion of mTMHs 6.3.3. Repositioning in the membrane One turn in an ideal α-helix employs ≈ 3.6 amino acids per turn with one turn having the height of 5.4 Å. To span the hydrophobic core of a lipid layer approximately 20 residues are needed to form a sufficiently long helix. However, the length of transmembrane helices vary between 12 and 40 residues promoting reorganization of both proteins and lipids (49,100,137,182) . One of such adaptations is the tilting of longer helices relative to the membrane normal (36) . While tilting of transmembrane domains can occur as a consequence of hydrophobic mismatch, it is also possibly that tilting is induced during membrane protein folding due to packing interactions. It may also enable polar and charged side chains to become transmembrane, allowing them to get buried in the protein rather than being exposed to lipids (see Figure 5 D, Figure ?? and Paper III) (186) . The glutamate transporter homologue from Pyrococcus horikoshii serves as an example. Its three-dimensional structure is complicated with both mTMHs and transmembrane helices disrupted by coils. In a previous study, the primary sequence was aligned with known secondary structures and a theoretical hydrophobicity profile. Overlapping regions with more hydrophobic character were identified and at least one those segments was shown to be more prone insert into the membrane (98) . Similar behavior has been reported in the human Band 3 protein and the KAT1 voltage dependent K+ -channel (161,187) where polar interactions within the protein enables membrane integration of transmembrane segments (188) . Reentrant regions are segments that penetrate the lipid bilayer without traversing it, having their N- and C-terminus on the same side. They are enriched in alanine, glycine and proline residues and are mostly found 6.3.1. Positive-inside rule Flanking loops and neighboring helices can be important for the membrane integration of mTMHs (133) . In accordance to the positive-inside rule, arginines and lysines in cytoplasmic loops contribute towards the apparent free energy of membrane insertion by ≈ 0.5 kcal/mol of a transmembrane helix, see Figure 5A (122) . However, cytosolic loops with positively charged residues do not always improve insertion of a mTMH (133) . Characteristics in one transmembrane helix can influence the insertion propensity of another. This phenomenon was first observed for the human band 3 protein, where the strong orientational preference of a transmembrane helix resulted in membrane integration of an upstream region (135) . Previous studies have implicated both positive charges and hydrophobicity in the likelihood of a transmembrane helix adopting a certain topology (84,138,140,182) . A recent, systematic study showed how orientational preference of a neighboring helix could both increase and decrease the insertion of a mTMH (166) (see Figure 5B). What then, gives a transmembrane helix an orientational preference - apart from cytoplasmic positively charged amino-acids residues? Long and hydrophobic helices may favor Nout − Cin orientation, while short and less hydrophobic helices instead adopt an Nin − Cout topology (137–139) . A hydrophobic transmembrane helix may spend a shorter time inside the translocon, thus not having enough time to reorient. A less hydrophobic transmembrane helix, on the other hand, may not move out from the translocon as fast allowing re-orientation. This “hydrophobicity signal” can however be overriden by, positively charged residues at the N-terminus and 12 in water and ion channels (117) . Their integration to the membrane can occur both co- and post-translationally (188,189) . with rigid molecules of varying size fused to known Sec-substrates. SecYEG allowed the unrestricted passage of constructs up to 22-24 Å (193) , which is larger than seen in crystal structures or estimated by molecular dynamics simulations (199) . Given the conflicting results and the huge thermodynamic driving force for membrane protein insertion (13,51,110) a rather interesting model has been proposed. Perhaps the transmembrane helices never fully enter the translocon channel but interact with the cytoplasmic membrane interface and slide into the membrane utilizing the translocon lateral gate (200) . This would sit well with the observations of neighboring helices enabling the membrane integration of mTMHs. 6.4. Can translocon influence hydrophobicity threshold or topology? The translocon itself may influence the insertion and topology of transmembrane helices. Conserved residues both in the translocon pore ring and the later gate can influence the insertion of signal sequences (190,191) . Additionally, the arginine to glutamate substitution at the plug domain of yeast translocon weakens the positiveinside rule (129) . Substitution-sensitive residues have also been identified in the lateral gate, where they could both increase and reduce the threshold hydrophobicity for transmembrane segment. However, this impact is strong only for single-spanning membrane proteins and on the first transmembrane domain of a polytopic membrane protein (192) . Whether the translocon is big enough to allow a polypeptide to invert has remained controversial (58,61,68,193,194) . Since transmembrane helices can stay in close proximity to the translocon (136,195) it has been proposed that reorientation occurs adjacent to the translocon complex even though not in it (84) . Then again, the high protein content (68–70,79,80) alone might favor insertion and re-orientation of membrane protein domains (11,136) . The time point and location where transmembrane helices can form interactions with each other have also gained a lot of attention. Helical hairpins might form as early as in the ribosome “folding vestibule” (196) and might allow membrane insertion of a mTMH (164) . Can the translocon accommodate more than one polypeptide chain? Experimental studies suggest it might (165,197) . In fact, many membrane proteins do linger within or close by the channel (136,198) . The translocon associated proteins TRAM and TRAP have both been cross-linked to nascent polypeptides emerging from the translocon affecting both membrane insertion (75,77) and topology (78) of their substrates. The size of the translocon protein conducting channel is important considering the popular model for membrane protein insertion (57,58,88) . While the cytoplasmic cavity of M. jannaschii SecY has a diameter of 20 to 25 Å, the narrowest part of the channel ≈ 5 − 8 Å wide. In order to accommodate an α-helix, the channel would have to expand (58) . Interestingly, the central pore appear wider (≈ 10 Å) in the Thermotoga maritima SecY structure, where SecY interacts with SecA and SecG (194) . The channel size has also been assessed experimentally. In one study, SecYEG was challenged 6.5. The membrane environment The topology of a membrane protein can be remarkably dynamic and can be altered by changing the environment. When the zwitterionic membrane lipid PE is depleted from E. coli, the topology of LacY exhibits a partial and reversible topological inversion (158) . This re-assembly is independent on any cellular machineries (13) . While the behavior of LacY may be surprising, it should not be considered an anomaly. Lipid-dependent topological reorganizations have been shown to occur for other proteins as well (13,126) and membrane lipids are important for both insertion and stability of many membrane proteins (reviewed in (21,22) ). Further, since most eukaryotic membrane proteins are initially inserted into the endoplasmic reticulum and subsequently targeted to the membranes of other organelles this might also be reflected in membrane protein folding. The lipid composition in the different membranes is reflected in the composition of transmembrane segments (34) and while the the ER membrane is mainly composed of phosphatidylcholine, PE, and phosphatidylinositol, the plasma membrane is in turned enriched in phosphatidylcholine, sphingomyelin and contains cholesterol (17,201) . Perhaps this is also reflected in membrane protein topology (168–170) ? 13 Figure 5: Local sequence context can help insert a mTMH. A. Positive charges in cytoplasmic loops can contribute towards the free energy for membrane integration and promote membrane insertion of a mTMH. B. Orientational preference, induced by e.g. positive charges, can pull a mTMH into the membrane. C. Specific interactions between a mTMH and an other transmembrane helix can allow insertion of the helix-pair. D. Repositioning can allow a segment of lower hydrophobicity to enter the membrane, even though a more hydrophobic part is initially recognized by the translocon. Figure 4: Topological rearrangements in AQP1. AQP1 is initially inserted into the membrane as a four-helix intermediate (top). TMH2 is marginally hydrophobic and can not be inserted into the membrane initially. This results in TMH3 obtaining the wrong orientation and TMH4 can not insert into the membrane due to a strong positive-charge bias. For AQP1 to obtain its 6 transmembrane domain topology (bottom), the reorientation of TMH3 is required. This reorientation requires residue Arg93 to cross the membrane (marked with a red plus). 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