Atransmembrane protein is a type ofintegral membrane protein that spans the entirety of thecell membrane. Many transmembrane proteins function asgateways to permit the transport of specific substances across the membrane. They frequently undergo significantconformational changes to move a substance through the membrane. They are usually highlyhydrophobic and aggregate and precipitate in water. They requiredetergents or nonpolar solvents for extraction, although some of them (beta-barrels) can be also extracted usingdenaturing agents.
Thepeptide sequence that spans the membrane, or thetransmembrane segment, is largely hydrophobic and can be visualized using thehydropathy plot.[1] Depending on the number of transmembrane segments, transmembrane proteins can be classified assingle-pass membrane proteins, or as multipass membrane proteins.[2] Some other integralmembrane proteins are calledmonotopic, meaning that they are also permanently attached to the membrane, but do not pass through it.[3]
There are two basic types of transmembrane proteins:[4]alpha-helical andbeta barrels. Alpha-helical proteins are present in the inner membranes of bacterial cells or the plasma membrane of eukaryotic cells, and sometimes in thebacterial outer membrane.[5] This is the major category of transmembrane proteins. In humans, 27% of all proteins have been estimated to be alpha-helical membrane proteins.[6]Beta-barrel proteins are so far found only in outer membranes ofgram-negative bacteria,cell walls ofgram-positive bacteria,outer membranes ofmitochondria andchloroplasts, or can be secreted aspore-forming toxins. All beta-barrel transmembrane proteins have simplest up-and-down topology, which may reflect their common evolutionary origin and similar folding mechanism.[7]
In addition to the protein domains, there are unusual transmembrane elements formed by peptides. A typical example isgramicidin A, a peptide that forms a dimeric transmembrane β-helix.[8] This peptide is secreted bygram-positive bacteria as anantibiotic. A transmembranepolyproline-II helix has not been reported in natural proteins. Nonetheless, this structure was experimentally observed in specifically designed artificial peptides.[9]
This classification refers to theposition of the protein N- and C-termini on the different sides of thelipid bilayer. Types I, II, III and IV aresingle-pass molecules. Type I transmembrane proteins are anchored to the lipid membrane with a stop-transfer anchor sequence and have their N-terminal domains targeted to theendoplasmic reticulum (ER)lumen during synthesis (and the extracellular space, if mature forms are located oncell membranes). Type II and III are anchored with a signal-anchor sequence, with type II being targeted to the ER lumen with its C-terminal domain, while type III have their N-terminal domains targeted to the ER lumen. Type IV is subdivided into IV-A, with their N-terminal domains targeted to the cytosol and IV-B, with an N-terminal domain targeted to the lumen.[10] The implications for the division in the four types are especially manifest at the time of translocation and ER-bound translation, when the protein has to be passed through the ER membrane in a direction dependent on the type.[citation needed]
Membrane protein structures can be determined byX-ray crystallography,electron microscopy orNMR spectroscopy.[12] The most commontertiary structures of these proteins are transmembranehelix bundle andbeta barrel. The portion of the membrane proteins that are attached to the lipid bilayer (seeannular lipid shell) consist mostly of hydrophobic amino acids.[13]
Membrane proteins which have hydrophobic surfaces, are relatively flexible and are expressed at relatively low levels. This creates difficulties in obtaining enough protein and then growing crystals. Hence, despite the significant functional importance of membrane proteins, determining atomic resolution structures for these proteins is more difficult than globular proteins.[14] As of January 2013 less than 0.1% of protein structures determined were membrane proteins despite being 20–30% of the total proteome.[15] Due to this difficulty and the importance of this class of proteins methods of protein structure prediction based on hydropathy plots, the positive inside rule and other methods have been developed.[16][17][18]
Transmembranealpha-helical (α-helical) proteins are unusually stable judging from thermaldenaturation studies, because they do not unfold completely within the membranes (the complete unfolding would require breaking down too many α-helicalH-bonds in the nonpolar media). On the other hand, these proteins easilymisfold, due to non-native aggregation in membranes, transition to themolten globule states, formation of non-nativedisulfide bonds, or unfolding of peripheral regions and nonregular loops that are locally less stable.[19]
It is also important to properly define theunfolded state. Theunfolded state of membrane proteins in detergentmicelles is different from that in the thermaldenaturation experiments.[citation needed] This state represents a combination of folded hydrophobic α-helices and partially unfolded segments covered by thedetergent. For example, the "unfolded"bacteriorhodopsin inSDS micelles has four transmembrane α-helices folded, while the rest of the protein is situated at the micelle-water interface and can adopt different types of non-nativeamphiphilic structures. Free energy differences between such detergent-denatured and native states are similar to stabilities of water-soluble proteins (< 10 kcal/mol).[citation needed]
Refolding of α-helical transmembrane proteinsin vitro is technically difficult. There are relatively few examples of the successful refolding experiments, as forbacteriorhodopsin.In vivo, all such proteins are normally folded co-translationally within the large transmembranetranslocon. The translocon channel provides a highly heterogeneous environment for the nascent transmembrane α-helices. A relatively polar amphiphilic α-helix can adopt a transmembrane orientation in the translocon (although it would be at the membrane surface or unfoldedin vitro), because its polar residues can face the central water-filled channel of the translocon. Such mechanism is necessary for incorporation of polar α-helices into structures of transmembrane proteins. The amphiphilic helices remain attached to the translocon until the protein is completely synthesized and folded. If the protein remains unfolded and attached to the translocon for too long, it is degraded by specific "quality control" cellular systems.[citation needed]
Stability of beta barrel (β-barrel) transmembrane proteins is similar to stability of water-soluble proteins, based on chemical denaturation studies. Some of them are very stable even in chaotropic agents and high temperature. Their foldingin vivo is facilitated by water-solublechaperones, such as protein Skp. It is thought that β-barrel membrane proteins come from one ancestor even having different number of sheets which could be added or doubled during evolution. Some studies show a huge sequence conservation among different organisms and also conserved amino acids which hold the structure and help with folding.[20]
Note:n andS are, respectively, the number of beta-strands and the "shear number"[22] of thebeta-barrel