Peripheral membrane protein

Peripheral membrane proteins are proteins that adhere only temporarily to the biological membrane with which they are associated. These molecules attach to integral membrane proteins, or penetrate the peripheral regions of the lipid bilayer. The regulatory protein subunits of many ion channels and transmembrane receptors, for example, may be defined as peripheral membrane proteins. In contrast to integral membrane proteins, peripheral membrane proteins tend to collect in the water-soluble fraction during protein purification. An exception to this rule are proteins with GPI anchors, whose purification properties may be similar to those of integral membrane proteins.

Schematic representation of the different types of interaction between monotopic membrane proteins and the cell membrane: 1. interaction by an amphipathic α-helix parallel to the membrane plane (in-plane membrane helix) 2. interaction by a hydrophic loop 3. interaction by a covalently bound membrane lipid (lipidation) 4. electrostatic or ionic interactions with membrane lipids (e.g. through a calcium ion)
The membrane is represented in light brown.

Classification

Peripheral membrane proteins can be classified as follows:

• Ordinary water-soluble proteins that do not interact directly with the lipid bilayer but attached to integral membrane proteins.
• Amphitropic proteins that associate directly with lipid bilayers and can exist in two alternative states: a water-soluble and a lipid bilayer-bound, unlike integral proteins existing only in the membrane-bound state. It is noteworthy that protein-protein and protein-lipid interactions are not mutually exclusive. For example, G-proteins and some protein kinases associate with lipid bilayers and transmembrane receptors simultaneously. It was also found that many polypeptide hormones, antimicrobial peptides, and neurotoxins accumulate at the lipid bilayer surface prior to binding to their transmembrane protein targets. Such peptides and proteins are also amphitropic molecules.

Some water-soluble proteins can associate with lipid bialyers irreversibly and form transmembrane alpha-helical or beta-barrel channels. This happens with pore forming toxins (colicin A, alpha-hemolysin, and others), BcL-2 like proteins involved in apoptosis, some amphiphilic antimicrobial peptides, and certain annexins. These proteins can be also described as amphitropic or "non-permanent", because one of their conformational states is water-soluble or loosely associated with membranes.

The boundary between peripheral and typical water-soluble proteins is blurred. Some proteins normally found in the cytoplasm (e.g. albumin, ribonuclease, lysozyme, or hemoglobin) can associate with lipid bilayers under certain experimental conditions in vitro. Some proteins associate strongly and even irreversibly with lipid bilayers if they are partially unfolded or form the molten globule state. Association of proteins with membranes can also be triggered by pH changes.

Membrane binding mechanisms

Association of peripheral proteins with membranes is a complicated process that may involve significant conformational changes, folding, or refolding of the membrane-associated proteins or peptides, formation or dissociation of protein quaternary structures or oligomeric complexes, and specific binding of ions, ligands, or regulatory lipids. Typical amphithropic proteins must interact strongly with the lipid bilayer to perform their biological functions, such as the enzymatic processing of lipids or other hydrophobic substances, membrane anchoring, or binding and transport of nonpolar compounds. These proteins can be anchored to the bilayer by hydrophobic interactions of exposed nonpolar residues, by specific non-covalent binding of regulatory lipids, or through covalently-bound lipid anchors.

Non-specific hydrophobic association

Amphitropic proteins associate with lipid bilayers through various hydrophobic anchors, such as amphiphilic α-helixes, exposed nonpolar loops, post-translationally acylated or lipidated amino acid residues, or acyl chains of specifically bound regulatory lipids such as phosphatidylinositol phosphates. The hydrophobic interactions are important even for highly cationic peptides and proteins of natural origin, such as the polybasic domain of MARCKS protein or histactophilin, when their natural hydrophobic anchors are present.

Covalently bound lipid anchors

Lipid anchored proteins are covalently attached to different fatty acid acyl chains in the cytoplasmic leaflet of cell membrane (palmitoylation, myristoylation, or prenylation), or to the entire lipid molecules (such as in GPI or cholesterol), at the exoplasmic side of cell membrane. Association with membranes through acylated residues is still reversible, because the acyl chain can be buried in a hydrophobic binding pocket of a protein when it dissociates from the membrane, as in the beta-subunits of G-proteins. The lipid anchors are usually bound to the highly flexible segments of proteins that are not resolved in the protein crystal structures.

Specific protein-lipid binding

Some cytosolic proteins are recruited to different cellular membranes by recognizing certain types of lipids, which are present mostly in the corresponding membranes. The binding occurs through membrane-targeting domains that have specific binding pockets for head groups of their cognate lipids. This is a typical protein-ligand association, which is stabilized by intermolecular H-bonds, van der Waals interactions, and hydrophobic interactions between the protein and ligand. Such complexes are also stabilized by ionic bridges between Asp or Glu residues of the proteins and lipid phosphates through intermediate Ca2+ ions.

Protein-lipid electrostatic interactions

Any positively charged protein will be attracted to a negatively charged membrane by nonspecific electrostatic interactions. However, not all peripheral peptides and proteins are cationic, and only certain sides of membrane are negatively charged: the inner leaflet of plasma membranes, the outer leaflet of outer bacterial membranes and mitochondrial membranes. Therefore, electrostatic interactions play an important role in membrane targeting of electron carriers (e.g. cytochrome c), cationic toxins (e.g. charybdotoxin), and specific membrane-targeting domains (e.g. some PH domains, C1 domains, and C2 domains).

Electrostatic interactions are strongly dependent on the ionic strength. They are relatively weak at the physiological ionic strength (0.14M NaCl): ~3 to 4 kcal/mol at the physiological ionic strength for small cationic proteins, such as cytochrome c, charybdotoxin or hisactophilin.

Membrane penetration depth

The lipid bilayer consists of the hydrophobic core region formed by the acyl chains of the lipids, and membrane interfacial regions that are formed by the polar head groups of lipids. The interfacial regions of model phospholipid bilayers have thickness of 8 to 10 Å, although they can be wider in biological membranes that include large amounts gangliosides or lipopolysaccharides The thickness of hydrocarbon core region of typical biological membranes estimated by small-angle X-ray scattering vary from 27 to 32 Å The boundary between the hydrocarbon core region and the water-saturated interface is very narrow (~3Å) and defined by the effective concentration of water that changes exponentially from nearly zero to ~2M. The corresponding hydrocarbon membrane boundary plane passes through the carbonyl groups of phospholipids. The phosphate groups of phospholipids are completely hydrated and situated ~5 Å outside the hydrocarbon boundaries.

Orientations and penetration depths of many amphitropic proteins and peptides with respect to the hydrocarbon membrane boundary were studied using the site-directed spin labeling, chemical labeling, membrane binding measurements for protein mutants, fluorescence quenching, solution and solid-state NMR spectroscopy, ATR FTIR spectroscopy, and other methods.

At least two distinct membrane-association modes of the proteins have been identified. Some of them remain completely in the aqueous solution and do not penetrate into the lipid head group region ("S" proteins). Such proteins avoid the energetic penalties associated with perturbation of the lipid bilayer and interact with the lipid bilayer only electrostatically. The examples are poly-lysine, ANTH domain, and water-soluble domains of many lipid-anchored or transmembrane proteins. However, typical amphitropic proteins can penetrate through the interfacial region and reach the hydrocarbon interior of membrane, which provides some gain in the hydrophobic interactions with membrane ("H" type). An intermediate "I" type was suggested for proteins that penetrate into membrane interfacial region but only "touch" the hydrophobic core, as possibly in the case of cytochrome c. The different types of protein-membrane complexes have distinct thermodynamic parameters of binding

Surprisingly, even unfolded peptides can penetrate deep across the lipid head group region and reach the hydrocarbon interior of membrane if these peptides have a few nonpolar residues. Such behavior is also typical for amphiphilic α-helical peptides.

Membrane binding affinity

Association of amphitropic proteins with lipid bilayers depends on various experimental conditions, primarily the specific lipid composition of the membrane. For example, the presence of negatively charged lipids can improve the binding of peripheral proteins to model membranes. This effect may be due to a number of factors, including electrostatic attraction of a cationic protein to the negatively charged membrane surface, specific binding of anionic lipid ligands to the protein cavities, reduced membrane lateral pressure, or increased hydration of the membrane interfacial region due to strong electrostatic repulsions between the negatively charged head groups of lipids.

Different categories of peripheral proteins

Enzymes

These enzymes may participate in metabolism of different membrane components, such as lipids (phospholipases and cholesterol oxidases), cell wall oligosaccharides (glycosyltransferase and transglycosidases), or proteins (signal peptidase and palmitoyl protein thioesterases). They can also digest lipids that form micelles or nonpolar droplets in water (pancreatic lipases).

• Alpha/beta hydrolase superfamily (bacterial, fungal, gastric and pancreatic lipases, palmitoyl protein thioesterases, cutinase, and cholinesterases) [1]
• Secretory phospholipases A2 [2]
• FabD/lysophospholipase-like (including cytosolic phospholipase A2) [3]
• Cholesterol oxidases [4]
• Carotenoid oxygenase [5]
• Lipoxygenases [6]
• Alpha toxins [7]
• Phospholipase C [8]
• Sphingomyelinase C [9]
• Transglycosidases [10]
• Ferrochelatase [11]
• Myotubularin-related protein [12]
• Glycosyltransferase MurG [13]
• Dihydroorotate dehydrogenases and glycolate oxidase [14]
• Vitelline membrane outer protein-I [15]

Structural domains

Structural domains mediate attachment of other proteins to membranes. Their binding to membranes can be mediated by calcium ions (Ca²⁺) that form bridges between the acidic protein residues and phosphate groups of lipids, as in annexins or GLA domains.

• Annexins [16]
• GLA-domains of blood coagulation system [17]
• Influenza virus matrix M1 protein [18]
• Hisactophilin-1 [19]
• Seminal plasma protein [20]
• Translocation ATPase SecA [21]
• Exocyst complex component Sec5 [22]
• Synapsin I [23]
• Synuclein [24]
• Epididymal secretory proteins and Rho GDP-dissociation inhibitors [25]
• Rab GDP dissociation inhibitor alpha [26]
• Phosducin [27]
• Spectrin and α-actinin-2 [28]
• Peroxin pex5 and vesicular transport protein sec17 [29]

Membrane-targeting domains (“lipid clamps”)

Membrane-targeting domains associate specifically with head groups of their lipid ligands embedded into the membrane. These lipid ligands are present in different concentrations in distinct types of biological membranes (for example, PIP3 can be found mostly in membranes of early endosome, PIP(3,5) in late endosome, and PIP4 in Golgi). Hence, each domain is targeted to its own membrane.

• C1 domains [30] bind diacylglycerol and phorbol esters.
• C2 domains [31] bind phosphatidylserine or phosphatidylcholine
• Pleckstrin homology domains [32], PX domains [33], and Tubby domains [34] bind different phosphoinositides
• FYVE domains [35] are more specific for PIP3
• ENTH domains bind PIP(3,4) or PIP(4,5).
• ANTH domain binds PIP(4,5)
• Proteins from ERM (ezrin/radixin/moesin) family [36] bind PIP(4,5)

• Other phosphoinositide-binding proteins include phosphotyrosine-binding domain [37] and certain PDZ domains. They bind PIP(4,5).

• Discoidin domains of blood coagulation factors [38]
• ENTH, VHS and ANTH domains [39]

Transporters of hydrophobic substances

These peripheral proteins function as carriers of non-polar compounds between different types of cell membranes or between membranes and cytosolic protein complexes. The transported substances are phosphatidylinositol, tocopherol, gangliosides, glycolipids, sterol derivatives, retinol, or fatty acids.

• Glycolipid transfer proteins [40]
• Lipocalins including retinol binding proteins and fatty acid-binding proteins [41]
• Polyisoprenoid-binding protein [42]
• Ganglioside GM2 activators [43]
• α-Tocopherol and phosphatidylinositol sec14p transfer proteins [44]
• Sterol carrier proteins [45]
• Phosphatidylinositol transfer proteins and STAR domains [46]
• Oxysterol-binding protein [47]

Electron carriers

• Cytochrome c [48]
• Cupredoxins [49]
• High potential iron protein [50]
• Adrenodoxin reductase [51]
• Electron transfer flavoproteins [52]

Polypeptide ligands

Different hormones, toxins, inhibitors, or antimicrobial peptides usually interact specifically with large TM protein complexes. They can be also accumulated at the lipid bilayer surface, prior to binding their protein targets. Such polypeptide ligands are often positively charged and interact electrostatically with anionic membranes.

• α-Helical peptide hormones [53]
• Tachykinin peptides [54]
• Saposin B and NK-lysin [55]
• Heat-stable enterotoxin B [56]
• Conotoxins, spider toxins, insect toxins, albumin 1, and leginsulin [57]
• Scorpion venom toxins [58]
• Snake venom toxins [59]
• Neurotoxin III [60]
• Defensins and sea anemone sodium channel toxins [61]
• Poneratoxin and mastoparan [62]
• α-Conotoxins [63]
• Macrocyclic bacteriocins: subtilosin and microcin J25 [64]
• Tricyclic peptide RP71935 [65]
• Gramicidin S [66]
• Antimicrobial peptide HP [67]
• Lactoferricin B [68]
• Cyclic lipopeptide antibiotics: daptomycin and tsushimycin [69]
• Cyclotides [70]
• Leucocin-like bacteriocins [71]

Channel-forming proteins and peptides

Channel-forming polypeptides undergo oligomerization and significant conformational transitions and therefore can associate with membranes irreversibly. Structure of the membrane-bound channel has been determined only for α-hemolysin. In other cases, experimental structures represents a water-soluble conformation that weakly interacts with the lipid bilayer.

• Apoptosis regulator Bcl-2 [72]
• Colicin A [73]
• δ-Endotoxins [74]
• Anemone pore-forming cytolysins [75]
• Perfringolysin [76]
• Botulinum neurotoxin B [77]
• Crambin, γ-purothionin, and hellethionin [78]
• Bacteriocin AS-48 [79]
• Ectatomin [80]
• Magainin [81]
• Peptaibols [82]
• Insect defensins [83]
• Plant defensins [84]
• Moricins [85]
• Pleruocidin [86]
• Lantibiotic peptides: Actagardine, mersacidin and nisin [87]

General references

• Protein-lipid interactions (Ed. L.K. Tamm) Wiley, 2005.
• Cho, W. and Stahelin, R.V. (2005). "Membrane-protein interactions in cell signaling and membrane trafficking". Annual Review of Biophysics and Biomolecular Structure. 34: 119–151. doi:10.1146/annurev.biophys.33.110502.133337. Retrieved 2007-01-23. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
• Goni F.M. (2002). "Non-permanent proteins in membranes: when proteins come as visitors" (PDF). Mol. Membr. Biol. 19: 237–245.
• Johnson J, Cornell R (1999). "Amphitropic proteins: regulation by reversible membrane interactions (review)" (PDF). Mol Membr Biol. 16 (3): 217–35. PMID 10503244.
• Seaton B.A. and Roberts M.F. Peripheral membrane proteins. pp. 355-403. In Biological Membranes (Eds. K. Mertz and B.Roux), Birkhauser Boston, 1996.
• Benga G. Protein-lipid interactions in biological membranes, pp.159-188. In Structure and Properties of Biological Membranes, vol. 1 (Ed. G. Benga) Boca Raton CRC Press, 1985.
• Kessel A. and Ben-Tal N. 2002. Free energy determinants of peptide association with lipid bilayers. In Current Topics in Membranes 52: 205-253.
• Malmberg N, Falke J (2005). "Use of EPR power saturation to analyze the membrane-docking geometries of peripheral proteins: applications to C2 domains". Annu Rev Biophys Biomol Struct. 34: 71–90. PMID 15869384.
• McIntosh T.J. and Simon S.A. 2006. Roles of bilayer material properties in function and distribution of membrane proteins. Annu. Rev. Biophys. Biomol. Struct. 35: 177-198.
• McIntosh T, Simon S (2006). "Roles of bilayer material properties in function and distribution of membrane proteins". Annu Rev Biophys Biomol Struct. 35: 177–98. PMID 16689633.

See also

• Membrane proteins
• Lipoproteins
• Integral membrane proteins
• Transmembrane proteins
• Antimicrobial peptides

External links

• Orientations of Proteins in Membranes (OPM) database 3D structures of some peripheral proteins and their calculated orientations in membranes
• DOLOP Genomics-oriented database of bacterial lipoproteins
• Peptaibol database
• Antimicrobial Peptide Database