Membrane proteins are crucial players in cell function, embedded in or associated with the lipid bilayer. They come in various types, including integral and peripheral proteins, each with unique roles in transport, signaling, and structural support.

Understanding membrane proteins is key to grasping cellular processes. Their structure-function relationships, targeting mechanisms, and diverse functions make them essential components in maintaining cellular homeostasis and facilitating communication between cells and their environment.

Membrane protein types and roles

Integral and peripheral membrane proteins

• Integral membrane proteins are embedded within the lipid bilayer
  • Classified as transmembrane proteins or lipid-anchored proteins based on their interaction with the membrane
  • Transmembrane proteins span the entire lipid bilayer (ion channels, receptors)
  • Lipid-anchored proteins are covalently attached to lipid molecules in the membrane (GPI-anchored proteins)
• Peripheral membrane proteins are loosely associated with the membrane surface
  • Interact through electrostatic interactions or hydrogen bonding with lipid head groups or integral membrane proteins
  • Can dissociate from the membrane under certain conditions (changes in pH, ionic strength)

Functional roles of membrane proteins

• Transport proteins facilitate the movement of ions and molecules across the membrane
  • Channels allow passive diffusion of specific ions or small molecules (potassium channels, aquaporins)
  • Carriers actively transport substrates against their concentration gradient using energy (ATP) (sodium-potassium pump, glucose transporters)
• Receptor proteins bind to specific ligands and initiate intracellular signaling cascades
  • Ligands include hormones, neurotransmitters, and growth factors (insulin receptor, nicotinic acetylcholine receptor)
  • Binding induces conformational changes that trigger downstream signaling events (activation of G proteins, phosphorylation cascades)
• Enzymatic proteins catalyze chemical reactions at the membrane surface or within the lipid bilayer
  • Examples include phospholipases, proteases, and glycosyltransferases (phospholipase A2, γ-secretase)
  • Reactions can modify lipids, proteins, or other molecules in the membrane or extracellular space
• Cell adhesion proteins mediate cell-cell and cell-matrix interactions
  • Integrins link the extracellular matrix to the cytoskeleton and facilitate cell migration and signaling (α4β1 integrin)
  • Cadherins form homophilic interactions between adjacent cells and maintain tissue integrity (E-cadherin)
• Structural proteins maintain the shape and stability of the membrane
  • Cytoskeletal anchors connect the membrane to the cytoskeleton (ankyrin, spectrin)
  • Scaffolding proteins organize and stabilize membrane protein complexes (PSD-95 in postsynaptic densities)

Structure-function relationships of membrane proteins

Hydrophobic regions and membrane interactions

• Hydrophobic regions of integral membrane proteins interact with the hydrophobic core of the lipid bilayer
  • Composed of α-helices or β-barrels
  • α-helical transmembrane segments are common in receptors and transporters (rhodopsin, glucose transporters)
  • β-barrel structures are found in porins and some enzymes (OmpF porin, VDAC)
• Hydrophilic regions face the aqueous environment on either side of the membrane
  • Contain charged and polar amino acids that interact with water and other molecules
  • Form the functional domains of the protein (ligand-binding sites, catalytic sites)

Protein topology and orientation

• The arrangement and number of transmembrane segments determine the overall topology and orientation of the protein
  • Single-pass proteins have one transmembrane segment (receptor tyrosine kinases, glycophorin A)
  • Multi-pass proteins have multiple transmembrane segments (GPCRs, ion channels)
• The orientation of the protein (N-terminus and C-terminus on opposite sides of the membrane) is determined by the distribution of charged residues in the transmembrane segments
  • Positive-inside rule: positively charged residues are more abundant on the cytoplasmic side of the membrane

Amino acid sequence and tertiary structure

• The specific amino acid sequence and tertiary structure of a membrane protein dictate its function
  • Ion channels have selectivity filters formed by specific amino acid residues that determine ion selectivity (potassium channels, sodium channels)
  • Enzymes have catalytic sites with specific amino acid arrangements that facilitate substrate binding and catalysis (acetylcholinesterase)
• Post-translational modifications can modulate the function and stability of membrane proteins
  • Glycosylation adds carbohydrate moieties to specific residues and can affect protein folding, stability, and interactions (N-linked glycosylation of influenza hemagglutinin)
  • Phosphorylation of specific residues can regulate protein activity and interactions (phosphorylation of GPCRs by GRKs)

Conformational changes and protein dynamics

• Conformational changes in membrane proteins are crucial for their function
  • Ligand binding or changes in membrane potential can induce conformational changes (opening and closing of ion channels, activation of GPCRs)
  • Conformational changes can expose or hide functional sites, modulate interactions with other proteins, or alter the protein's activity
• Membrane proteins are dynamic and can undergo various movements
  • Lateral diffusion within the plane of the membrane (diffusion of receptors in the plasma membrane)
  • Rotation around the axis perpendicular to the membrane (rotation of ATP synthase during catalysis)
  • Conformational fluctuations that are important for protein function (gating of ion channels, alternating access mechanism in transporters)

Membrane protein targeting and insertion

Signal recognition and targeting

• The signal recognition particle (SRP) recognizes and binds to the signal sequence of nascent membrane proteins as they emerge from the ribosome
  • Signal sequences are hydrophobic stretches of amino acids that target proteins to the ER membrane
  • SRP binding to the signal sequence pauses translation and targets the ribosome-nascent chain complex to the ER membrane
• The SRP-ribosome complex is targeted to the SRP receptor on the ER membrane
  • The SRP receptor is a heterodimeric protein complex that binds to the SRP and facilitates the transfer of the nascent protein to the translocon complex
  • GTP hydrolysis by the SRP and its receptor releases the SRP and allows translation to resume

Translocon-mediated insertion

• The translocon complex, composed of Sec61 proteins, forms a channel that allows the insertion of the nascent membrane protein into the ER membrane
  • The Sec61 complex is a heterotrimeric protein complex that forms a protein-conducting channel across the ER membrane
  • The ribosome binds to the cytosolic side of the translocon, and the nascent protein is threaded through the channel into the ER lumen or lipid bilayer
• The orientation of the membrane protein is determined by the distribution of charged residues in the signal sequence and the presence of stop-transfer sequences
  • The signal sequence is cleaved off by signal peptidase after insertion into the ER lumen
  • Stop-transfer sequences are hydrophobic segments that halt translocation and anchor the protein in the membrane
  • The orientation of the protein (Ncyt/Cexo or Nexo/Ccyt) is determined by the charge distribution of the flanking regions around the transmembrane segments

Folding and post-translational modifications

• Chaperone proteins assist in the folding and assembly of membrane proteins within the ER lumen
  • BiP (binding immunoglobulin protein) is an Hsp70 chaperone that binds to hydrophobic regions of unfolded proteins and prevents aggregation
  • Calnexin and calreticulin are lectin chaperones that bind to glycosylated proteins and promote proper folding
• Post-translational modifications occur in the ER and contribute to the proper folding and stability of membrane proteins
  • N-linked glycosylation adds oligosaccharides to asparagine residues in the consensus sequence Asn-X-Ser/Thr
  • Disulfide bond formation between cysteine residues stabilizes the tertiary structure of membrane proteins
  • Glycosylphosphatidylinositol (GPI) anchors are added to the C-terminus of some proteins and anchor them to the extracellular leaflet of the membrane

Techniques for studying membrane proteins

Structural biology techniques

• X-ray crystallography involves the crystallization of purified membrane proteins and the analysis of the diffraction patterns generated by X-rays to determine the atomic structure
  • Requires large amounts of pure, homogeneous protein and the formation of well-ordered crystals
  • Has been used to determine the structures of various membrane proteins (potassium channels, GPCRs)
• Cryo-electron microscopy (cryo-EM) enables the visualization of membrane proteins in their native lipid environment
  • Samples are rapidly frozen in liquid ethane to preserve their native structure
  • Electron microscopy is used to image the frozen samples and generate 3D reconstructions
  • Has revolutionized the field of membrane protein structural biology due to its ability to study large, dynamic, and heterogeneous protein complexes (respiratory complexes, ion channels)
• Nuclear magnetic resonance (NMR) spectroscopy provides information on the dynamic structure and interactions of membrane proteins in solution
  • Requires isotopically labeled (15N, 13C) protein samples and measures the magnetic properties of atomic nuclei
  • Can provide information on protein dynamics, ligand binding, and protein-protein interactions
  • Limited to relatively small membrane proteins due to the complexity of the spectra

Fluorescence and electrophysiological techniques

• Fluorescence spectroscopy techniques can be used to study the dynamics and interactions of membrane proteins in living cells
  • Förster resonance energy transfer (FRET) measures the distance-dependent energy transfer between two fluorophores and can be used to study protein-protein interactions and conformational changes (GPCR activation, protein oligomerization)
  • Fluorescence recovery after photobleaching (FRAP) measures the lateral diffusion of fluorescently labeled proteins in the membrane and can provide information on protein mobility and interactions (diffusion of receptors in the plasma membrane)
• Electrophysiological techniques allow the measurement of ionic currents through individual ion channels in real-time
  • Patch-clamp recording involves the formation of a high-resistance seal between a glass micropipette and the cell membrane, allowing the measurement of currents through single channels or whole cells
  • Voltage-clamp and current-clamp techniques can be used to study the gating properties and kinetics of ion channels (voltage-gated sodium channels, ligand-gated ion channels)

Biochemical and computational approaches

• Biochemical assays provide insights into the function and interactions of membrane proteins
  • Ligand binding assays measure the affinity and specificity of protein-ligand interactions using radiolabeled or fluorescently labeled ligands (radioligand binding assays for GPCRs)
  • Enzyme kinetics experiments measure the catalytic activity of membrane-bound enzymes and can provide information on substrate specificity and inhibition (kinetics of acetylcholinesterase)
  • Cross-linking experiments use bifunctional reagents to covalently link interacting proteins and can provide information on protein-protein interactions and oligomeric states (cross-linking of GPCR dimers)
• Computational modeling and simulation approaches can complement experimental data and provide a detailed understanding of membrane protein dynamics and interactions
  • Molecular dynamics simulations use classical mechanics to simulate the motions of atoms and molecules over time and can provide insights into protein dynamics and ligand binding (simulations of ion channel gating, lipid-protein interactions)
  • Homology modeling and threading techniques can be used to predict the structure of membrane proteins based on the structures of related proteins (modeling of orphan GPCRs)
  • Docking simulations can be used to predict the binding mode and affinity of small molecule ligands to membrane protein targets (virtual screening of GPCR ligands)