#membrane proteins

Basics of Cell Biology

Which cellular structure is associated with the Freeze-Fracture technique?

Freeze-fracture technique, developed by Branton and refined by Pinto da Silva, revolutionized visualization of membrane interior by physically splitting frozen lipid bilayers. Live cells are rapidly frozen in liquid propane at -180C, fractured with cold knife under high vacuum, fracture plane preferentially passes through hydrophobic interior of membranes where van der Waals forces are weakest, splitting bilayer into exoplasmic and protoplasmic leaflets exposing intramembrane particles representing integral proteins including band 3 anion exchanger, aquaporins, connexin gap junction hexamers, and rhodopsin. Platinum-carbon replica shadowing captures topography, original biological material dissolved with acid, replica examined by transmission EM revealing random or crystalline particle distribution, quantifiable density changes upon hormonal stimulation. While mitochondria cristae, nuclear pores, and even ribosome organization can be studied, classic application was plasma membrane demonstrating proteins embedded within lipid matrix, supporting Singer-Nicolson fluid mosaic, quantifying tight junction strand complexity, and visualizing exocytosis fusion rosettes, establishing direct structural evidence for mosaic protein arrangement.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 10: Freeze-Fracture and Membrane Proteins.

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Properties of Plasma Membrane

Which of the following statements about membrane proteins is true?

Fluid mosaic model does not imply unrestricted random diffusion all components; regulated anchoring is prevalent and functionally critical. Many membrane proteins undergo co translational or post translational lipidation targeting membranes microdomains. N myristoylation by NMT attaches fourteen carbon myristate via amide to N terminal glycine exposing after methionine removal in Src Yes Lck non receptor kinases localizing to plasma membrane rafts. S palmitoylation by DHHC PAT family forms reversible thioester on cysteine in Ras PSD95 SNAP25 enhancing raft partitioning and stabilizing membrane orientation regulated by APT thioesterases. Prenylation farnesyl fifteen or geranylgeranyl twenty via FTase GGTase on CaaX cysteine of Rab Rho ensures transient association regulated by GDI. GPI anchoring in ER involves transamidase replacing C terminal signal with ethanolamine phosphate mannose three glucosamine PI core tethering alkaline phosphatase Thy1 CD59. Cytoskeletal fencing ankyrin spectrin actin further corrals diffusion. Hence some proteins tethered by lipids distribution patchy thickness regulated hydrophobic mismatch rather than protein abundance alone concept essential for exams understanding membrane organization beyond fluid mosaic simplicity.

Ref: Lingwood & Simons, Science 2010, Lipidation membrane domains anchoring. Alberts Chapter 10.

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Plasma Membrane Proteins

Which component helps in proper folding of β-barrel membrane proteins?

Biogenesis of beta-barrel outer membrane proteins requires specialized folding and insertion systems preventing aggregation of beta-strands rich in hydrophobic residues. After Sec dependent translocation across inner membrane nascent unfolded chain enters periplasm where holdase chaperones SurA peptidyl prolyl isomerase and Skp trimeric cavity chaperone maintain unfolded state, DegP protease quality control degrades misfolded species. Targeting to BAM beta-barrel assembly machinery comprising central BamA sixteen-strand beta-barrel itself with five N-terminal POTRA domains that bind substrates and lipoproteins BamB-E essential for outer membrane permeability. BamA lateral gate between strand one and sixteen separates allowing substrate barrel insertion via hybrid barrel mechanism where membrane thins locally. In mitochondria analogous SAM complex Sam50 plus small TIM chaperones assemble VDAC Tom40. In chloroplasts OEP80 mediates. ATP not directly utilized by BAM but by SecA pushing. Actin filaments eukaryotic cortical cytoskeleton not present bacterial envelope, hydropathy index predictor not folding factor. Thus chaperones and assembly complexes indispensable for proper beta-barrel folding and membrane integration ensuring channel function.

Ref: Wimley, The Versatile Beta-Barrel Membrane Protein Folding, Curr Opin Struct Biol 2003.

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Plasma Membrane Proteins

Which of the following statements about membrane proteins is FALSE?

Fluid mosaic model postulates membrane proteins diffuse laterally, associate with cytoskeleton, function as enzymes receptors carriers and structural anchors. Transverse movement of proteins between leaflets termed flip-flop faces enormous energetic barrier because large hydrophilic extramembranous domains containing charged residues lysine arginine aspartate glutamate would need dehydration and passage through hydrocarbon core twenty to thirty angstroms low dielectric constant incurring free energy tens kilocalories plus disruption of established orientation determined during biogenesis at Sec61 translocon following signal anchor and positive-inside rule where cytosolic Lys Arg enriched. Experimental tracking using fluorescent labeled Band 3 or glycophorin in erythrocytes shows no spontaneous flipping over days; turnover occurs via endocytosis lysosomal degradation and recycling rather than flipping. Phospholipid flip-flop catalyzed by flippases floppases scramblases half-life minutes hours regulated. Therefore assertion that membrane proteins can flip between leaflets without assistance is false highlighting conservation of topology and asymmetry essential for vectorial transport and signaling orientation preserved throughout lifetime.

Ref: Alberts et al., Molecular Biology of the Cell, 6th ed., Chapter 10, Membrane Protein Topology and Asymmetry.

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Plasma Membrane Proteins

Which of the following proteins uses a GPI anchor to attach to the membrane?

Classification of lipid-anchored proteins includes GPI anchor addition in endoplasmic reticulum lumen via multi-step pathway conserved across eukaryotes. GPI precursor synthesis starts on cytoplasmic face adding N-acetylglucosamine to phosphatidylinositol by PIG-A, deacetylation by PIG-L, flipping to luminal side by flippase, addition of three mannose residues from dolichol-phosphate-mannose by PIG-M PIG-V PIG-B, addition of phosphoethanolamine by PIG-O PIG-F, final transfer en bloc to C-terminal GPI signal peptide cleavage site containing omega site small residues by GPI transamidase comprising PIG-K catalytic cysteine protease forming amide bond between protein carboxyl and phosphoethanolamine linked to terminal mannose glycan mannose core attached via glucosamine to inositol diacylglycerol embedding in outer leaflet. Proteins remain tethered high lateral mobility localized lipid rafts cleavable by phospholipase C or D releasing soluble forms. Examples hydrolytic enzymes alkaline phosphatase ecto-5 nucleotidase, adhesion molecules Thy-1 CD90, prion PrPc, regulators CD55 CD59. Multipass proteins aquaporin six helices Band 3 fourteen helices GPCR seven helices cross bilayer via peptide helices not lipid tether, distinguishing GPI-linked class.

Ref: Mayor and Riezman, Sorting GPI-Anchored Proteins, Nature Reviews Mol Cell Biol 2004.

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Plasma Membrane Proteins

Which RBC membrane protein is essential for Cl⁻ transport?

Physiology of carbon dioxide delivery relies on chloride bicarbonate exchange across erythrocyte membrane rather than G protein signaling. Authentic transporter Band 3 anion exchanger 1 SLC4A1 abundant integral protein fourteen transmembrane helices catalyzing electroneutral one to one antiport of chloride for bicarbonate at high turnover supporting conversion of CO2 to bicarbonate via carbonic anhydrase for plasma carriage known as chloride shift Hamburger phenomenon. At pulmonary capillaries exchange reverses enabling CO2 exhalation. GPCR family members are seven-transmembrane receptors coupling to heterotrimeric G proteins generating second messengers cAMP inositol trisphosphate diacylglycerol activating kinases but not transporting chloride as primary mode. Glycophorin major single-pass sialoglycoprotein provides glycocalyx negative charge and pathogen receptor lacking transport activity, spectrin filamentous tetrameric cytoskeletal protein maintains biconcave elasticity lacking ion translocation. Therefore scientifically validated chloride transporter is Band 3 although answer choice listed as GPCR in this dataset reflects probable curation inconsistency noted here, emphasizing need to recognize Band 3 as genuine chloride shift mediator for acid base and gas transport physiology.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 15, AE1 Band 3 Chloride-Bicarbonate Exchanger.

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Plasma Membrane Proteins

Which of the following properties affects membrane protein mobility?

Diffusion behavior of membrane proteins governed by hydrodynamic and cytoskeletal factors. Saffman-Delbruck model relates diffusion coefficient D to membrane thickness h viscosity mu m and protein radius r through D proportional to natural log of membrane to protein size. Number of transmembrane domains increases effective radius and frictional drag because each helix engages annular lipids diffusing as complex and more extensive hydrophobic surface contacts viscous acyl chains. Single-pass glycophorin diffusion coefficient about five times ten to minus nine centimeters squared per second faster than seven-pass GPCR about one times ten to minus nine and fourteen-pass Band 3 about zero point five. Additional slowing originates from hop diffusion model developed by Kusumi showing actin cytoskeleton fences create compartments eighty to two hundred nanometers where proteins diffuse rapidly then hop between compartments limited by transient fence and picket interactions. Binding to immobilized ankyrin spectrin network reduces mobile fraction to fifty percent. Size of extracellular loops influences extracellular matrix interactions but less than transmembrane count, hydropathy index predicts insertion, pH charge limited effect. Number of transmembrane domains therefore primarily affects mobility.

Ref: Kusumi et al., Paradigm Shift in Membrane Protein Diffusion and Corral Model, Annu Rev Biophys.

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Plasma Membrane Proteins

Which of the following is a common feature of transmembrane β-barrel proteins?

Beta-barrel membrane proteins display characteristic functional and structural attributes correlating with aqueous pore formation. Architecture comprises even number amphipathic beta-strands eight to twenty four organized antiparallel hydrogen bonded sheet closed cylindrically first strand bonded last strand strands tilted thirty to sixty degrees relative to barrel axis. Even residues hydrophobic facing lipid core, odd hydrophilic facing lumen generating water-filled channel diameter about seven to fifteen angstroms variable. Extracellular loops often long folding into pore constriction loop three in OmpF forming eyelet governing size exclusion approximately six hundred daltons cutoff and charge selectivity via acidic glutamate aspartate and basic arginine lysine lining. These porins facilitate passive diffusion of small polar nutrients including sugars, amino acids, phosphate, nucleosides, and antibiotics down concentration gradient rates up to million per second without energy, essential for Gram-negative bacterial survival. Trimeric assembly stabilizes. Eukaryotic mitochondria VDAC transports ATP ADP metabolites. Alpha-helical transporters active secondary carriers elsewhere, beta-barrels exclusively allow diffusion of small polar molecules through relatively nonselective aqueous pore.

Ref: Nikaido, Molecular Basis of Bacterial Outer Membrane Permeability, Microbiol Mol Biol Rev 2003.

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Plasma Membrane Proteins

Which technique can be used to determine if a membrane protein is inside-out or right-side-out?

Determining orientation of membrane proteins after reconstitution into artificial vesicles essential for establishing vectorial transport competence. Proteoliposomes generated by removing detergent via dialysis may incorporate proteins randomly right-side-out exposing original extracellular domains outward or inside-out exposing cytoplasmic domains outward leading to mixed activity. Sidedness assay exploits membrane impermeability of antibodies one hundred fifty kilodaltons IgG unable to cross sealed bilayer. Intact vesicles incubated with monoclonal antibody against known extracellular epitope such as glycophorin N-terminus or Band 3 loop three undergo binding exclusively when epitope faces external medium detectable by secondary gold-conjugated antibody electron microscopy, flow cytometry, or after pelleting by immunoblotting pellet. Permeabilization with low concentration Triton X-100 zero point one percent exposes total epitopes providing total protein control. Alternative protease protection where extracellular trypsin cleaves only outward loops. Hydropathy plots predict number of spans not orientation in liposomes, SDS-PAGE separates subunits irrespective of sidedness, FRAP measures lateral mobility. Thus antibody labeling using impermeant probe provides definitive sidedness determination for inside-out versus right-side-out vesicles.

Ref: Rohde et al., Determination of Membrane Protein Orientation by Antibody Labeling, J Mol Biol Methods.

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Plasma Membrane Proteins

Which of the following sequences is highly conserved in Aquaporins?

Highly conserved sequence motif in aquaporin family defines aqueous pore architecture and selectivity. Loop B half-helix and Loop E half-helix each dip into membrane from opposite sides each containing invariant tripeptide Asn-Pro-Ala forming two NPA boxes meeting at center of channel creating electrostatic barrier and orienting water molecules in opposite NPA asparagine carbonyl hydrogen bond donors. Proline introduces kink allowing asparagine side chain to project into pore, alanine stabilizes packing between half-helix and transmembrane helices. Structural alignments show two NPA motifs generate constriction forcing water reorientation one hundred eighty degrees disrupting continuous hydrogen bonded chain required for proton hopping via Grotthuss mechanism, while arginine selectivity filter excludes ions. Mutation NPA to NPG or NPS in aquaporin-2 causes nephrogenic diabetes insipidus reducing water permeability and autosomal dominant cataract. Database search using NPA signature identifies over three hundred aquaporin orthologs across Bacteria E coli aquaporin Z, Archaea, plants tonoplast intrinsic proteins, mammals, distinguishing water channels from glycerol facilitators and ion channels lacking motif.

Ref: Fujiyoshi et al., Structure of Aquaporin Water Channel and NPA Motif, Nature 2002.

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Plasma Membrane Proteins

Which statement about Glycophorin A is correct?

Detailed topology of glycophorin A major human erythrocyte sialoglycoprotein clarifies single-pass characteristics. Gene GYPA on chromosome four encodes one hundred fifty residues including cleavable signal peptide. Mature protein seventy kilodalton apparent due to extensive glycosylation: seventy residue extracellular domain heavily O-glycosylated fifteen O-linked tetrasaccharides NeuAc alpha2-3 Gal beta1-3 GalNAc plus one N-linked complex chain adding sialic acid dense negative charge defining MNS blood group M and N antigens and preventing rouleaux formation. Hydrophobic anchor residues seventy three to ninety five forms nineteen residue alpha helix containing GXXXG dimerization motif facilitating high affinity dimerization free energy minus twelve kilocalories measured by analytical ultracentrifugation, widely used model for helix-helix interactions. Cytosolic tail thirty six residues acidic interacts with protein 4.1R FERM domain linking to spectrin actin junctional complex near actin protofilament, regulating lateral mobility and mechanosensing. Unlike fourteen-pass Band 3 anion transporter, glycophorin does not transport chloride nor span multiple times nor bind actin directly via actin binding domain, therefore single-pass nature best describes architecture and enables studies of membrane protein folding energetics and Plasmodium invasion receptor function.

Ref: Alberts et al., Molecular Biology of the Cell, 6th ed., Chapter 10, Glycophorin A Structure and Topology.

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Plasma Membrane Proteins

Which experimental technique is used to study the lateral diffusion of membrane proteins?

Fluid mosaic behavior requires quantitative assessment of lateral mobility within plane. Fluorescence recovery after photobleaching FRAP technique labels membrane proteins via GFP fusion or fluorescein-conjugated antibodies, uses focused high-intensity laser pulse to bleach micrometer spot irreversibly photodestroying fluorophores, then monitors fluorescence intensity return as unbleached molecules diffuse from surrounding membrane into bleached area over seconds to minutes recorded by photomultiplier or camera. Recovery kinetics fitted to diffusion equation yields effective diffusion coefficient typically ten to minus nine to ten to minus eleven centimeters squared per second and mobile fraction reflecting cytoskeleton immobilized fraction. Controls using latrunculin depolymerizing actin increase mobility demonstrating corral model hop diffusion between actin fences eighty nanometers. Cholesterol depletion disrupts lipid rafts accelerating recovery. In contrast X-ray crystallography reports static atomic coordinates of crystallized molecules, Western blotting detects specific protein presence without dynamics, SDS-PAGE separates denatured subunits by mass. Therefore FRAP remains classical technique to study lateral diffusion of membrane proteins and lipids in live cells.

Ref: Axelrod et al., Mobility Measurement by FRAP, Biophysical Journal 1976.

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