Plasma Membrane Proteins

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 protein is responsible for maintaining the biconcave shape of RBCs?

Maintenance of biconcave disc shape maximizing surface to volume ratio for deformability and gas exchange depends on membrane skeleton elasticity and organization. Spectrin consists of alpha two hundred eighty kilodalton and beta two hundred forty six kilodalton subunits each containing twenty triple-helical spectrin repeats of one hundred six residues forming elongated flexible anti-parallel heterodimer one hundred nanometers long, two heterodimers associate head to head via helical bundle forming tetramer about two hundred nanometers acting as entropic spring with persistence length about ten nanometers. Tetramers interconnected at junctional complexes comprising thirteen subunit short F-actin filaments capped by adducin and tropomodulin plus protein 4.1R dematin tropomyosin forming pseudohexagonal lattice of about thirty five thousand nodes per cell beneath lipid bilayer. Linkage to bilayer via ankyrin-Band 3 and protein 4.1R-glycophorin C ensures force transmission. Hereditary mutations in SPTA1 encoding alpha spectrin and SPTB encoding beta spectrin cause hereditary spherocytosis and elliptocytosis with fragile cells. GPCR seven-pass signaling and aquaporin channel not structural, confirming spectrin role maintaining biconcave geometry and mechanical elasticity under shear stress circulation.

Ref: Lux and Palek, Erythrocyte Membrane Skeleton and Spectrin Elasticity, Blood Cells.

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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 of the following describes the function of hydrophobic photoaffinity labeling?

Mapping membrane-embedded regions benefits from probes restricted to hydrophobic phase. Hydrophobic photoaffinity labeling utilizes synthetic phospholipid analogues bearing photoactivatable moieties including aryl azide generating nitrene upon UV about three hundred fifty nanometers, benzophenone generating ketyl radical, diazirine generating carbene highly reactive inserting into C-H bonds within three to five angstroms. Probes partition spontaneously into acyl chain region due to hydrophobicity, concentration in bilayer interior high. Upon irradiation during brief pulse, covalent adducts form between probe and amino acid side chains in contact with lipid-facing surfaces corresponding to transmembrane alpha helices or beta strands. Extramembranous extracellular and cytoplasmic domains exposed to aqueous phase remain unlabeled because probe absent there. After labeling, proteins digested with trypsin chymotrypsin, labeled peptides isolated by chromatography, residues identified by mass spectrometry sequencing defining membrane boundaries. Water soluble photoaffinity reagents label opposite. Technique historically mapped bacteriorhodopsin seven helices and glycophorin helix before crystallography, confirming transmembrane region identification approach essential for topology modeling.

Ref: Brunner, Photolabeling and Hydrophobic Core Mapping, Annu Rev Biochem 1993.

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Plasma Membrane Proteins

Which of the following molecules can be transported by Aquaporin?

Selectivity of aquaporin pores illustrates precise molecular sieving. Aquaporin-1 monomer six helical bundle creates hourglass pore length approximately twenty angstroms with two constrictions: extracellular aromatic arginine filter formed by Arg195 guanidinium, His180 imidazole, Phe56 phenyl providing size filter diameter two point eight angstroms allowing water kinetic diameter two point zero angstroms but excluding hydrated sodium diameter seven point two angstroms, glucose eight angstroms, urea larger, and electrostatic barrier preventing proton conductance via positive Arg repulsion breaking continuous water wire. Second NPA constriction at center forces water reorientation interrupting Grotthuss hopping. Water moves single file driven by osmotic gradient direction high to low chemical potential up to billions per second maintaining kidney proximal reabsorption ninety percent filtered water, red cell volume regulation, lung alveolar fluid clearance. Sodium requires ENaC epithelial sodium channel, glucose requires SGLT and GLUT transporters, ATP requires ABC transporters. Therefore water is uniquely transported molecule by classic aquaporins distinguishing from ionic and metabolite transporters with larger pores and coupled mechanisms.

Ref: Verkman et al., Aquaporin Water Channels – Physiology and Selectivity, Nature Reviews Mol Cell Biol.

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