#biochemistry question

Lipid Link Protein

How many carbon atoms are present in the farnesyl lipid modification?

Prenylation adds isoprenoid lipids derived from mevalonate pathway to cysteine in CAAX motif. Two major forms exist: 15-carbon farnesyl comprising three isoprene units and 20-carbon geranylgeranyl comprising four isoprene units. Farnesyl pyrophosphate synthesized from isopentenyl pyrophosphate and dimethylallyl pyrophosphate serves donor for farnesyltransferase FTase, while geranylgeranyl pyrophosphate serves donor for geranylgeranyltransferase I and II. Farnesyl group contains 15 carbon atoms arranged as 2E,6E-farnesyl with three double bonds providing hydrophobic moiety about 10 angstrom len

Ref: Zhang & Casey, Annu Rev Biochem 1996, Prenyl groups; Wang & Casey 2016, Prenylation.

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Lipid Link Protein

Which lipid-linked protein modification is not reversible?

Lipid modifications vary in stability and enzymatic reversibility enabling dynamic membrane association. Palmitoylation S-acylation via DHHC family palmitoyl acyltransferases forming thioester linkage can be removed by acyl protein thioesterases APT1, APT2 and ABHD17 family allowing cycles of membrane binding and cytosolic redistribution crucial for neuronal signaling proteins like PSD-95 and H-Ras. Prenylation thioether linkage is irreversible chemically stable requiring proteolysis for reversal; however protease cleavage of prenylated C-terminus does not regenerate unmodified cysteine. GPI a

Ref: Resh, Nat Chem Biol 2013, Reversibility of lipid modifications; Chamberlain & Shipston, Physiol Rev 2015.

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Lipid Link Protein

Which of the following statements is true regarding GPI-anchored proteins?

Glycosylphosphatidylinositol anchored proteins are synthesized in endoplasmic reticulum via en bloc transfer of preassembled GPI moiety to C-terminus after cleavage of hydrophobic signal, trafficked through Golgi where lipid remodeling occurs, and delivered to extracellular leaflet of plasma membrane where they reside in ordered lipid rafts enriched in cholesterol and sphingolipid. Attachment chemistry involves phosphoethanolamine bridging peptide carboxyl to glycan tetramannosyl glucosamine core linked via phosphodiester to phosphatidylinositol lipid with two fatty acyl chains inserted into o

Ref: Kinoshita & Fujita, Biochim Biophys Acta 2016, GPI-anchored proteins localization and bonds.

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Lipid Link Protein

What is the critical micelle concentration (CMC)?

Amphipathic detergents in aqueous solution exhibit concentration-dependent self-assembly thermodynamically governed by hydrophobic effect. Monomers exist freely at low concentration with hydrophobic tails partially exposed to water energetically unfavorable. Upon reaching threshold called critical micelle concentration CMC, tail sequestration drives spontaneous formation of spherical or ellipsoidal aggregates called micelles where hydrophobic tails cluster inward forming oily core while hydrophilic heads face water outward, minimizing free energy. CMC is characteristic for each detergent influ

Ref: Garavito & Ferguson-Miller, J Biol Chem 2001, Detergent micellization; le Maire et al., 2000.

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Lipid Link Protein

Which detergent is most likely to maintain a protein’s native structure during extraction?

Extraction of integral membrane proteins for functional study demands detergent that disrupts lipid bilayer but avoids complete unfolding of polypeptide. Ionic strong detergents like SDS bind cooperatively to protein backbone at about one SDS per two amino acids, imparting negative charge, disrupting secondary structure and denaturing enzymes, suitable for electrophoresis but not activity assays. Non-ionic detergents Triton X-100, Nonidet P-40, dodecyl-beta-D-maltoside DDM and octyl glucoside are milder; they replace lipids around transmembrane helices forming protein-detergent micelles, maint

Ref: Privé, Methods 2007, Detergent choice; Garavito & Ferguson-Miller, J Biol Chem 2001.

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Lipid Link Protein

Which of the following detergents is non-ionic?

Detergents classify based on head group charge: ionic anionic like sodium dodecyl sulfate SDS and sodium deoxycholate, cationic like hexadecyltrimethylammonium bromide and quaternary ammonium compounds, and non-ionic bearing uncharged polar head like polyoxyethylene ethers. Triton X-100 octyl phenol ethoxylate contains hydrophilic polyethylene oxide chain and hydrophobic tert-octylphenyl group without ionizable group, thus non-ionic and relatively mild preserving native protein interactions. SDS possesses negatively charged sulfate, strongly denatures proteins by binding hydrophobic regions an

Ref: le Maire et al., Biochim Biophys Acta 2000, Detergent classification; Privé, Methods 2007.

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