Protein-Ligand Binding_L1-2 Flashcards
What are the desired traits of the bioconjugate techniques?
(1) Add new functionalities to proteins with reagents that:
1. Covalently modify biomolecules in specific, desired sites
2. Produce minimal side reactions
3. Work in ‘gentle’ solution conditions (i.e. aqueous, near neutral pH)
4. Are safe to use outside ‘chemistry’ labs: do not require equipments like fume cupboard and PPE
5. Are commercially available
6. Have been approved in different systems – research papers
(2) Modification of cysteines, primary amines, carboxylic acids:
1. Crosslinking of macromolecules for structural/functional studies
2. Label transfer - Crosslinking for identification of PPIs
3. Cell surface crosslinking
4. Labelling for fluorescence detection
How is the amine modified?
(1) amine conversion to NHS esters(N-hydroxy succinimide)
<1> Assays:
1. Carry out reactions at pH 7.2-8.5 for 0.5-4h at RT or 4ºC.
2. Exclude buffers containing primary amines (e.g. Tris).
3. Use Tris or glycine buffer to stop the reaction: as the Tris competes against the primary amines, in some cases, stopping the esterification is favored.
4. Hydrophobic crosslinker - able to cross membranes
<2>reagent: NHS ester+ protein-bound primary amine
product: NHS+amide in stable conjugates formed from the attack of the amine to the ester bond
(2) Modification of amines – sulfo-NHS:
<1> The –SO3 (sulfonate) group increases the water solubility of compounds containing them and has no effect on the reaction chemistry.
<2> Sulfonate also prevents sulfo-NHS crosslinkers from permeating cell membranes - use in cell surface crosslinking methods.
<3> reagent: amine-reactive sulfo-NHS ester conjugated with the protein 1 + protein 2-bound primary amine: the addition of sulphate does not affect the primary amide attack
product: two proteins joined by amide bond+ sulfo-NHS molecule
How to generate the biotinylated protein based on the primary amine modification?
The incoming sulfo-NHS-LC-biotin, a molecule formed from the the sulfo-NHS and biotin joined by the spacer, chemically converts the primary amine bound to the protein to the amide bond, leavig the sulfo-NHS as the leaving group.
How is the cysteine modified?
(1) Maleimides target thiol groups (-SH) to form thioethers: -S-
(2) Select residues in peptides or surface residues in proteins for X → C substitutions.
(3) Assay: Exclude thiol-containing compounds, e.g. b-mercaptoethanol (BME), dithiothreitol (DTT) and Tris(2 carboxyethyl)phosphine (TCEP) from rxn buffers - they compete for coupling sites.
(4) Maleimide reagent+sulfhydryl on protein-> stable conjugate by the thioether bond(pH 6.5/7.5)
* The thiol-containing compounds are ordered in an increase in the reducing power
elaborate on the property of maleimides – crosslinking of superoxide dismutase
(1) DTME(dithio-bismaleimidoethane) is the dimer formed from two maleimidethanes.
(2) DTME cross-linked SOD1(superoxide dismutase) is stabilised by the crosslinking of the two attached maleimidethanes(at the cysteine site) as a disulphide bridge, the two proteins are separated by equal distance from the centre and the whole molecule is symmetric. The central disulphide bond can be reduced but not the ones at the ends.
How is the fluorescent label be added on the protein?
Maleimides facilitates the addition of fluorescent labels to the target protein: after the maleimide has already been added on the protein in the cystine-modification process, the fluorescein is fused to the maleimide.
By combing the maleimides and sulfo-NHS, how could the enzyme-antibody conjugates be generated?
(1) Reaction scheme for labeling reduced Ab fragments with maleimide-activated enzymes
(2) enzyme with its end being primary amine reacts with the sulfo-MSCC-crosslinker to result in the maleimide-activated enzyme with the reactive section which is ready for the attack from the sulfhydryl-activated antibodies, the enzyme-protein conjugates are thereby produced.
what is the photoaffinity labeling(PAL)
(1) Photo-reactive reagents are chemically inert compounds - reactive when exposed to UV or visible light
(2) Proteins modified with PAL reagents can covalently bind their target(s) after activation by light
(3) Unlike other methods, PAL can label prey protein in low abundance and low affinity proteins
(4) Active field of research, with increasing number of commercially available compounds
What are the ideal properties of PAL?
(1) Stable in the dark in a range of pHs
(2) Activation at wavelengths that do minimal damage to biomolecules(prey protein)
(3) Still capable to generate intermediates that react and form stable adducts
(4) Minimal side reactions
What are the three important components in PAL?
(1) photoactive moiety: permanent, photo-inducible, able to attach on the secondary target
(2) affinity/specificity unit: bind to the prey protein
(3) identification/reporter tag: e.g. fluorescent dye, radioisotope or partner for a specific binding event
How does sulfo-SBED play its role in bait protein trapping site?
Sulfo-SBED is composed of three sections, each serving different functions: the biotin(purification), photoactivatable aryl azide(react with the primary amide after bing photoreactive) and sulphate NHS. The linker between the sulphate NHS and aryl azide contains a cleavable disulphide bond which can be easily formed or reduced. The zryl azide is promoted to be active with the photon interaction, forming short-lived nitrens which can undergo the ring expansion and react with the primary amides. The amide trapped here is the interacting protein, known as prey protein used to explore the protein-protein interaction. The sulpahte NHS can also react with the primary amide at pH 7 to 9. The captured protein at NHS site is refered as the bait protein which interacts with the prey protein. Once the protein-protein interaction is estabolished and stabilised, the disulphide bond in the linker can be broken, the prey-bait complex is covalently attached to the biotin which acts as the purification enzymatic tag in the pulldown experiment. The streptavidin is fixed on the beads surface in the purification.
What is the CAN/Nup 214 NPC-filament protein’s importance in the cells in terms of tackling the viral infection
(1) Adenovirus type 2 (Ad2) imports its DNA genome through the nuclear pore complex (NPC) of cells in interphase for viral production.
(2) The NPC-filament protein CAN/Nup214 acts as a docking site for the incoming Ad2 capsid, CAN/Nup 214 recognises the capsid Ad2 protein for DNA attchament to the the viral particle formation
What is the workflow of PAL?
(1) sulfo-SBED binds to the bait protein under the UV light induction
(2) a range of prey protein can be selected by the bait protein to form triplex
(3) the triplex can be reduced to cleave the disulphide , weakly bound prey proteins are eliminated by the step.
(4) purification by the streptavidin
(5) proteolytic cleavage and run the MS
(6) peptide-protein identification
What is the diazirines and how can its property be used?
(1) The most commonly used photoaffinity group - excellent chemical stability
(2) Highly resistant towards e.g. temperature, acidic and basic conditions
(3) Activated at 350–355 nm (reduces potential to damage biomolecules): 350-355 nm is the range indicating the soft UV light
(4) Carbene intermediate is extremely reactive and has a short half-life, reacting quickly with the compound in close approximity.
(5) diazirine reagent containing the doubly bonded nitrogen couple is excited by the 350nm UV light, nitrogen is released and carbene intermediate is formed. Protein comes along to form stable conjugate with the radical carbene.
What property of the diazirines can be applied to cultivate the photo amino acids?
(1) photo-Met and photo-Leu can be dissolved in the cell media for cell cultivation at 37 degrees, 5% CO2 in 24 hours, cells acquire amino acid supply from the media and build up protein with the photoreactive amino acids
(2) cells then are explored to the UV light at 350nm. The processed cell clusters are harvested, lysed and the cell extraction is used as Western blot sample. Bands on the gel are revealed for the identification of the prey protein by using the antibodies, as well as the monomers which utilise the modified amino acid as building block.
