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Protein-Ligand Interactions Flashcards

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1
Q

Characteristics of Techniques to measure protein-ligand interactions

A
  1. quantitative
  2. label free (not modifying surface or binding properties)
  3. true in solution
  4. sensitive with minimal sample size
  5. high-throughput
  6. atomic resolution (identify residues of the binding site)
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2
Q

Yeast 2 Hybrid

A
  • genetic screening technique
  • permits wide search for potential binding partners
  • links ligand proteins to their genes
  • allows identification of a ligand from a large library
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3
Q

Yeast 2 Hybrid Principle

A
  • exploits modular nature of gene activator proteins (eg. GAL4)
  • use recombinant DNA techniques to create two fusion contacts
    1. DBD-bait (protein of interest) in one plasmid
    2. Prey-AD (binding partner taken from library) in one plasmid
  • if the bait binds the prey the DBD and AD are reunited and switch on a reporter gene
    = binding drives RNApol transcription of reporter
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4
Q

Yeast 2 Hybrid Method

A
  • the prey: DNA coding for the AD fused to DNA coding for a selection of proteins using cDNA generated from mRNA extractions
  • can create a cell library of AD prey fusions but each yeast picks up only one prey molecule
  • select for the interactions
  • reporter gene allows selectivity (positive antibiotic selection)
  • the only colonies growing are those with the interaction
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5
Q

Yeast 3-Hybrid

A
  • protein:RNA interactions
  • same principle as Y2H
  • uses library of RNAs binding to a fused protein
  • This system also makes it possible to identify those regions of an RNA or protein that are required for a known interaction and to test the combinations of RNA and protein to confirm whether they interact in vivo.
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6
Q

Yeast 3 Hybrid Example

A
  • The first hybrid protein is made up of an RNA binding protein (RBD) fused to a DNA binding domain (DBD). The second fusion protein molecule contains a second RNA binding protein fused to the transcriptional activation domain (AD). The third hybrid part is an RNA molecule which bridges above two fusion proteins by providing two specific RNA targets for the RNA binding proteins. When this tripartite constituent forms at a promoter, the reporter gene is turned on, even transiently. And the expressed reporter products can be recognized by simple biochemical or phenotypic assays.
  • MS2 dimer is fused to the LexA DNA binding protein
  • MS2 binds to MS2 RNA connected to RNAX
  • the AD is fused to protein Y
  • if the protein binds to the RNA the activation domain causes reporter gene expression and positive selection
  • can also be used for RNA-RNA interactions using a RNA activator domain fused to a RNAY instead of protein
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7
Q

Improvements of Yeast 3 Hybrid

A
  • reduces false positives
  • stabilise stems of displayed RNA to avoid MS2 RNA interactions vs G-C clamp or the T cassette
  • dimerise the MS2 to increase RNA affinity
  • increase expression of Protein-AD fusion
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8
Q

Yeast 2/3 Hybrid Advantages

A
  1. scalable
  2. direct ID of interacting prey from DNA sequence of recovered colonies
  3. prone to false positives (preys binding nonspecifically causing activation)
  4. no affinity quantification
  5. yeast is a good model organism
    - just an indication of binding
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9
Q

GST PullDown

A
  • another fusion technique
  • Glutathione S-transferase fused to known bait
  • method relies on binding of GST to glutathione conjugated beads
  • used for proteins
  • need to purify the GST fusion and label the binding partner
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10
Q

GST Method

A
  • fuse GST with a protein
  • conjugate onto beads
  • bait will bind to the protein
  • wash away impurities and elute protein X with bound ligand
  • glutathione solution elutes fusion protein with its interacting partners
  • used mainly for confirmation of binding as you have to elute the protein complexes and analyse with SDS Page
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11
Q

GST Example Data

A
  • need a positive control to show all fragment are present
  • need negative control to show specific DBT binding to fragments
  • test for interaction of 5 different deletion mutants of protein X with GST-PTB protein
  • mutant proteins are radiolabelled and the gels are autoradiograms
  • the fragments which have bound to PTB-GST fusion will be eluted and ran on the gel to show which bind best/which are not present
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12
Q

GST Pros vs Cons

A
  1. quick and easy
  2. radiolabelling means the fusion protein is not seen
  3. not quantitative
  4. need controls
  5. GST-bait fusion susceptible to proteolysis
  6. larger amounts of target/prey proteins needed for identification
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13
Q

Gel Shift Assay

A
  • measures protein-NA interactions
  • DNA/RNA is radiolabelled (incorporate radioactive phosphate nucleotides during 3’ fill in reaction or by 5’ end labelling with radiolabelled ATP)
  • mix reactants (NA and protein) and run on native gel of acrylamid/agarose
  • negative charge of NA drives migration in the gel - analyse result on autoradiogram
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14
Q

Gel Shift Assay Results

A
  • as the protein concentration increases, the gel shows a stronger band of the RNA:protein complex higher up on the gel
  • denaturing gels break the interaction so the native gels must be used
  • can give evidence of stoichiometry (number of binding sites) based on number of bands in the gel as well as their affinity
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15
Q

Gel Shift Assay Pros vs Cons

A
  1. easy to visualise
  2. stoichiometric information
  3. not fast
  4. cannot measure dissociation constant
  5. weak interactions not measured
  6. can compare different proteins or different nucleic acids to the same protein easily (identify binding residues)
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16
Q

Enzymatic Tagging

A
  • biotinylation using E. Coli BirA
  • formation of amide bonds via thioester intermediates using sortase
  • tags covalently modify molecules in specific sites
  • produce minimal or no effect on the protein structure
  • work in gentle solution conditions
  • are widely available
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17
Q

Biotinylation

A
  • Avitag is a peptide substrate for the biotin ligase enzyme (BirA)
  • add the avitag sequence to the termini of a protein to make it a BirA substrate
  • use the ligase, ATP, and biotin to biotinylate protein-Avitag fusion
  • in vitro: purified BirA
  • in cell: co-express BirA with protein-Avitag fusion of interest and purify a biontinylated POI product
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18
Q

Avidin

A
  • biotin binding protein
  • found in egg white
  • glycosylated and 66 kDa
  • avidin-biotin complex is the strongest known non-covalent interaction between protein-ligand
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19
Q

Avidin Variants

A
  1. neutrAvidin: avidin process to remove carbs (less unspecific binding)
  2. streptavidin: bacterial homologue, non0glycosylated
  3. captavidin: reduced binding affinity for biotin. biotinylated substrates released at less harsh conditions than normal avidin needs
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20
Q

Biotin-Avidin Interaction

A
  • stable to extremes of pH, temperature, solvents, etc

- resistance to SDS-Page denaturation

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21
Q

Desthiobiotin

A
  • biotin analogue binding less tightly to biotin-binding proteins and easily displaced by biotin
  • displacement shown by using labelled antibodies in the cell
  • allows avidin purification and removal
  • contains an extra methyl group and a methyl replacement of a S in the ring
  • BirA evolved to use this as a substrate as well
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22
Q

Gram Negative vs Positive Cell Walls

A

Gram Negative: two lipid layers with peptidoglycan in the periplasmic space. uses disulphide bonds
Gram Positive: one lipid layer with peptidoglycan outside. uses disulphide and isopeptide bonds

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23
Q

Disulphide Bond Formation

A
  1. secretion into the periplasm
  2. oxidation by DsbA (protein’s disulphide is reduced to thiol groups)
  3. DsbB reoxidation to reform disulphides
  4. isomerization of substrate if misoxidized by DsbC
  5. regeneration of Dsb disulphides
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24
Q

Isopeptide Bonds

A
  • An isopeptide bond is an amide bond that can form for example between a carboxyl group of one amino acid and an amino group of another.
  • NOT the primary a-groups though (side chains)
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25
Q

Disulphide Bonds and Protein Stability

A
  • eg. fimbrial proteins use for domain stabilization
  • eg. EGFR extracellular dimer is used for stability (25 disulphides per domain)
  • there is an increase in S-S bonds with increase in growth temperature (correlation with protein stability)
  • Thermophiles contain protein disulfide oxidoreductase suggests these proteins need the bonds
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26
Q

Engineering of Disulphide Bonds

A
  • bonds stabilise proteins by reducing conformational entropy of the denatured state
  • using B factors to select regions of high mobility, mutate these residues
  • change in thermal stability associated with S-S bonds correlated with the change in mobility of the mutated residue pair
  • eg. can engineer a Cysteine residue to form disulphides in a dimer or can increase oligomer stability
  • this increases thermal stability greatly
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27
Q

Isopeptide Bond Formation

A
  • amide bond formed between carboxyl or amide group of one amino acid and amino/carboxyl group of another
  • at least one group comes from a side chain
  • common in surface proteins of Gram positive bacteria
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28
Q

Isopeptide Bond Stability

A
  • gram positive bacteria have spontaneous formation of intramolecular isopeptide bonds
  • happens in hydrophobic core
  • learn mechanism
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29
Q

SpyTag and SpyCatcher

A
  • The SpyTag/SpyCatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide SpyTag (13 amino acids) spontaneously reacts with the protein SpyCatcher (12.3 kDa) to form an intermolecular isopeptide bond between the pair.[1] DNA sequence encoding either SpyTag or SpyCatcher can be recombinantly introduced into the DNA sequence encoding a protein of interest, forming a fusion protein. These fusion proteins can be covalently linked when mixed in a reaction through the SpyTag/SpyCatcher system.
30
Q

SpyTag/Catch Applications

A
  1. vaccination production: By fusing either SpyTag or SpyCatcher to self-assembling molecules such as virus-like particles, antigens fused to the other pair can be decorated onto the molecule via the isopeptide bond formed.[10][11][12][13] This enables fast production of vaccines as the central self-assembling molecule can be stocked up beforehand, whilst the antigen can be easily produced under optimal conditions to achieve proper protein folding.
  2. enzyme cyclisation: Cyclization of enzymes by fusing the N- and C-termini of the protein helps elevate the stability of the enzyme against heat. By having SpyTag and SpyCatcher together in the enzyme at the termini, the enzyme will undergo spontaneous cyclization by forming the isopeptide bond. Cyclized beta-lactamase, phytase, firefly luciferase, and xylanase (to name a few) have shown retained enzyme activity even after being subjected to heat at 100 °C
31
Q

Isopeptide Bonds: Anchoring

A
  • sortase is an enzyme anchoring secreted proteins on the external cell membrane of Gram+ bacteria
  • Cleaves LPXTG motif on C terminus of proteins and forms isopeptide bonds between the terminus and peptidoglycan via acyl intermediate thioester
    1. precursor surface protein is attached to a sortase via the acyl intermediate
    2. motif is cleaved by peptidoglycan leaving a cell wall anchored surface protein (covalent linkage via amide bond)
    The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the sortase enzyme.
  • essentially can act as a labelling protein
32
Q

Isopeptide Bonds: Oligomerisation

A
  • isopeptide bonds used in oligomerisation of protein domains on the surface (one example is pilin)
  • the pilin sortase recognises the LPxTG motif & the housekeeping sortase recognises the LAxTG motif
  • the pilin specific sortase binds to its motif in the pilin domain and can continue adding more domains with this motif
  • then a specific domain with the LAxTG motif is added, causing housekeeping sortase recognition and cleavage onto peptidogylcan to form the oligomerized pilin polypeptide chain
33
Q

Thioesters

A
  • form spontaneously on protein surface
  • carboxylic acid + thiol = thioester
  • thioester + primary amine = amide + thiol
34
Q

Chemical Harpoons

A
  • thioesters are prevalent in surface proteins of Gram positive bacteria
  • these reactive groups may be used to covalently link bacteria and their host (humans for example)
  • therefore, thioesters may react with primary amine groups to form stable amide bonds and bind the bacteria to the surface of host cells
35
Q

Ubiquitin ligases and transglutaminase

A
  • enzyme catalyzed intermolecular reactions
  • both enzymes contain a side chain -SH of cystein that forms a thioester with substrate proteins
  • eg. transglutaminase -SH attacks the amide group of the substrate to form a thioester
  • the thioester is vulnerable to attack and transfers substrate one to substrate two (conjugation via the thioester intermediate)
36
Q

Sortase Tagging

A
  • can form different conjugates with acyl donors or acceptors
  • sortase catalyzed ligation
37
Q

Advantages of Bioconjugate techniques

A
  • add new functionalities to proteins with reagents that covalently modify biomolecules in specific sites
  • produce minimal side reactions
  • gentle solutions
  • safe to use
  • widely available
38
Q

Bioconjugate techniques

A
  • modification of cysteins, primary amines, carboxylic acids
  • crosslinking macromolecules for structural and functional studies
  • label transfer
  • fluorescence labelling
39
Q

NHS esters

A
  • amine modification
  • NHS ester reagent is reacted with the primary amine on a protein to for a stable conjugate with an amide bond
  • NHS is the leaving group
  • primary amine group is a nucleophile breaking the bond
  • is a hydrophobic crosslinker
40
Q

Sulfo-NHS

A
  • amine modification
  • amine reactive sulfo NHS ester added to primary amine of a protein to form amide bond conjugate and sulfo-NHS leaving group
  • sulfonate increases water solubility of compounds
  • prevents crossing of cell membranes so is used in cell surface crosslinking methods / protein purification
41
Q

Example of Sulfo-NHS

A
  • biotinylation of primary amines

- Sulfo-NHS-Biotin reacts with primary amine to create biotinylated molecule

42
Q

Maleimides

A
  • cysteine modification
  • maleimides target thiol groups to form thioethers
  • select residues on the protein for cysteine substitution
  • sulhydryl reacts to form stable conjugate with the maleimide (thioether)
  • can be fluorescent to add label to protein
43
Q

Enzyme-Ab Conjugates

A
  • combining maleimides and sulfo-NHS
  • label reduced Ab fragments with maleimide activated enzymes
  • Sulfo-SMCC crosslinker reacts with the primary amine of an enzyme
  • sulfydryl activated antibody reacts with the maleimide activated enzyme to form to conjugate
  • by tagging the Ab with an enyzyme you can detect it in a blot
44
Q

Peptide Conjugation

A
  • peptide conjugation to carrier proteins for antibody production
  • carrier protein with free amine reacted with Sulfo-SMCC crosslinker
  • maleimide activated carrier protein reacts with sulfhydrl group of a peptide to form carrier peptide conjugate
45
Q

PhotoAffinity Labeling

A
  • photo-reactive reagents are chemically inert compounds
  • reactive when exposed to UV or visible light
  • proteins modified with PAL reagents covalently bind their targets after light activation
  • PAL can label low abundance and low affinity proteins
    (interaction of the bait against the prey in a large complex)
46
Q

Properties of PAL reagents

A
  • stable in the dark at many pHs
  • activation at wavelengths not damaging the molecules
  • capable of forming intermediates reacting to form stable adducts with prey
  • minimal side reactions (want specificity with prey)
47
Q

PAL reagents

A
  1. photoreactive moiety: permanent attachment to 2ndary target (prey)
  2. affinity/specificity unit: reversible binding to target protein
  3. identification/reporter tag: allows purification
48
Q

Sulfo-SBED

A
  1. photoreactive moiety is phenyl azide
  2. affinity unit is sulfo-NHS with cleavable disulfide
  3. identification tag is streptavidin
    - incubate with bait
    - free amine in the bait with react with the sulfo-NHS
    - add cell extract with suspected prey
    - prey will bind
    - expose solution to UV for crosslinking prey to photoreactive group
    - reducing agent removes bait protein
    - purify and identify the prey
49
Q

Ad2 Nuclear Pore Interactions

A
  • Ad2 imports DNA genome via nuclear pore complex of cells for viral production
  • NPC-filament protein is a docking site for the capsid
  • use Sulfo-SBED to test if the filament protein becomes biotinylated (western blot) as this only happens if it interacts with the Ad2
50
Q

Diazirines

A
  • most commonly used photoaffinity group: carbon bound to two nitrogen atoms, which are double-bonded to each other
  • excellent chemical stability and versatility
  • used as functional group in amino acids and other compounds
  • N2 leaves to form carbene that reacts with protein to form conjugate
51
Q

Photo-reactive amino acids

A
  • photo leucine or methionine
  • add photo-reactive amino acids to cell media
  • expose cells to UV light to activate
  • harvest and lyse cells
  • run gel and analyse using specific Ab
  • see interactions that a protein established with other things
52
Q

Diazirines Applications

A

eg. protegrin: antimicrobial peptide made by leukocytes
- identify targeting bacteria this peptide binds to
- identify antimicrobial effect of mimetics (antibacterial effects)
- add diazirines to look at fluorescence
- measure DNA released (photoamino acids?) after peptide treatment : lysis is caused

53
Q

Advantages of Photoamino acids

A
  • minimal structural changes
  • cell penetration
  • selective labelling in cell free expression systems
  • protein modification by protein-peptide:PAL fusions
  • wide range of applications
54
Q

Fluorophores

A
  • absorb light of one color (wavelength) and emit the energy at a different wavelength
  • visual probes
  • can be intrinsic, covalently attached, or remain in solution
  • fluorescence is sensitive to environmental changes and increases in rigid/hydrophobic environments
55
Q

Fluorescence and energy levels

A
  • ground state electrons absorb light to move to higher orbital (excited state)
  • radiationless decay to lowest vibrational state of excitation orbitals
  • transition back to ground state
  • emission as photons of a longer wavelength due to to energy loss via heat
56
Q

Fluorescein

A
  • type of fluorophore
  • absorption at 494mn
  • emission at 523nm
57
Q

FRET

A
  • interaction between excited states of two fluorescent molecules detects proximity between the labeled biomolecules
  • the first emission wavelength overlaps with the second absorption wavelength
  • no photon emission needed for the excitation transfer
  • donor and acceptor need to be close : reports on binding interactions
58
Q

Example of FRET

A
  • excitation of CFP at 430nm gives fluorescence at 475-500 nm which excites YFP to fluoresce at 525-550 nm if they are close enough
  • need fusion proteins of CFP and YFP with your bait and prey (protein of interest)
59
Q

FRET Method

A
  • protein X fused to blue fluorescent protein
  • protein Y fused to green fluorescent protein
  • protein X emits blue light that excites GFP for green light emission
  • if protein interaction occurs green rather than blue light will be detected
60
Q

Fluorimeter

A
  • light from the sample is emitted in all directions but is measured at 90 degrees from the incident beam
  • reduces noise to measure only light emitted from sample (not background)
  • monochromators are used to select wavelengths that can pass through
61
Q

Fluorescence measurements

A

Measure fluorescence intensity for

  1. free ligand (F0)
  2. bound ligand (Fmax)
  3. protein ligand complexes at a range of protein concentrations (F vs [P])
    - vary concentration of non-fluorescent binding partner
    - fraction bound (Y) = F - F0 / Fmax - F0
    - plot Y vs protein concentration to determine dissociation constant
62
Q

Differential Scanning Fluorimetry

A
  • relies on changes in thermal stability of a protein upon ligand binding
  • simple (similar to qPCR)
  • can test binding of peptides, NA, sugars, etc
  • useful to optimise solution conditions of a protein
  • screens libraries of small ligands (drug discovery)
63
Q

Principle of DSF

A
  • protein is incubated with the soluble dye (minimal interaction
  • the molecule becomes fluorescent when bound to hydrophobic patches of the protein
  • as temperature increases the protein unfolds to expose more of these patches, leading to more color
  • Tm can be determined (look at increase in stability)
  • fluorescence hits a max then decreases due to aggregation of denatured proteins/dye
64
Q

DSF and myosin tail interacting protein

A
  • protein has a calmodulin fold with 2 domains
  • clamps around the ligand using aspartic acid from an ion pair
  • DSF looked at the effect of mutants and ligands of different lengths
  • mutants break an ion pair and decrease stability
  • Tm shows increase or decrease of stability in the protein
65
Q

DSF Evaluation

A
  • inexpensive and quick
  • high throughput
  • easy to visualize
  • identify weak interactions
  • quantitatve
  • widely applicable
  • false negatives possible
  • cannot use proteins with a reduced hydrophobic core such as elongated proteins
66
Q

Microscale Thermophoresis

A
  • thermophoresis is the directed movement of molecules in a temperature gradient
  • IR laser radiation is focused on specific point at sample in a capillary tube
  • fluorescent signal change generated from the 2nL volume being heated
  • determines Kd in 30 mins
67
Q

Microscale Thermophoresis setup

A
  • optical system warms sample but also has a light source to excite the chromophore
  • dichroic mirror letting certain wavelength pass and not others
  • results show a T jump decrease in fluorescence after IR laser is on
  • this is caused by diffusion limited thermophoresis
  • measure difference between point of IR on and IR off (difference is less when bound)
  • transform into plot of [ligand] vs difference in F
68
Q

Principle of MST

A
  • IR laser on decreases fluorescence
  • increase in temperature at the center of IR irradiation will drive away fluorescent molecules (increased Ke): large change
  • increasing concentrations of a binding partner slows migration of the fluorescent ligand : smaller change
  • warming sample increases random motion of all molecules : smaller diffuse faster but when bound cannot
69
Q

Evaluation of MST

A
  • principle of thermophoresis not well understood
  • ligand screening isn’t high throughput
  • upper limit of Kd restricted by solubility of high Mw binding partner
  • maybe not easily analysable in terms of binding and affinity constants
  • highly sensitive and quantitative analysis of protein interactions
  • no immobilization
  • low sample consumption
  • fast KD determination
70
Q

Evaluation of Fluorescence Methods

A
  • sensitive
  • can be quantitative
  • examine in vivo protein interactions
  • may need specific reagents or GFP fusion proteins
  • widely available

Protein Science (11 decks)

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