Protein-Ligand Binding_L1-1 Flashcards
What properoties are required as ideal technique?
(1) Increasingly quantitative: accurate and reproducible
(2) free label: labels on the protein surface affects the studies
(3) true ‘in-solution’: attaching a protein to the solid surface influences the binding surfaces
(4) sensitive->small sample size: small concentration/amount
(5) high-throughput: measure one ligand at one input
(6) fast: the outcome obtained in 30min to 2 hours
(7) atomic resolution: identify the residues at the active site
introduce genetic screening technique briefly
(1) Permits a wide search for potential binding partners for known or unknown proteins
(2) Links proteins to their genes
(3) Once the interaction has been identified, the gene coding for the ligand can be figured out as well.
What is the principle of the yeast two-hybrid screening technique?
(1) the fusion construct composed of the DBD-bait(usually known) and AD-prey(can be either known or unkown) is built up by recombinant DNA technique. Only when the two sections re-unit can the reporter protein be activated and expressed as a sign of the two module joining. The technique exploits the modular nature of the gene activator protein, such as GAL4
(2) the bait consists of DBD and target protein, and the prey consists of binding partner and the transcriptional activation domain. The binding partner complements with the target protein.
(3) genes coding for the prey and bait are introduced into the yeast cell, when the two components bind with each other, the transcriptional activation domain recruits the RNA polymerase which transcribes the reporter gene
* DBD: DNA-binding domain
* AD: activation domain
What is the common practice in the yeast two-hybrid screening technique?
(1) Transform yeast with your “bait” of choice
Bait = DNA coding for the DBD fused to DNA for your protein of interest)
(2) Prepare the prey
= DNA coding the AD fused to DNA for a selection of proteins (e. g. using cDNA generated from mRNA extracted from a particular cell type)
(3) Transform yeast cells with the prey DNA
→ whole library of AD-prey fusions
Each yeast cell picks up only one prey DNA molecule
Select for interactions
What features can the reporter gene possess to show the presence of both bait and prey?
Use a “reporter gene” that allows selectivity (antibiotic resistance or growth in the absence of a specific nutrient)-> positive selection: In this case, the only yeast colonies that grow are those containing an interacting bait and prey, cells containing the interacting bait-prey complex survive as the reporter gene can code for the antibiotic resistant proteins
introduce the yeast three-hybrid: protien-RNA interactions?
(1) same principle as the Y2H screening
(2) prey protein is dimerised from two monomers which comprise of the MS2 domain and LexA domain, the MS2 specifically fuses the LexA for transcribing the operator gene. MS2 can also binds to the MS2 RNA at the binding cleft which connects the prey protein with the bait protein by extension. The prey protein contains an activation domain for transcribing the reporter gene, as well as the the Prot Y. The RNA segment binding to the Prot Y is referred as RNA X. Activation domain transcribes the reporter gene.
How does the conjugating RNA manage to display accurate interaction in order to reduce the false positives?
(1) The secondary structure at the G-C clamps stabilise stems of displayed RNA to avoid interactions with MS2 RNA
(2) The MS2 domains dimerise to increase the affinity for particular RNA
(3) the expression of the Prot Y-AD fusion protein increases to improve the binding possibility with the RNA X
introduce yeast 3-hybrid: RNA-RNA interaction
the bait remains constant, the RNA fragment at the bait protein binding cleft is RNA X which interacts with the RNA Y. RNA Y is attached to the m26 AD which directly acts as activation domain. A range of m26 AD can be covalently linked to the RNA X in a complex to transcribe the reporter gene as an RNA polymerase
what are the pros and cons of yeast 2-hybrid screening?
pros:
(1) Scalable
1. can screen bait vs many prey (e.g. from cDNA library) per experiment
2. can screen cDNA library vs cDNA library (used to figure out “interaction maps” for organisms)
(2) Direct identification of interacting prey from DNA sequence of recovered colonies
cons:
(1) Method prone to “false positives” - interactions identified by Y2H should be tested by alternative methods, because one bait protein can have multiple binding partners.
(2) No quantitation of affinity
describe the GST pull-downs
(1) Another fusion technique
GST = glutathione S-transferase (binds glutathione) is fused to known bait protein
(2) Method relies on:
binding of GST to glutathione-conjugated beads
Method works more at the protein level (in contrast to Y2H)
(3) Recombinant DNA technology fuses the protein X and glutathione S-transferase(GST) for the hybrid protein to attach onto the glutathione-cojugated beads at the glutathione.
(4) All the proteins are bio-radically labelled.
(5) Mix “prey” proteins sample with GST-bait fusion protein and glutathione beads. Separate bound proteins by centrifugation. Elute protein complexes with glutathione and analyse by SDS-PAGE. Used mainly to confirm a suspected interaction (with a known protein)
Or to identify binding partners from a soup of proteins (e.g. cell lysate). Binding partners may be determined by direct protein sequencing (i.e. Mass Spec)
What will the real data of GST pulldown experiment be like?
for the three groups, markers are loaded onto the gel.
(1) positive control: the prey protein fragmentised for gel electrophoresis, the band intensity can therefore be compared with the last group.
(2) negative control: the bait protein and the non-specifically binding protein are mixed and loaded, by addition this step, the specificity between the bait and the prey can be verified.
(3) the complex formed between the bait and prey, the band intensity comparison with the positive control reflects the affinity of each fragment with the bait protein.
(4) Aim: to find out which fragment of X binds PTB
Protein X mutants are radiolabelled (i.e. 35S Met).
Gels are autoradiograms (GST-PTB: the bait protein not visible, only in combination with the radiolabelled prey protein can be visible)
pros and cons of GST pulldown experiment?
pros:
(1) Quick and easy (if you have a clone of your bait protein)
(2) if target or prey proteins are radiolabelled, GST-fusion bait protein is invisible on the autoradiogram
cons:
(1) Not very quantitative
(2) Careful controls needed
(3) GST-bait fusion may be susceptible to proteolysis
Larger amounts of target/prey proteins needed for identification (if not already known)
What principles and uses do the gel shift assay have?
(1) Used to measure protein-NA(nucleotide) interactions (DNA or RNA; ss or ds: either singly stranded or doubly stranded)
(2) NA is radiolabelled (transcription or end-labelling):
Incorporate an [γ-³²P]dNTP during a 3’ fill-in reaction in DNA replication using Klenow fragment or by 5’ end labelling using [γ-³²P]ATP and T4 polynucleotide kinase
(3) Non-radioactive versions now available
(4) Mix reactants and run on a native gel (acrylamide or agarose)
(5) Negative charge of NA drives migration in the gel - analyse result on autoradiogram
What information can be deduced from the gel shift assay?
(1) protein-RNA interaction
(2) if the protein is radiolabelled, very low concentration of protein substrate is required in the assay
(3) the RNA concentration remains constant for all the lanes but the substrate concentration increases gradually.
(4) From the native gel patterns, the free RNA concentration which is represented by the lower-mass band drops, inversely, the RNA-protein complex concentration rises. The free RNA concentration goes down to 0 if no RNA is bound.
What are the ideal results expected from the gel shift assay?
(1) Free NA migrates fastest (furthest)
(2) Complexes are retarded
(3) Technique gives information on stoichiometry
(4) For one-binding site protein, the bands are separated into two rows with inverse change in the concentration.
(5) For two-binding site protein, the bands are arranged into three rows, the free RNA concentration drops with the protein concentration increase, the other two binding sites of different affinities display different trends in substrate binding. As one protein binidng site can be relatively weaker than the other.
