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Imperial Y2 - Protein Science > Ernesto Cota - Protein-Ligand Interactions > Flashcards

Ernesto Cota - Protein-Ligand Interactions Flashcards

1
Q

Goal of Lecture series?

A

Continuation of the Structural Biology course - Look at techniques to identify ligands for our protein of interest

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2
Q

What are the different techniques that we are going to look at to investigate Protein-ligand interactions?

A
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3
Q

When thinking of protein-ligand interactions experimental techniques, what are we ideally looking for?

A
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4
Q

What is the Yeast-2-hybrid technique?

A

Yeast 2-hybrid method

Genetic screening technique - allows us to identify a ligand from a library of thousands of ligands

Permits a wide search for potential binding partners for known or unknown proteins

Links proteins to their genes - once we identify the interaction, we have a gene that expresses that ligand

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5
Q

What is the principle behind the Yeast 2-hybrid method?

A
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6
Q

Outline in more detail how the Yeast-2-Hybrid method is carried out? i.e. how do we get the fusion proteins into the cell?

A

Yeast 2-hybrid - typical practice

  1. Transform yeast with your “bait” of choice

Bait = DNA coding for the DBD fused to DNA for your protein of interest

  1. Prepare the prey - DNA coding the AD fused to DNA for a selection of ligands (e. g. using cDNA library generated from mRNA extracted from a particular cell type of interest)
  2. Transform yeast cells with the prey DNA - whole library of AD-prey fusions –> Ideally each yeast cell picks up only one prey DNA molecule
  3. Check for reporter gene expression –> identifying ligand that binds to protein - remember we know and have the gene of the ligand in our cDNA library
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7
Q

What type of reporter gene is typically used in the Yeast-2-hybrid method?

A
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8
Q

What is the Yeast-3-hybrid method?

A
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9
Q

How can we modify the Yeast-3-hybrid system to reduce the number of false positives?

A
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10
Q

How can the Yeast-3-hybrid method be modified to examine RNA-RNA interactions?

A
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11
Q

Pros and cons of Yeast-2-hybrid method?

A
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12
Q

What is a GST-pull down method?

A

Used mainly to confirm a suspected interaction (with a known protein)

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13
Q

Explain the GST pull down method

A
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14
Q

Analyze the results from a GST pull down? What does it tell you?

Hint - testing different deletion mutants of ligand/prey so that they can figure out which region binds to bait

A
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15
Q

GST pull-down pros and cons?

A

GST pull-downs - pros, cons

Pros:

  • Quick and easy (if you have a clone of your bait protein)
  • If target or prey proteins are radiolabelled, GST-fusion bait protein is invisible on the autoradiogram

Cons:

  • Not very quantitative - i.e. binding constants?
  • Careful controls needed
  • GST-bait fusion may be susceptible to proteolysis
  • Larger amounts of target/prey proteins needed for identification (if not already known) - low signal
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16
Q

What is a Gel Shift Assay or Electrophoretic Mobility Shift Assay (EMSA)?

A
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17
Q

What does this gel-shift assay native gel show us?

A
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18
Q

Why not use SDS-PAGE for Gel Shift Assays?

A

We cannot denature the protein as we require the protein-NA interaction. Hence, a simply acrylamide gel is used

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19
Q

What would ideal Gel Shift Assay results look like?

A
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20
Q

How would you interpret the following gel-shift assay?

A
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21
Q

Gel-shift assay pros and cons?

A
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22
Q

Outline two examples enzymatic tagging of a protein of interest that commonly used and why they are commonly used?

A
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23
Q

Outline how we are able to biotinylate a protein of interest?

A
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24
Q

What are some examples of biotin binding partners that are commericially used?

A
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25
Q

Is the biotin-avidin interaction stable?

A
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26
Q

What role does the enzyme sortase nornally perform in gram +ive bacteria?

A

Sortase – function

Enzyme used to anchor secreted proteins on the external cell membrane of Gram+ bacteria

  1. Recognizes and cleaves the LPXT-G motif on the C terminus of secreted proteins - forming acyl-enzyme intermediate
  2. Transfer - Forms isopeptide (amide) bonds between C-terminus of secreted proteins and NT of peptidoglycan (lipid II)
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27
Q

How can sortases be used to create a fusion protein?

A
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28
Q

Real life application of sortases in red blood cells?

A

Engineer erythroid precursors → new RBCs that expressed a protein called Kell with a modified C-terminus (LPXTG-HA) → sortase recognizes motif and binds - sortase modified RBCs

Addition of target protein with an aminoglycine nucleophile - nucleophilically attack and replace sortase – tethering it to the cell surface

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29
Q

Why would we want to modify our proteins - create bioconjugates?

A

Most amino acids are neutral pH are relatively chemically inert, but we can…

Add new functionalities to proteins using 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
  5. Are commercially available
  6. Have been tested in different systems – research papers
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30
Q

What groups are we commonly modifying when creating bioconjugates?

And

Reason for creating bioconjugates?

A
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31
Q

How can we modify primary amines on a protein of interest for conjugation?

A
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32
Q

When performing modifications of amines, why would we want to use sulfo-NHS?

A
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33
Q

Outline what is going on in this example of amine modification?

A
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34
Q

Outline how cysteine residues (-SH) can be used for bioconjugation/modification?

A
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35
Q

Explain how Maleimides were used for crosslinking of superoxide dismutase?

A
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36
Q

Are Maleimides used to add fluorescent labels?

A
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37
Q

How can maleimides and sulfo-NHS be combined?

A
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38
Q

Outline how maleimides and sulfo-NHS conjugation is used for an ELISA?

A
  1. Nucleophilic attack of primary amine on ester of Sulfo-SMCC cross linker
  2. Activated Maleimide ring double bond reacts with HS-antibody to conjugate antibody to carrier
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39
Q

What is photoaffinity labelling (PAL)?

A

Photoaffinity Labeling (PAL) – only reactive when exposed to light

  • Photo-reactive reagents are chemically inert compounds - reactive when exposed to UV or visible light
  • Proteins modified with PAL reagents can covalently bind their target(s) after activation by light
  • Unlike other methods, PAL can label low abundance and low affinity proteins

Active field of research, with increasing number of commercially available compounds

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40
Q

Outline the ideal properties of a PAL reagent?

A
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41
Q

What are the three moeities on a PAL reagent?

A
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42
Q

Outline how the PAL reagent (Sulfo-SBED) can be used to investigate protein-ligand interactions?

A
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43
Q

Outline an example of Sulfo-SBED used in action?

A
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44
Q

After running PAL (sulfo-SBED), what did they find out about the interaction between Ad2 capsid and CAN NPC domain?

A
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45
Q

What does a normal PAL workflow look like?

A
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46
Q

What are Diazirines used for in bioconjugation?

A
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47
Q

How are photo-amino acids used to identify new ligand-protein interactions?

A
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48
Q

Apart from diazirines, what other cross-linking reagent can be used and how does it compare?

A

STAT3 and HSP90 - binding partners

Ku70 and ku80 - binding partners

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49
Q

Outline how diazirines were included into a study that looked at the antimicrobial peptide produced by leukocytes?

Srinivas et al., 2010

A
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50
Q

Using the cross-linking capabilities, what protein was the peptidomimetic peptide resembling protegrin binding to?

Srinivas et al., 2010

A
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51
Q

Give one reason why diazirines are useful and three applications of diazirines?

A
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52
Q

Apart from understanding which proteins & ligands interact, what else are we interested in finding out?

A

We need to have a theoretical understanding of protein-ligand interactions

Basically, we need to codify binding behavior mathematically –> understand binding mathematicall/binding data - DG, Kon/off, etc.

We will examine models for:

  1. Single site binding
  2. Multiple site binding (independent sites)
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53
Q

What is a single binding system and what assumptions are made?

A

Single site binding model - We measure the formation of ML – readout

Assumptions

  1. Binding is dynamic, reversible – non-covalent
  2. Measurements when thermodynamic eq. is reached - i.e. Conc. of complex doesn’t change over time

Important to support assumptions with structural data:

  1. Number of ligand binding sites?
  2. Any Cofactors?
  3. Inhibitors present?
  4. Conformational changes upon ligand binding – are they reversible?
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54
Q

What are the rate equations for the forward and reverse reaction of a single site binding model and how can this be rearranged to form an equation in terms of Ka?

A

Ka - equilibrium association constant

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55
Q

How do interpret Ka values? What does a large or small value mean?

A
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56
Q

What is the relationship between Ka and Kd?

A
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57
Q

What are the units for Kon and Koff and how are they calculated?

A
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58
Q

How can DG be calculated from DH & DS? How can DG be calculated using Ka?

A
59
Q

How can we express DG in terms of KD?

A
60
Q

What is fractional saturation and how is it calculated?

A
61
Q

How can we express fractional saturation (Y) in terms of ligand concentration - derive the equation

A
62
Q

What graph do we get when we plot fractional saturation (Y) against Ligand concentration? How can this be used to figure out Kd?

A
63
Q

When plotting Plotting Y vs [L], what graphical transformation is normally applied and why?

A
64
Q

What are the two main types of error that could possibly influence our results?

A
65
Q

What is the derivation of the straight line equation required for the Scatchard Plot?

A
66
Q

What does a Scatchard Plot look like and what type of information can be obtained from the plot?

A
67
Q

Is the Scatchard Plot useful to identify any deviations from our binding model?

A
68
Q

What happens when we have multiple independant sites of equal affinity? What happens to the equilibrium equations?

A
69
Q

How can we express of K1 and K2 equations (multiple site binding) in terms of KD?

A
70
Q

For multiple independent sites of equal affinity, how can we create a single expression for fractional saturation?

A
71
Q

How can we re-arange our fractional saturation equation (multiple binding sites) into the linear Scatchard form? What does the plot look like?

A
72
Q

What normally happens to Scatchard plots in real life?

A
73
Q

Outline how a KD expression for single site binding model can be rearanged to form a quadratic equation allowing us to calculate [ML]

Starting equations below

A
  1. Create equations for total and free protein and Ligand – rearrange to my M/LFree subject
  2. Insert into KD equation
  3. Rearrange
  4. Expand brackets
  5. Move KD[ML] to the RHS to make the equation = 0
  6. Factorize [ML]
  7. Substitute into quadratic formula to solve for [ML] – our x value
74
Q

For our quadratic expression to solve for [ML], how can we introduce a response measured variable Q?

A
75
Q

Why is streptavidin added before purification?

A

Strong non-covalent interaction - Useful for purification allows strong binding to biotin tag

76
Q

In practice how can we distinguish what the bait-prey complex is, if the yeast picks up more than 1 prey DNA?

A

Rarely happens – transformation efficiency in yeast cells is low

Plus when scanning a whole library, only a small selection of prey will bind the bait – hence, increasing the unlikely nature of transfecting two plasmids with prey that bind the bait

77
Q

When running a GST pull downs, you mentioned that the beads will be centrifuged and then run using SDS-PAGE. Wouldn’t SDS disrupt the binding between the prey to the bait & also the GST to the beads?

A

Yes we use SDS-Gel since we don’t really care about keeping the complex intact, rather the ligands will be radiolabelled and can thus be identified in the autoradiogram

78
Q

In the GST pulldown method, is the bait or the prey radiolabelled?

A

The prey is only radiolabelled – identify ligand

79
Q

In GST pull downs, what does working more at protein level mean in contrast to Y2H?

A

GST - we need to identify ligand identity via mass spec

Y2H - simply sequence the DNA of the inserted plasmid

80
Q

How do each of the yeast 3 hybrid improvements help reduce number of false positives?

A

a) Separation of RNAX and MS2 in space – preventing their interaction
b) Dimerization – increases effective concentration + reducing KD of interaction (two binding regions on the same protein)
c) Increase expression of ProtY-AD fusion - increases signal

Generally, more efficient and likely to produce a true signal

81
Q

For fractional saturation, why are we using KD and not KA?

A

Can use either but most of the time KD is used + easier units (M instead of M-1)

82
Q

What are the two fluorescence methods of studying Protein-ligand interactions that are going to be examined?

A

Examine fluorescence methods with particular focus on…

Differential Scanning fluorimetry

Microscale thermophoresis

83
Q

What is the principal behind Fluorescence methods?

A
84
Q

What is the principal behind fluorescence (energy levels)?

A
85
Q

What is fluorescein?

A
86
Q

What is FRET? How does it work?

A
87
Q

What does this real FRET-spectra show?

A
88
Q

Principal of using FRET to study protein-ligand interaction?

A
89
Q

How can FRET be used in cells?

A
90
Q

What does a Fluorimeter set-up look like?

A
91
Q

How can we obtain KD by making fluorescence measurments? - Hint fractional saturation

A
92
Q

Can we fluorescently label the protein instead of ligand?

A
93
Q

What is Differential Scanning Fluorimetry?

A
94
Q

Outline the principle behind Differential Scanning Fluorimetry, how does it work?

A
95
Q

In a DSF plot, what does a curve that shifts to the right indicate?

A
96
Q

Pros and cons of Differential Scanning Fluorimetry (DSF)?

A
97
Q

What is Microscale Thermophoresis (MST)?

A
98
Q

What does the Microscale thermophoresis instrumental setup look like?

A
99
Q

What is the procedure for a Microscale thermophoresis experiment?

A
100
Q

What happens to the ligands/protein present in our capillary tube as we rapidly heat it up with IR?

A
101
Q

What do we do with fluorence data obtained from MicroScale Thermophoresis (MST)?

A
102
Q

Microscale thermophoresis advantages and disadvantages?

A
103
Q

In general, Fluorescence methods are…

A
  1. Sensitive
  2. Can be quantitative - KD and Tm
  3. Equilibrium measurements (in vitro)
  4. Use to examine protein-protein interactions in living cells (FRET)
  5. May require special reagents
  6. GFP-fusion proteins
  7. Chemical attachment of fluorophores to your protein
104
Q

What are the most widely used techniques (gold standard), when it comes to studying protein-ligand interactions?

A
105
Q

What is the range of dissociation constants for NMR, SPR and ITC - rough range?

A
106
Q

What is the principle behind Isothermal Titration Calorimetry (ITC)?

A
107
Q

What does the ITC instrument setup look like?

A
108
Q

What does ITC raw data look like? What are the three binding phases?

A
109
Q

What equation for single-site binding can be used to determine DH and KD using the data obtained from ITC?

A
110
Q

How can we determine DH and binding constant (KB = KA) from ITC data?

A
111
Q

What did Douse et al. (2012) show using the ITC technique?

A
112
Q

Before starting an ITC experiment, what should you think about?

A
113
Q

What parameter limits the range of binding constants that can be measured using ITC?

A
114
Q

What parameters are obtained from ITC?

A
115
Q

What is meant by Thermodynamic discrimination?

A
116
Q

What are the pros and cons of ITC?

A
117
Q

What is Surface Plasmon Resonance (SPR)?

A
118
Q

Outline the principle + setup behind Surface Plasmon Resonance (SPR)?

A
119
Q

How does binding of prey to bait influence resonance angle?

A
120
Q

Upon prey binding to bait in SPR, what changes occur in the detector?

A
121
Q

What does SPR data look like? What valuable data can be obtain?

A

RU = resonance units

122
Q

How are Kon and Koff obtained from the SPR data?

A
123
Q

What is happening in this real life example of SPR data?

Note - curves represent varying prey conc.

A
124
Q

Pros and cons of SPR?

A
125
Q

How is NMR used to study Protein-ligand interactions?

A

Nuclear Magnetic Resonance (NMR)

Modern methodology – in constant development

Provides a series of techniques to identify PLI’s..

a) Combines binding constants and structural information
b) Able to observe/measure interactions with a wide range of affinities

Useful for non-crystallisable complexes or as complement to XRD/EM studies

Three different techniques using NMR will be discussed

126
Q

What is chemical shift pertubation (first analytical technique)?

A
127
Q

How can CSP be used to give residue-specific information?

A
128
Q

What is one problem associated with CSP binding site assignments?

A

Problem - Not possible to separate CSPs from direct ligand binding and CSPs due to allosteric/ conformational changes upon ligand binding (possible with other NMR techniques) - especially for big dynamic proteins

129
Q

Why and how do we obtain a single CSP value from resonances of proton and the attached atom (N or C) of a given peak?

A
130
Q

How can KD be obtained from CSP analysis?

A
131
Q

If we assume that Kon is diffusion controlled, what equation can we setup that relates Koff and KD?

A
132
Q

When we get a chemical shift pertubation/change, what two exchange regimes are possible and what do they tell us?

A
133
Q

How can the different exchange regimes at NMR timescale inform us about Koff and KD?

A
134
Q

Outline what Proton/Deuterium (H/D) exchange is?

A
135
Q

What is H/D exchange useful for?

A

Using this technique as we can map the residues involved in binding the ligand and thus create an interaction surface

136
Q

What is NMR cross-saturation (third technique)?

A
137
Q

How can we identify the interaction interfaces from NMR cross-relaxation?

A
138
Q

How can we increase our signal to noise in cross relaxation data?

A
139
Q

What does the attach image show?

A
140
Q

Why is there a gradient of light seen in the detector of a Surface Plasmon resonance?

A

As you move away from the resonance angle, less light will be absorbed by film and instead it is reflected onto the detector - resulting in gradient

141
Q

What do you mean by HSQCs are virtually artefact free?

A

Lack of experimental phenomenon that may reduce the reliability of data e.g. Insensitive to problems like protein/ligand aggregation - no visible on the spectra

Hence, Chemical shift change/peak comes from soluble proteins

Note - important factor that you have to control is pH as it effects protonation state – especially in amides

142
Q

In measuring cross saturation, how to only deuterate H attaching to C but not to N?

A

Example, grow in E. Coli cells in a deuterated media

Result? - Everything becomes deuterated BUT upon purification we use normal buffers (normal hydrogen) and all the amides will readily exchange their deuterated hydrogen whereas Carbons will not

If you want to exchange the amides within the core of the proton (still deuterated) you would have to (partially) unfold the protein in order to allow for exchange in normal buffer

143
Q

What type of question will Ernesto be asking during the exam?

A

Problem based

  1. Have a strategy to find out what the ligand is for a newly identified protein
  2. Once identified, how can you validate the ligand binding
  3. After that outline how you can determine binding constants and binding sites

Imperial Y2 - Protein Science (4 decks)

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