Protein-Protein Interactions

Question 1

Name in vitro method to detect proteins

Answer 1

1. Liquid chromatography techniques
2. Isothermak titration calorimetry (ITC)
3. Surface plasmin resonance spectroscopy (SPR)
4. Nuclear magnetic resonance spectroscopy (NMR)

Question 2

Name in vivo methods

Answer 2

1. Yeast-/Bacterial 2-hybrid
2. Förster resonance energy transfer (FRET)
3. Proximity labeling an chemical cross linking + Mass spectronomy

Question 3

Does the FeS carrier IscA interacts with periplasmic aldehyoxidoreductase PaoABC?

Example of a Protein-Protein Interaction investigation

Answer 3

• To study PPI in vitro we first need to purify the involved proteins
• Assumed starting point: Cell-free solution of a mix of proteins
• To purify a protein –> Ammonium sulfate precipitation is requiered
  1. Ammonium sulfate has a high solubility in aqueous solutions
  2. Proteins precipitate at high salt concentrations and can be recovered later by dialysis

Advantage: No protein tag are needed
Disadvantage: Other proteins precipitate as well and impurties remain in the protein solution

Question 4

HIS-Tag and affinity chromatography

Protein purification method

Answer 4

**The His-Tag and affinity chromatography **

• N/C-terminal addition of histidine- residues (mostly Hexa-His His6)
• His-tag binds to nickel ions on a Nitrilotriacetic acid (Ni-NTA) matrix
• Wash steps with low imidazole concentrations remove unspecifically bound proteins
• Elution of the His-tagged protein with high imidazole concentration

1. Add His-Tag to specific Protein by genetic engeneering. (expression vector with open reading frame)
2. Equilibration: Buffer is added to column ( Buffer is compartible with protein of intererst)
3. Binding: Our taged protein is abled to bind to matrix –> with several non covalent interactions
4. Washing: Non specific and untagged Proteins are washed away. Wash buffer conatins components that disturb weak interactions but retain specific interactions.
5. Elution: Collect Protein that is bound in the column. Wash buffer is changing the composition of the buffer
6. Check: SDS-Page or western plot

**Disadvantages **(in general for all protein tags):
- Possible changes in 3D structure, steric impairment, improper folding, interference with interaction
- Removal: Cleavage by either Thrombin or Tobacco-Etch-Virus (TEV)-protease

Question 5

Ion exchange and Gel filtration chromatography

Answer 5

**1. Gel filtration chromatography **
–> Size Exclusion CHromatogrpahy (SEC)
* Sample run through porous matrix, smaller proteins enter beads
* Fractionation of eluting proteins – larger proteins elute first

**2. Ion exchange chromatography **
–> Seperation by electric charge of the proteins

Question 6

Liquid chromatography techniques

in-vitro

Answer 6

• used for seperation, identification and quantififcation of chemical components in a mixture
• Separation by size, charge or interaction with a bound ligand
• Different affinity techniques:
  e.g: - Antibodies – Pull down assay (Co- immuno precipitation)
• Streptavidin with Biotin
• His-tag with Ni-Nitrilotriacetic acid (Ni-NTA)

**Immunoprecipitation: **:
* Based on the binding of a specific antibody to a protein of interest (POI), in order to purify proteins that might be in a complex with the POI
* The antibody is attached to a resin material, e.g. magnetic beads
* Eluted fractions are then further analyzed and the sequence can be determined by mass spectrometry

Question 7

Isothermal Titration Caliometry ITC

in-vitro

Answer 7

• Basic Setup:
  –> 2 Cells (Reference cell and Sample cell)
  –> Injector
  –> Stirrer
  –> surrounded by adiabatic jacket to keep it at same temperatur *
• Reference cell contains
• Sample Cell contains Protein and Ligand, Ligand is injected by automatic Syringe
  Cells are always kept at same temperatur, temperature change comes from protein-ligand or ligand-ligand interaction
• Set volumes of solution are added to the sample cell.
• In the beginning most of the ligand binds (larger peaks), at the end the binding partner is saturated and only heat of dilution is measured (small peaks).
• Peaks are integrated and plotted against the molar ratio of the binding partners
• heat plotted against time
• From ITC data the binding enthalpy, entropy, heat capacity, and binding stoichiometry (number of binding sites) can be determined
• • Measures the binding affinity in terms of the dissociation **constant KD from weak affinities **(millimolar range) **to tight affinities **(single digit nanomolar range)

Question 8

Advantaged and disadvantaged of ITC

Answer 8

Advantages:
* No immobilization or tagging needed
* Measures the dissociation constant KD, the number of binding sites and the binding enthalpy ΔH
* Influence of buffer conditions on the binding can easily be tested

Disadvantages:
* Relatively high amounts of protein needed (in the mg range)
* Often only interactions with medium affinities can be precisely measured. The concentration of the binding protein should be in the range of 10-100x the dissociation constant, but the lower cutoff for detection is around 1nMol protein, making measurement of high affinity interaction impossible. Very low affinities on the other hand mean that not enough heat is produced per interaction, in order to detect it, except when highly concentrated samples are used, which tend to create background noise due to aggregation

Question 9

Surface Plasmon Resonance Spectroscopy (SPR)

In-vitro

Answer 9

• Protein of interest/Ligand is immobilised on a gold film at the interface between a prism and an aqueous solution
• A beam of light is directed at the prism
• The resonance angle is affected by changes in the composition of the molecule layer attached to the gold film, e.g. binding events
• A solution of proteins/Ligands or other molecules flows past the sensor chip. In case of a** binding event**, this is detected as a change in resonance angle
• With this technique the association rate k_on and dissociation rate k_off of the interacting proteins can determined in real time

Question 10

Advantages and Disadvantages of SPR

Answer 10

Advantages:
* Label-free: Proteins dont have to modified with possibly interfering tags
* Real time: Measuring of binding kinetics possible Study of weak and fast interactions possible
* Also low affinities (KD down to 10 μM) can be measured
* Low sample quantities are needed (only few μL)

Diadvantages:
* The bait protein has to survive the covalent coupling to the gold surface
* After each measurement bound prey protein has to be wash off thoroughly
* The molecular weight of the binding partner in solution cannot be very low, as the change in mass must be high enough to be detected
* Quite expensive equipment

Question 11

Nuclear Magnetic Resonance Spectroscopy (NMR)

In-vitro

Answer 11

• Nuclei of certain elements and their isotopes have an intrinsic magnetic moment (nuclear spin ≠ 0)
  (Nuclei = odd number of protons or odd number of neutrons e.g: H,C,N,F,P)
• A strong outer magnetic field aligns these spins
• A second weak oscillating magnetic field perpendicular to the first perturbs the aligned spins at the resonance frequency of the respective nucleus. The frequency depends on the chemical surrounding of the nucleus.
• One-dimensional 1H-NMR spectra of ethanol and a short protein fragment; the complexity increases, peaks overlap
• Nuclear Overhauser effect spectroscopy (NOESY) gives information about nuclei in close proximity (less than 5 Å) but not necessarily in protein sequence, i.e. either because of folded protein structure or at interaction sites of different proteins or ligands.
• A change in the NOESY signals can be used to infer information about interactions between proteins and conformational changes upon binding.

Question 12

Advantage and Disadvantage of NMR

Answer 12

Advantages
* One can infer structural information about the protein complex formed
Disadvantages:
* Expensive specialized equipment
* Rather limited to small to medium sized proteins
* Highly concentrated samples needed
* Need for isotopic labelling of samples

Question 13

Yeast and Bacterial 2-hybrid (Y2H and B2H)

Answer 13

• Uses Transcription activation system
• reporter genes indicate interaction
• If 2 Proteins interact the reporter gene gets expressed
• If the Proteins not interact –> no expression of reporter gene

**Transcription activation (Backround) **
* Promoter sequence upstream of gene sequence
* Transcription factors bind to promoter region
* TF recruits RNA Polymerase II
* RNA Polymerase II reads gene sequence and synthesize RNA

**Yeast-2-Hybrid System: **
* Split TF into 2 domains
1. DNA binding domain (DBD)
2. Activaton domain (AD)
* Both Proteins of interest are fused each to one of the domains
* DBD always binds to Promoter
* AD just binds to DBD if proteins interact
* If Proteins interact –> RNA Polymerase II can be recruited and synthesizes RNA (transcription)
* If Proteins dont interact –> only DBD binds to Promoter, DBD and AD are to far apart to interact and to recruit RNA Polymerase II –> No transcription

Bait and Prey and His RNA

Question 14

Advantage and disadvantage of Y2H and B2H

Answer 14

Advantages
* Does not require expensive equipment
* Is independent of the type of the screened interaction
* Can be used in a high throughput approach

Disadvantages
* False negatives:
–> Interaction might be dependent on whether proteins are bait or prey
–> Steric hindrance of interaction by fusion DBD or AD
–> Transient interactions might not be detected
* False positives:
–> Colocalization of proteins, that normally would not be in the same cell compartment
–> Unspecific interaction between bait and prey

Question 15

Förster Resonance Energy Transfer (FRET)

Answer 15

• different fluorophores are tagges to proteins of interest
• Donor: Protein X is tagged with CFP (blue)
• Acceptor: Protein Y is tagged to YFP (Yello fluorescent protein)
• Proteins need to be very close to each other ( < 10nm)
  1. Excitation of CFP (specific wave length)
  2. CFP emits blue light
  3. If Proteins interact, Förster energy transfers fro CFP to YFP
  4. YFP emits light (Yellow)
  5. Emission spectrum of CFP overlaps with excitation spectrum of YFP –> YFP accepts Energy
• If Proteins dont interact
  1. Excitation of CFP
  2. CFP emits blue light
  3. No energy transfer
  4. No yellow light from YFP
• Two candidate proteins are expressed as fusions to two fluorochromes, with different absorption and emission maxima
• the emission maximum of the first must overlap with the absorption maximum of the second fluorochrome.
• The fluorochromes have to be in close proximity (i.e. 1 – 10nm) for FRET to occur
• The transfer of energy between the fluorochromes is non-radiative*

Question 16

Advantages and Disadvantages of FRET

Answer 16

Advantage:
* Can be used in a high throughput approach
* Allows for localization of the interaction inside the cell when used with fluorescence microscopy

Disadvantages
* Fluorescent proteins are often sensitive to changes in pH, ionic concentrations, oxidation and temperature
* A low signal-to-noise ratio, making longer exposure times necessary sometimes
* photobleaching
* autofluorescence

Question 17

Proximity dependet labeling base techinques

Answer 17

1. A protein of interest (POI) is fused to engineered biotin ligase BirA (BioID) or ascorbate peroxidase APX (APEX)
2. Growth of cell culture in presence of biotin/biotin phenol
3. cloud of activated biotin is released and attaches covalently to surrounding lysine (BioID) and tyrosine (APEX) residues
4. Covalent nature of the tag allows harsh purification conditions without risking to break up labile interactions
5. The cloud of released activated biotin spans roughly 10-20 nm
6. Sub-cellular structures are dynamic and biotinylated protein will spread over time
7. To get a snapshot of the proteins our bait protein interacts with, one should keep incubation times low, but sufficient signal- to-noice ratios also have to be taken into account.
8. By using several bait proteins, one can cross check putative interaction partners and idealy come up with an protein interaction network

Question 18

Chemical corsslinking

Answer 18

• There is a great variety of different crosslinking reagents:
  –> Varying spacer length
  –> Varying reactive groups targeting different amino acid residues on the protein surface
  –> Cross linkers with additional tags for purification to enrich the linked protein complex for analysis
• N-hydroxysuccinimide (NHS) ester based crosslinkers are common (see blue box in figure (b))

Question 19

mass spectrometry

Answer 19

• Isolated proteins are enzymatically digested to form peptides of managable length
• Trypsin is a common choice. It cuts at the C-terminal ends of arginine and lysine residues
• For LC-MS/MS approaches, the tryptic peptides are fed into the MS via an HPLC.
• The peptides get ionized and enter the 1. out of 3 chambers, where they are seperated
• The second containes an inert gas like helium. By collision with gas molecules, the peptides get fragmented
• These fragments are separated in the third chamber