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Binding Quantification with Thermophoresis. Measurements. Speaker: Christian Niederauer. Outline. Protein-Protein Interactions Grb2 Dimerization β- Lactamase TEM1 Binding to its I nhibitor BLIP Protein-Peptide Interaction: AMA1 and RON2

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binding quantification with thermophoresis

Binding QuantificationwithThermophoresis

Measurements

Speaker: Christian Niederauer

slide2

Outline

  • Protein-Protein Interactions
    • Grb2 Dimerization
    • β-Lactamase TEM1 Binding toitsInhibitor BLIP
  • Protein-Peptide Interaction: AMA1 and RON2
  • Analyzing GPCR Membrane Proteins NTS1 & A2aR
  • Cooperative binding: Synaptotagmin
outline
Outline
  • Protein-Protein Interactions
    • Grb2 Dimerization
    • β-Lactamase TEM1 Binding toits Inhibitor BLIP
  • Protein-Peptide Interaction: AMA1 and RON2
  • Analyzing GPCR Membrane Proteins NTS1 & A2aR
  • Cooperativebinding: Synaptotagmin
protein protein b inding grb2 grb2 dimerization
Protein-Protein Binding: Grb2-Grb2Dimerization

Grb2 dimers may control

activity of FGFR2

Dimerization of Grb2 is analyzed

Dimerizationaffinitytoo high for ITC approach

DLS yieldsreliableresultswecancompareto MST!

fixed concentration of labeled grb2
Fixed ConcentrationofLabeledGrb2

KD=0.65 ± 0.08 µM

monomer

dimer

MST allowsusageofproteinconcentrationsfarbelowactualKD

DLS:

  • Monomer predominantupto 0.4 µM
  • DLS: Dimer predominantfrom 0.4µM upwards

consistentwith MST measurements

slide6

Outline

  • Protein-Protein Interactions
    • Grb2 Dimerization
    • β-Lactamase TEM1 Binding toits Inhibitor BLIP
  • Protein-Peptide Interaction: AMA1 and RON2
  • Analyzing GPCR Membrane Proteins NTS1 & A2aR
  • Cooperativebinding: Synaptotagmin
protein protein b inding lactamase tem1 blip
Protein-Protein Binding: β-Lactamase TEM1 -BLIP

TEM-1:

anti-antibioticenzyme

BLIP:

inhibits TEM-1

Contributionofdifferent aminoacids?

Investigation withproteinmutants

fixed c oncentration of labeled wt tem 1
Fixed ConcentrationofLabeledwt-TEM-1

wt-BLIPistitrated

  • KD=3.8 ± 1 nM(check: 3.2 ± 0.6nM)
fixed c oncentration of l abeled wt tem 1
Fixed ConcentrationofLabeledwt-TEM-1

W112A-BLIPistitrated

  • KD=0.5 ± 0.1 µM (check: 0.36 µM)

position112

tryptophanalanine

fixed concentration of labeled wt tem 1
Fixed ConcentrationofLabeledwt-TEM-1

W150A-BLIPistitrated

  • KD=1.7 ± 0.4 µM (check: 3.8 ± 0.6 µM)

position150

tryptophanalanine

mirrored a ssay reversed titration protocol
Mirrored Assay: Reversed Titration Protocol

Fixed ConcentrationofLabeledwt-BLIP

mirrored assay r eversed titration protocol
MirroredAssay: Reversed Titration Protocol

Fixed ConcentrationofLabeledwt-BLIP

  • wt-TEM: KD= 4.8 ± 1.7 nM (check:KD=3.8 ± 1 nM)
  • wt-TEM in lysate: KD=10 ± 4 nM

R243A-TEM: KD= 190 ± 50 nM

position243

argininealanine

slide14

Outline

  • Protein-Protein Interactions
    • Grb2 Dimerization
    • β-Lactamase TEM1 Binding toits Inhibitor BLIP
  • Protein-Peptide Interaction: AMA1 and RON2
  • Analyzing GPCR Membrane Proteins NTS1 & A2aR
  • Cooperativebinding: Synaptotagmin
protein peptid b inding ama1 ron2
Protein-Peptid Binding: AMA1 –RON2
  • Prakash Srinivasa, et al. : Binding of Plasmodiummerozoite proteins RON2 and AMA1 triggers commitment to invasion, Proceedingsofthe National Academy ofSciences 2011
protein peptid b inding ama1 ron21
Protein-Peptid Binding: AMA1 –RON2
  • Plasmodium falciparum invades red blood cells and causes malaria
  • invasion is critically dependend on RON2-AMA1-interaction
  • quantification of AMA1-RON2 binding via MST
  • RON2 has two cysteine residues forming a disulfide bridge:
  •  essential for binding to AMA1
  •  RON2 mutation/alkylation experiments show abolished binding

unlabeled

slide17

Fixed Concentration of Labeled RON2

MST: Single binding event with KD= 28 ± 2 nM

Check: SPR withKD = 13 ± 1 nM

closer look at the two binding events
Closer Look at the Two Binding Events
  • each half of datapoints is fitted
  • high affinity KD= 62 ± 16 nM
  • lowaffinityKD= 1.4 ± 0.2 µM
  • fit of equation including two events
  • high affinity KD= 81 ± 21 nM
  • low affinity KD= 1.2 ± 0.1 µM

high affinity KD varies because the fit is restricted to the first half of data

 saturation plateau is missing because low affinity event is superimposing

protein peptid binding ama1 ron2
Protein-Peptid Binding AMA1 –RON2
  • shows importance to perform the assay in both ways
    • AMA1-titration only shows high-affinity binding event
    • RON2-titration reveals low-affinity binding event
  • high affinity KD has to be inferred from AMA1-titration
  • unlabeled RON2 peptide might have two forms in solution:

with and without disulfide bond (cyclized and linear form)

slide21

Outline

  • Protein-Protein Interactions
    • Grb2 Dimerization
    • β-Lactamase TEM1 Binding toits Inhibitor BLIP
  • Protein-Peptide Interaction: AMA1 and RON2
  • AnalyzingGCRP Membrane Proteins NTS1 & A2aR
  • Cooperativebinding: Synaptotagmin
analyzing gcrp membrane proteins
Analyzing GCRP Membrane Proteins

largest class of membrane proteins encoded in the human genome

 target of approximately 40% of all modern medicinal drugs!

  • seven transmembrane helices
  • connected by flexible loops
  • great structural diversity at the extracellular ligand binding site
gcrp neurotensin receptor 1 nts1
GCRP: Neurotensin Receptor 1 (NTS1)

Neurotensin:

found in neural tissue and possibly related to mental disorders

 binding of Neurotensin to its receptor examined

 influence of NTS1 antagonist SR48692 interesting

label-free approach possible due to Trp in NTS1

binding of nts to nts1
Binding of NTS to NTS1

Titrating NT to unlabeled NTS1:

KD 20nM

Titrating NTS1 to labeled NT:

KD= 21 ± 20 nM

high error: NTS1 amount was limited

 saturation could not be reached

  • SPR check:
  • 1-2 nM for both unlabeled
  • 7 ± 3 nMfor TAMRA-labeledNeurotensin
  • 1.4 nMfor Cy5-labeled Neurotensin

KD dependence on fluorophore

effect of antagonist on nts1
Effect of Antagonist on NTS1

Label-free SR48692 titrated to NTS1:

KD= 15 ± 11 nM(literature check: 3-10 nM)

Competition Assay: SR titrated to pre-saturated (1µM NT) receptor:

KD= 640 ± 50 nM(shift of one order of magnitude!)

 competition for same binding pocket

slide26

Effect of Antagonist on NTS1

  • binding of SR yields opposite effect in thermophoretic depletion
  • NT promotes conformational changes in NTS1 (100 kDa)
  • SR locks NTS1 in an inactive conformation

 different hydration shell  thermophoretic properties change

a denosin a2a receptor
Adenosin A2A Receptor
  • A2aR is regulates myocardial blood flow
  • binding is observed with label-free MST:

orthosteric antagonists:

caffeine (19µM)

theophylline (14µM)

ZM241385 (1.2nM)

allosteric ligand: amiloride (12µM)

a2ar binding to orthosteric antagonists
A2aR Binding to Orthosteric Antagonists
  • Caffeine: 40± 17 µM
  • Theophylline : 5± 2µM
  • ZM241385: 43nM

comparably small change in thermophoretic mobility

influence of amiloride to a2ar binding
Influence of Amiloride to A2aR Binding

Caffeine @250µM amiloride

40µM  84 ± 10µM

[email protected]µM amiloride

5 nM 27 ± 6µM

Amiloride (KD = 52 ±7µM) induces conformational change:

receptor activity and hydration shell change

  • thermophoretic mobility increases strongly and changes signs

MST can also be used to investigate allosteric binding effects!

slide31

Outline

  • Protein-Protein Interactions
    • Grb2 Dimerization
    • β-Lactamase TEM1 Binding toits Inhibitor BLIP
  • Protein-Peptide Interaction: AMA1 and RON2
  • Analyzing GPCR Membrane Proteins NTS1 & A2aR
  • Cooperativebinding: Synaptotagmin
synaptotagmin syt1
Synaptotagmin (syt1)

Ca2+-sensor involved in neurotransmitter-release

cooperative binding synaptotagmin
Cooperative Binding: Synaptotagmin

Synaptotagmin binds to both Ca2+ and membranes with incorporated PIP2

  • Ca2+
  • liposomes containg PIP2
  • Synaptotagmin

two possible pathways:

A: Ca2+ binds to syt1, complex binds to liposome

B: syt1 binds to liposome, Ca2+ binds to syt1

cooperative b inding c a 2 a nd pip2
Cooperative Binding: Ca2+ andPIP2

KD,Ca=13± 3µMwith Ca2+

KD= 50± 10µM without

binding of syt1 to liposomes with PIP2 rises in presence of Ca2+

 reverse assay: Ca2+binding to syt1 with different PIP2 concentration

slide35

Cooperative Binding: Ca2+ and PIP2

PIP2: added directly

syt1-PIP2 saturates if [PIP2] > 10µM

syt1- Ca2+ saturates if [Ca2+] > 50µM

slide36

Cooperative Binding: Ca2+ and PIP2

Pathway A: syt1 saturated with Ca2+: KD for PIP2 decreases from 20µM to < 2µM

Pathway B: syt1 saturated with PIP2: KD for Ca2+ decreases from 220µM to 3.3µM

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