Transcript Comparison of the ligand interactions of truncated and full length

Protein-small molecule interactions for
durg discovery
• Molecules that bind to enzymes and decrease their activity by
stopping the substrate entering the active site
• Blocking an enzyme’s activity can kill a pathogen or correct a
metabolic imbalance
• Activating or Blocking receptors or ion channels and influencing
signal cascades
• Inhibitor binding is either reversible or irreversible
• High specificity and potency ensure that a drug will have few
side effects and thus low toxicity
Surface plasmon resonance
biosensor
•
Changes on the sensor chip surface can be detected by changes of
the refractive index
•
Enables to study protein interactions
Determination of kinetic parameters
•
Kinetic values and the affinity can be calculated from the injection
of a concentration series.
Sensor chip surface
Immobilisation of the enzymes
GABA-receptor
-Receptor is overexpressed in
Sf9-cells
-Lysis of cells
-Immobilisation of lipid vesicle
from cell lysate
Immobilistation
Immobilization of GABA on L1-Chip
Time: 15 min
Flow: 2 µL/min
Empty spot: 2 serving as reference
Estimation of expected binding level from interaction with histamine:
100Da
* 7,500RU  3RU
250,000Da
Sample spot: 1 or 2, 4,000 – 7,500
kRU
 approx. 4 – 7.5 ng(protein)/mm2
 Immobilization level very low for detection of mass-dependent change of
R max 
2017-04-08
refractive index
Interaction analyses with
histamine
...
0.20 mM
25.0 mM
50.0 mM
100 mM
• Preparing different concnetrations of Histamin in 10 mM Tris-HCl,
150 mM NaCl, 0,02 % Protease Inhibitor, pH 8.5
• Studying interaction with prepared surface (ta = 180s , Flow: 90
µL/min)
Evaluation of the sensograms
•Adjusting all sensorgrams to zero
•Subtracting signals from reference surface and
blank injections
•Estimating kinetic values