8
A P P L I C A T I O N N O T E
GLP1R – Exendin (9-39) peptide interaction
Exendin (9-39) (Avexitide), a truncated form of
the GLP1R agonist, exendin-4, is a specific GLP-1
receptor antagonist which is known to prevent
hypoglycemia and maintains stability of blood
glucose during a prolonged fast in individuals
with KATPHI (congenital hyperinsulinism owing
to inactivating mutations in the ATP-sensitive K+
channel) (23).
As illustrated in Figure 6A, the copolymer nan-
odisc-stabilized GLP1R had a hydrodynamic
radius (rH) of 5.51 ± 0.12 nm, which aligns with
the anticipated size of the GPCR-lipid-nanodisc
complex. The polydispersity index (PDI) was de-
termined to be 0.06 ± 0.02, indicating that the
purified sample was highly monodisperse. In
addition, DLS analysis of the same sample in a
thermal ramp also shows that the nanodisc-sta-
bilized GLP1R remained thermally stable up to
approximately 58°C.
In summary, the copolymer nanodisc-stabilized P2X4 exhibited excellent colloidal and ther-
mal stability which is essential for interaction analysis. The Spectral Shi method was se-
lected as the optimal approach for analyzing the binding of a small molecule (~355.19Da) to
a large nanodisc (~150 kDa trimeric P2X4 + surrounding lipids and copolymer belt) due to its
sensitivity and precision in detecting biomolecular interactions regardless of mass changes
upon binding. This case study highlights the potential of utilizing the aforementioned system
in a membrane protein ligand screening project.
The Cy5 analogue-labeled GLP1R nanodisc was
maintained at a constant concentration of 20
nM, while the concentration of Exendin (9-39)
was varied between 0.02 nM and 0.4 µM (Fig-
ure 6B). The Kd was determined by analyzing
the Spectral Shi signal yielding 4.51 nM, which
aligns with the published data.
Figure 5: Western blot and SDS PAGE of GLP1R sol-
ubilized with Native MP
TM
. GLP1R runs at a height of
its theoretical molecular weight of 56.17 kDa (marked
with arrows). Detection of proteins in SDS Page via
specific primar y Rho1D4 antibody and secondar y
HRP antibody.
