Background: Obesity has emerged as a global public health challenge associated with
increased risk of hyperlipidemia and hypertension. It contributes to high sympathetic activity and
increased catecholamine levels. The hypothalamic melanocortin system is known to regulate the energy
homeostasis. The role of melanocortin 4 receptor (MC4R) has been demonstrated pharmacologically
and in animal studies, which showed that severe obesity in MC4R knockout mice was
caused by increased food intake and decreased energy consumption. Over 70 multiple different mis-
-sense and nonsense mutations in hMC4R have been found at a high frequency of 2-8% in severe
early onset or hereditary obesity. The single amino acid variation (D90N) located in the second
transmembrane domain (TM2) of MC4R results in accelerated growth and childhood onset obesity.
Interestingly, the functional characterization of D90N hMC4R mutant TM2 (m-hMC4R-TM2) revealed
normal cell surface expression and binding with agonist similar to the hMC4R wild-type
TM2 (wt-hMC4R-TM2) but loss of signal transduction mediated via Gs/adenylyl cyclase activation.
It is essential to delineate the three-dimensional structure of MC4Rs in order to elucidate their
Objective: In this study, we demonstrate the optimized expression and isolation of wt/m-hMC4R-TM2
proteins under different chemical cleavage reaction times and purification procedures via SDS
precipitation. The solid-state NMR spectroscopy was carried out to study the structure of wt/m-hMC4R-
TM2 protein in the anisotropic phospholipid bicelles.
Methods: The KSI-wt/m-hMC4R-TM2 fusion proteins developed in cell culture with LB medium.
In order to isolate the expressed fusion protein from the cell, ultrasonication, Ni-NTA affinity chromatography,
dialysis, and lyophilization techniques were used. Then, to obtain a protein with higher
purity and higher yield, the CNBr chemical cleavage time was subdivided into 30 minutes, 1 h,
2 h, 3 h, and 4 h. Purification process was performed using FPLC, and 100 mM KCl and dialysis
were used to remove the SDS. CD spectrometer, MALDI-TOF, solution-state NMR, and solid-state
NMR were used to confirmed purity and structure of the wt/m-hMC4R-TM2.
Results: The precipitation method was used to remove the SDS bound to proteins as KCl-SDS. We
optimized the 2 h cleavage reaction times for both wt-hMC4R-TM2 and m-hMC4R-TM2 depending
on the purity based on mass spectra and 1H-15N HSQC spectra and the yield after final purification.
The 1D 1H-15N CP (Cross polarization) solid-state NMR spectra suggest that the wt/m-hMC4R-
TM2 undergo rotational diffusion around a perpendicular axis along the bilayer normal.
Conclusion: We expressed wt/m-hMC4R-TM2 in E.coli and optimized the isolation and purification
process, especially CNBr chemical cleavage time. The efficiency of KCl-SDS precipitation
was confirmed via MALDI-TOF MS and the pure proteins obtained using this method were characterized
by CD spectroscopy and solution-state NMR. The results of 1H-15N HSQC spectra in solution-
state NMR also show the probability for structural studies. The 1D 1H-15N CP solid-state NMR
spectra indicate that most of the residues in both the wt/m-hMC4R-TM2 peptides are integrated into