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 functional aspects.
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 solidstate 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, 4 h, 5 h. Purification process was
performed using FPLC, and 100 mM KCl and dialysis were used to remove the SDS. CD spectrometer, MALDITOF, solution-state NMR, and solid-state NMR were used to confirmed purity and structure of the wt/mhMC4R-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 MALDITOF MS and the pure proteins obtained using this method were characterized by CD spectroscopy and solutionstate 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-hMC4RTM2 peptides are integrated into the membrane.
Algorithms for automated assignment of solution-state and solid-state protein NMR spectra.