Purification and initiation of structural characterization of human peripheral myelin protein 22, an integral membrane protein linked to peripheral neuropathies.
Gene duplications, deletions, and point mutations in peripheral myelin protein 22 (PMP22) are linked to several inherited peripheral neuropathies. However, the structural and biochemical properties of this very hydrophobic putative tetraspan integral membrane protein have received little attention, in part because of difficulties in obtaining milligram quantities of wild type and disease-linked mutant forms of the protein. In this study a fusion protein was constructed consisting of a fragment of lambda repressor, a decahistidine tag, an intervening TEV protease cleavage site, a Strep tag, and the human PMP22 sequence. This fusion protein was expressed in Escherichia coli at a level of 10-20 mg/L of protein. Following TEV cleavage of the fusion partner, PMP22 was purified and its structural properties were examined in several different types of detergent micelles using cross-linking, near and far-UV circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy. PMP22 is highly helical and, in certain detergents, shows evidence of stable tertiary structure. The protein exhibits a strong tendency to dimerize. The 1H-15N TROSY NMR spectrum is well dispersed and contains signals from all regions of the protein. It appears that detergent-solubilized PMP22 is amenable to detailed structural characterization via crystallography or NMR. This work sets the stage for more detailed studies of the structure, folding, and misfolding of wild type and disease-linked mutants in order to unravel the molecular defects underlying peripheral neuropathies.
Mobley
Charles K
CK
Department of Biochemistry and Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8725, USA.
Myers
Jeffrey K
JK
Hadziselimovic
Arina
A
Ellis
Charles D
CD
Sanders
Charles R
CR
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010523:Peripheral Nervous System Diseases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization