Purification and Initiation of Structural Characterization of Human Peripheral Myelin Protein 22, an Integral Membrane Protein Linked to Peripheral Neuropathies

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• 作者：Charles K. Mobley ; Jeffrey K. Myers ; Arina Hadziselimovic ; Charles D. Ellis ; Charles R. Sanders
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：October 2, 2007
• 年：2007
• 卷：46
• 期：39
• 页码：11185 - 11195
• 全文大小：338K
• 年卷期：v.46,no.39(October 2, 2007)
• ISSN：1520-4995

文摘

Gene duplications, deletions, and point mutations in peripheral myelin protein 22 (PMP22)are linked to several inherited peripheral neuropathies. However, the structural and biochemical propertiesof this very hydrophobic putative tetraspan integral membrane protein have received little attention, inpart because of difficulties in obtaining milligram quantities of wild type and disease-linked mutant formsof 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. FollowingTEV cleavage of the fusion partner, PMP22 was purified and its structural properties were examined inseveral different types of detergent micelles using cross-linking, near and far-UV circular dichroism, andnuclear 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. Itappears that detergent-solubilized PMP22 is amenable to detailed structural characterization viacrystallography or NMR. This work sets the stage for more detailed studies of the structure, folding, andmisfolding of wild type and disease-linked mutants in order to unravel the molecular defects underlyingperipheral neuropathies.