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

Abstract

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.

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

“…A large body of literature supports the basic assumption of the model: For example, proteolysis of membrane proteins resulted in fragments containing entire TM sequences (Huang et al 1981), and chemically or recombinantly synthesized TM peptides spontaneously assembled thereby rescuing receptor activity (Kahn and Engelman 1992;Ridge et al 1995;Martin et al 1999;Wrubel et al 1994). Finally, peptides corresponding to the N and C terminus (Harmar 2001;O'Hara et al 1993), loop domains (Bennett et al 2004;Katragadda et al 2001a, b;Yeagle et al 2000) and transmembrane domains (Katragadda et al 2001a, b;Cohen et al 2008;Zheng et al 2006;Musial-Siwek et al 2008;Tian et al 2007;Lau et al 2008;Mobley et al 2007;Neumoin et al 2007) from GPCRs have been found to fold to distinct secondary structures which in certain cases resembled the structures of the corresponding regions of the intact receptor.…”

Making the Most of Fusion Tags Technology in Structural Characterization of Membrane Proteins

“…If the hydrophilic surface areas of membrane proteins are too small to contribute sufficiently to polar-polar protein interactions, it will be difficult to obtain high-quality protein crystals. These problems may be solved by using large hydrophilic fusion partners to enlarge the hydrophilic portion of membrane proteins [20,21]. [6,7], alkaline phosphatase (Pho) [6,8,9], b-lactamase [9,10], chloramphenicol acetyltransferase (CAT) [10] Fluorescent protein tag Topology identification, displaying protein interactions…”

“…His tag [19][20][21][22], MBP [23], GST [24], Strep tag [25] Protein tag Expression partner, protein stabilization Large size Ubiquitin [19], cytochrome b562 [26], Protein Z [27], thioredoxin [28], Bcl-XL [29] Peptide tag Detecting proteins, topology identification, displaying protein interactions;…”

“…Another example is the putative tetraspan integral membrane protein, peripheral myelin protein 22 (PMP22). Mobley et al [20] constructed a recombinant version of the PMP22 protein incorporating a His tag. After expression and purification of the recombinant fusion protein and cleavage of the fusion partner, they confirmed that PMP22 is highly helical and shows evidence of a having a stable tertiary structure.…”

“…For example, Zhuang et al used cytochrome b 562 as an insertional fusion tag by fusing it in the middle cytoplasmic loop of lactose permease LacY [20]. After expression and purification, the recombinant protein was reconstituted in the presence of phospholipids, yielding densely packed vesicles and wellordered 2D crystals from which structural information of LacY permease was obtained.…”