Journal of Biomolecular NMR 29: 545–546, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 545 Letter to the Editor: Backbone resonance assignments of the 18.5 kDa isoform of murine myelin basic protein (MBP) David S. Libich a , Valerie J. Robertson b , Martine M. Monette c & George Harauz a,∗ a Department of Molecular Biology and Genetics, and Biophysics Interdepartmental Group and b Department of Chemistry and Biochemistry, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada, N1G2W1; c Bruker BioSpin Ltd., 555 Steeles Avenue East, Milton, Ontario, Canada, L9T 1Y6. Received 13 February 2004; Accepted 1 April 2004 Key words: correlation spectroscopy, multiple sclerosis, myelin basic protein, NMR Biological context Myelin basic protein (MBP) is a family of developmen- tally-regulated and translocatable isoforms involved in formation of the myelin sheath of the central nervous system (Campagnoni and Skoff, 2001). The 18.5 kDa isoform of MBP is the most common in adult humans and exists as a series of highly post-translationally modified charge isomers (Kim et al., 2003). The in vivo environment of this isoform is the major dense line of myelin, where it maintains the cytoplasmic leaflets of the oligodendrocyte membrane in close ap- position. Deiminated MBP is a candidate autoantigen in multiple sclerosis. Other MBP isoforms potentially have roles in signalling pathways during myelin devel- opment, yet our understanding of these phenomena is limited by the lack of detailed structural knowledge. All MBP isoforms are ‘intrinsically unstructured’ to facilitate their interactions with diverse ligands (Hill et al., 2002). The protein is primarily a flexible coil in aqueous solution, but attains ordered secondary struc- ture in the presence of detergents and lipids, as well as in organic solvents such as trifluorethanol (TFE) (Liebes et al., 1975). Recently, site-directed spin labelling and electron paramagnetic resonance experi- ments on a recombinant form of the 18.5 kDa isoform of murine MBP (rmMBP, 176 residues, M r 19421.5 Da) in protein-vesicle pellets have defined how dif- ferent regions of the protein interact with myelin-like membranes (Bates et al., 2004). We have turned to solution NMR in order to define its tertiary struc- ∗ To whom correspondence should be addressed. E-mail: gharauz@uoguelph.ca. ture, and present here the first NMR assignment of the backbone of uniformly 13 C 15 N-labelled rmMBP. Methods and experiments The 18.5 kDa rmMBP (unmodified qC1 form, 176 residues including a C-terminal LEH 6 tag) was ex- pressed in E. coli BL21-CodonPlus(DE3)-RP cells (Stratagene, La Jolla, CA), and purified as previously described with some modifications to improve yield (Bates et al., 2002). Upon growth in M9 minimal media supplemented with 15 NH 4 Cl and 13 C 6 -glucose (Cambridge Isotope Laboratories – C.I.L., Cambridge, MA), a yield of 10 mg of purified, uniformly 13 C 15 N- labelled rmMBP was obtained per litre of culture. The protein was dissolved in 30% TFE-d 2 (C.I.L.) in water at a concentration of 2 mM, and NMR studies were undertaken at 25 ◦ C on a Bruker Avance spectrometer operating at a proton frequency of 600 MHz. Two- dimensional 1 H- 15 N HSQC data (Figure 1), and a combination of triple-resonance spectra using several complementary pulse sequences (HNCO, CBCANH, CBCA(CO)NH, HCC(CO)NH) were obtained (Sattler et al., 1999). The 1 H and 13 C chemical shifts were referenced to TSP (3-(trimethylsilyl)-propionic acid- d 4 sodium salt) in a capillary tube, and 15 N chemical shifts were referenced indirectly to liquid ammonia. Backbone assignment was performed using AURE- MOL (www.auremol.de) (Gronwald and Kalbitzer, 2004) and PACES (Coggins and Zhou, 2003) software.