Purification and spectroscopic characterization of the recombinant BG21 isoform of murine golli myelin basic protein

Abstract

A recombinant form of the murine Golli-myelin basic protein (MBP) isoform BG21 (rmBG21) has been expressed in E. coli, and isolated to 96% purity via metal chelation chromatography. Characteristic yields were 6-8 mg protein per liter of culture in either minimal M9 or standard Luria-Bertani media. Circular dichroism spectroscopy showed that rmBG21 had a large proportion of random coil in aqueous solution, but gained alpha-helix in the presence of monosialoganglioside G(M1) and PI(4)P, as well as in the membrane-mimetic solvent trifluoroethanol. Bioinformatics analyses of the amino acid sequence of rmBG21 predicted an N-terminal calmodulin (CaM)-binding site. It was determined by fluorescence spectroscopy and dynamic light scattering that rmBG21 and CaM interacted weakly in a 1:1 ratio in a Ca(2+)-dependent manner. Solution NMR spectra of uniformly [(13)C(15)N]-labeled protein in aqueous buffer were consistent with it being an extended protein; spectral quality was independent of temperature. Thus, like "classic" MBP and the Golli-MBP isoform J37, rmBG21 is intrinsically disordered, implying multi functionality, and that its conformation depends on its environment and bound ligands.

Binding of the Proline-Rich Segment of Myelin Basic Protein to SH3 Domains: Spectroscopic, Microarray, and Modeling Studies of Ligand Conformation and Effects of Posttranslational Modifications

“…Moreover, the interaction could involve also other structured regions in addition to the PPII segment, as is the case for the Nef protein (124,125). This consideration is plausible, as seems to be the case with calmodulin, where the classic MBP isoforms interact strongly but Golli-MBP isoforms (with almost identical predicted calmodulin targets on them) interact weakly (12,88,89,121). In addition, posttranslational modifications both locally and distally can modulate the interaction as they do for MBP interacting with calmodulin, actin, and tubulin (12,13,(15)(16)(17)71).…”

“…Circular Dichroism. Two 18-residue peptides encompassing the classic polyproline segment of MBP (outlined segment in Figure 1) were purchased from AnaSpec Inc. (San Jose, CA); the manufacturer denotes them as MBP (89)(90)(91)(92)(93)(94)(95)(96)(97)(98)(99)(100)(101)(102)(103)(104)(105) and MBP (89-98T-106). It should be noted that this manufacturer's designation is based on the human 18.5 kDa sequence numbering that includes the N-terminal methionyl residue as 1, even though it is actually cleaved posttranslationally (72,73), and that the numbering used in the literature always starts with the alanyl residue as 1 (2,3), which convention we shall follow here.…”

“…Numerous aromatic amino acids are present in the binding surface, viz., Tyr132, Trp119, Tyr137, Tyr91, and Tyr93. Cesareni et al (81) identified hydrophobic pockets formed by the residues AL(YF)D(YF) (89)(90)(91)(92)(93), WW (119)(120), and PXNY (134-137), although residues Ala89, Leu90, and Trp120 are quite far from the binding site. The first of these binding pockets in the SH3 domain is lined by negatively charged residuessGlu98, Asp99, and Asp100 in the RT loop; Glu116, Asp118, and Glu121 in the n-SRC loop; and Glu129 in the βD strand before the 3 10 helixsand can host a positively charged side chain flanking the core motif in the ligand (see below).…”

Phytolacca americana lectin (Pa-2; pokeweed mitogen): an intrinsically unordered protein and its conversion into partial order at low pH

“…Thus H + induced conformational changes of this unordered protein may be utilized to unveil its mechanism of action and mode of function in sugar binding and mitogenesis. It can be implicated that in vivo sugar binding to Pa-2 drives transconformation similar to what was observed in the case of rmBG21 where monosialoganglioside G M1 induces random coil → α-helix transition in vitro [39]. The above unusual characteristics of Pa-2 motivate us to carry out further structural studies to obtain a clear picture of unique folding pathway and phase transition of 'intrinsically unordered' protein.…”

The BG21 Isoform of Golli Myelin Basic Protein Is Intrinsically Disordered with a Highly Flexible Amino-Terminal Domain

“…Sample Preparation. The 21.6 kDa recombinant murine BG21 (rmBG21) was expressed in Escherichia coli, grown in M9 minimal media supplemented with [ 13 C]glucose and 15 NH 4 Cl, and purified as previously described (30). All measurements were done on a single 1 mM protein sample in 100 mM KCl and 90% H 2 O/10% D 2 O, pH 6.9 ( 0.1.…”