Biomedical subjects

Christopher M D Hill

Publications and source records attributed to Christopher M D Hill.

3 recordsLinked to original sources

PubMed2003

Myelin basic protein has multiple calmodulin-binding sites.

Myelin basic protein (MBP) has been shown to bind calmodulin (CaM) in a specific Ca(2+)-dependent manner via a primary target sequence at its C-terminus [Protein Sci. 12 (2003) 1507]. Upon deimination of MBP, the nature of the interaction changed significantly, suggesting either a new binding site or different conformers with different affinities for CaM. In order to resolve this issue, we investigated here the CaM-binding properties of N- and C-terminal deletion mutants of MBP using Trp fluorescence spectroscopy and mass spectrometry. We conclude that there is an additional CaM-binding site on MBP in a central segment (we posit murine residues 82-93) that forms an amphipathic alpha-helix.

Amino Acid Sequence↗

PubMed2003

Terminal deletion mutants of myelin basic protein: new insights into self-association and phospholipid interactions.

The 18.5kDa isoform of myelin basic protein (MBP) has strong and probably specific interactions with phosphoinositides that are of interest regarding this protein's function, and in effecting its two-dimensional crystallization for structural determination. We have designed and constructed truncation mutants of recombinant 18.5kDa murine myelin basic protein (rmMBP) lacking either the N- or C-terminal third, i.e. rmMBPDeltaN and rmMBPDeltaC, respectively. Both variants rmMBPDeltaC and rmMBPDeltaN generally had a reduced ability to aggregate lipid vesicles, compared to the whole protein, especially at lower protein/lipid ratios. Lipid vesicle cosedimentation showed that both truncated variants exhibited altered binding with phosphatidylinositol (PI). Incubation of these proteins under monolayers comprising PI and a nickel-chelating lipid yielded crystalline arrays of rmMBPDeltaC (but not rmMBPDeltaN) in the absence of high salt or osmolytes, which are required for crystallization of whole protein. This result suggests that the C-terminal segment of MBP is a significant source of conformational heterogeneity, and its removal will facilitate future planar or three-dimensional crystallization attempts. Incubation of rmMBPDeltaN and rmMBPDeltaC under monolayers comprising phosphatidylinositol-4-phosphate and a nickel-chelating lipid yielded tubular structures of opposite chirality, suggesting a synergistic effect of both termini of MBP in organizing myelin lipids.

Amino Acid Sequence↗

PubMed2003

Interaction of the 18.5-kD isoform of myelin basic protein with Ca2+ -calmodulin: effects of deimination assessed by intrinsic Trp fluorescence spectroscopy, dynamic light scattering, and circular dichroism.

The effects of deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) on its binding to calmodulin (CaM) have been examined. Four species of MBP were investigated: unmodified recombinant murine MBP (rmMBP-Cit(0)), an engineered protein with six quasi-citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP-qCit(6)), human component C1 (hMBP-Cit(0)), and human component C8 (hMBP-Cit(6)), both obtained from a patient with multiple sclerosis (MS). Both rmMBP-Cit(0) and hMBP-Cit(0) bound CaM in a Ca(2+)-dependent manner and primarily in a 1:1 stoichiometry, which was verified by dynamic light scattering. Circular dichroic spectroscopy was unable to detect any changes in secondary structure in MBP upon CaM-binding. Inherent Trp fluorescence spectroscopy and a single-site binding model were used to determine the dissociation constants: K(d) = 144 +/- 76 nM for rmMBP-Cit(0), and K(d) = 42 +/- 15 nM for hMBP-Cit(0). For rmMBP-qCit(6) and hMBP-Cit(6), the changes in fluorescence were suggestive of a two-site interaction, although the dissociation constants could not be accurately determined. These results can be explained by a local conformational change induced in MBP by deimination, exposing a second binding site with a weaker association with CaM, or by the existence of several conformers of deiminated MBP. Titration with the collisional quencher acrylamide, and steady-state and lifetime measurements of the fluorescence at 340 nm, showed both dynamic and static components to the quenching, and differences between the unmodified and deiminated proteins that were also consistent with a local conformational change due to deimination.

Acrylamides↗