Abstract
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-Cit0), an engineered protein with six quasi-citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP-qCit6), human component C1 (hMBP-Cit0), and human component C8 (hMBP-Cit6), both obtained from a patient with multiple sclerosis (MS). Both rmMBP-Cit0 and hMBP-Cit0 bound CaM in a Ca2+-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: Kd = 144 ± 76 nM for rmMBP-Cit0, and Kd = 42 ± 15 nM for hMBP-Cit0. For rmMBP-qCit6 and hMBP-Cit6, 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.
Original language | English (US) |
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Pages (from-to) | 1507-1521 |
Number of pages | 15 |
Journal | Protein Science |
Volume | 12 |
Issue number | 7 |
DOIs | |
State | Published - Jul 1 2003 |
Externally published | Yes |
Keywords
- Calmodulin
- Circular dichroism
- Citrulline
- Deimination
- Dynamic light scattering
- Fluorescence lifetime
- Intrinsic Trp fluorescence
- Multiple sclerosis
- Myelin basic protein
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology