Using mass spectrometry to study copper-protein binding under native and non-native conditions: beta-2-microglobulin.
A method based on metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) has been used to determine the Cu(II) binding sites in both native and unfolded conformations of beta-2-microglobulin (beta2m). Recent studies have shown that beta2m is destabilized and can form amyloid fibers in the presence of Cu(II). An increased affinity for Cu in unfolded states compared to that of the native state is suspected to facilitate overall protein destabilization. Cu-binding site information for native beta2m is difficult to obtain using traditional techniques because of its propensity to form amyloid fibers at relatively high protein concentrations in the presence of Cu and because of the nonspecific paramagnetic peak broadening observed in NMR analyses. In addition, Cu-binding information of unfolded beta2m is complicated by the high concentrations of denaturants (e.g., 8 M urea) needed to ensure protein unfolding. The MCO/MS approach has been successfully employed in this work to overcome these difficulties. The sensitivity of MS allowed the Cu-binding site of the native protein to be determined at the low concentrations of beta2m necessary to avoid amyloid fiber formation. Results indicate that the N-terminus of the protein and His31 are responsible for Cu(II) coordination in the native state. The MCO/MS method was also successful at determining the Cu-binding site in the presence of 8 M urea with the N-terminus, His31, His51, and His81 found to be Cu-bound in the unfolded state. This result supports the existence of a well-defined but different coordination structure in the unfolded state, which leads to the greater affinity for Cu(II) observed in the unfolded state of the protein. In general, it appears that the MCO/MS method is capable of providing Cu-binding site information for proteins that are difficult to study by traditional means.
Lim
Jihyeon
J
Department of Chemistry, University of Massachusetts-Amherst, Amherst, MA 01003, USA.
Vachet
Richard W
RW
D001665:Binding Sites; D002384:Catalysis; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D011489:Protein Denaturation; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000378:metabolism
D001613:beta 2-Microglobulin; D003300:Copper