Hydrogen/deuterium exchange mass spectrometry of actin in various biochemical contexts.
Hydrogen/deuterium exchange mass spectrometry (H/D MS) of monomeric actin (G-actin), polymeric actin (F-actin), phalloidin-bound F-actin and G-actin complexed with DNase I provides new insights into the architecture of F-actin and the effects of phalloidin and DNase I binding. Although the overall pattern of deuteration change supports the gross features of the Holmes F-actin model, two important differences were observed. Most significantly, no change in deuteration was observed in the critical "hydrophobic plug" region, suggesting this feature may not be present. Polymerization also produced deuteration increases for peptide fragments containing the ATP phosphate-binding loops, suggesting G-actin transitions to a more "open" conformation upon polymerization. However, polymerization produced decreases in deuteration mainly localized to the "inner", filament-axis side as predicted by the Holmes model. Mapping the phalloidin-induced decreases in F-actin deuteration onto the Lorenz binding site produced a single common patch straddling two monomers across the 1-start helix contact, again consistent with the Holmes architecture. Finally, both DNase I and phalloidin were able to alter the deuteration of regions distal to their respective binding sites. These results highlight the great opportunities for H/D MS to exploit high-resolution structures for detailed studies of the organization and dynamics of complex molecular assemblies.
Chik
John K
JK
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Dr NW, T2N 4N1, Calgary, Alta., Canada.
Schriemer
David C
DC
D000199:Actins / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003850:Deoxyribonuclease I / Q000737:chemistry; D003903:Deuterium; D004735:Energy Transfer; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D010590:Phalloidine / Q000737:chemistry; D011487:Protein Conformation; D011817:Rabbits
D000199:Actins; D010590:Phalloidine; D006859:Hydrogen; D000255:Adenosine Triphosphate; D003903:Deuterium; D003850:Deoxyribonuclease I