Structural studies of isolated native thick filaments from rabbit psoas muscle with AMP deaminase decoration

Abstract

AMP deaminase (adenylate deaminase; AMP aminohydrolase, EC 3.5.4.6), a large flat tetrameric enzyme found in skeletal muscle, binds strongly and specifically to the subfragment-2 region of rabbit skeletal muscle myosin. This allows its use as a structural probe in myosin and myosin rod aggregation studies. When mixed with a preparation of isolated native thick filaments, AMP deaminase decorates the entire filament backbone except for the central bare zone. Binding is particularly ordered in the banded region, where 11 stripes of about 43-nm spacing on either side of the bare zone have been observed in studies of isolated A-bands. No systematic absence of deaminase is seen here, indicating that the presence of the C-protein and H-protein bands does not make the binding site inaccessible to the tetramer. Optical diffraction patterns of the decorated filaments show the expected 42.9-nm periodicities and a reflection indexing at 28.6 nm. Because of the bulkiness of the tetramer relative to the number of available binding sites at each 14.3-nm interval along the filament shaft, the helix net is being sampled at a lower frequency than is the underlying myosin organization. As a result, reflections on layer lines other than orders of 42.9 nm are also observed (e.g., 57.2); these reflections strongly indicate a structure based on a 12/1 primitive helix. The results appear to eliminate the symmetric double two-fold and three-fold models of thick filament structure but are consistent with an asymmetric four-fold structure.