Effect of Nucleotides and Actin on the Intramolecular Crosslinking of Myosin Subfragment-1

Abstract

The heavy chain of myosin subfragment-1 (S1) is cleaved by limited trypsinolysis into three fragments, 27, 50, and 20 kDa--aligned in this order from the N-terminus. The tertiary structure of the molecule is essentially not affected by trypsinolysis. The spatial relations between the various regions of the molecule and the nucleotide- and actin-induced intramolecular movements were studied by cross-linking tryptic S1 with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), phenylenediglyoxal (PDG), and glutaraldehyde. The formation of cross-linked products was monitored by SDS-PAGE, using the fluorescent probes 9-anthronitrile and N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS), which specifically label the 27- and 20-kDa fragments, respectively. The reaction with the cross-linkers leads to the formation of 50-kDa/20-kDa, 27-kDa/20-kDa, 27-kDa/50-kDa, and 20-kDa/light chain cross-linked products. Of these, the most intensive was the formation of the 50-kDa/20-kDa products, which appeared as a doublet on the SDS-PAGE with all the cross-linkers. This indicates that the interface between the two fragments is rather extended. The presence of MgATP or MgADP promoted the formation of the 20-kDa/50-kDa cross-linked products, especially with the lower electrophoretic mobility band, when EEDQ was used as a cross-linker. With PDG as a cross-linker, MgATP also affected the cross-link formation between the 20-kDa fragment and the light chains whereas it had no influence on the formation of other products. On the other hand, the effect of actin on the cross-linking with the various cross-linkers was quite extensive, and it was manifested in the reduction of cross-link formation between the various S1 domains. It is concluded that both nucleotides and actin induce intramolecular movements in S1 and that the nucleotide-induced movements are more restricted than those induced by actin, which extend to larger regions of the molecule.

Interaction of the N-Terminal Part of the A1 Essential Light Chain with the Myosin Heavy Chain

“…The proteolysis of the cross-linked S1(A1) resulted in the formation of the 31-, 46-, and 47-kDa fluorescent products, whereas cleavage of the cross-linked S1(A2) generated fluorescent products of 40 kDa and 47 kDa. The 47-kDa products, obtained during digestion of both isoforms correspond to earlier identified products of the cross-linking between N-and C-terminal tryptic fragments of the heavy chain of S1, whereas the 46-kDa product in S1(A1) and the 40-kDa product in S1(A2) are formed by the cross-linking of the C-terminal heavy chain fragment to the respective light chains (20,21). Molecular mass of the 46-kDa product indicates that it contains the A1 light chain FIG.…”

“…Earlier studies have only demonstrated that both A2 light chain and A1 light chain devoid the N-terminal part can be cross-linked with EDC to the N-and C-terminal fragments of the heavy chain (20,21).…”

Cross‐Linking of Wheat Gluten Using a Water‐Soluble Carbodiimide

“…Preferentially intramolecular cross-linking of protein occurred which could increase the conformation stability. For this reason, these conditions were also used to introduce additional tertiary structure into proteins (Blotnick and Muhlrad 1994). Consequently, by changing reaction conditions, intermolecular cross-linking (resulting in a decreased protein solubility) or intramolecular cross-linking (mainly leading to conformation stabilization) can be achieved, thereby controlling the specific properties of the final product.…”