2,4-Dinitrophenol reduces the reactivity of Lys553 in the lower 50-kDa region of myosin subfragment 1

Abstract

2,4-Dinitrophenol (DNP) increases the affinity of myosin for actin and accelerates its Mg(2+)ATPase activity, suggesting that it acts on a region of the myosin head that transmits conformational changes to actin- and ATP-binding sites. The binding site/s for DNP are unknown; however similar hydrophobic compounds bind to the 50-kDa subfragment of the myosin head, near the actin-binding interface. In this region, a helix-loop-helix motif contains Lys553, which is specifically labeled with the fluorescent probe 6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS). This reaction is sensitive to conformational changes in the helix-loop-helix and the labeling efficiency was reduced when S1 was bound to actin, DNP or nucleotide analogs. The nucleotide analogs had a range of effects (PPi>ADP·AlF(4)(-)>ADP) irrespective of the open-closed state of switch 2. The greatest reduction in labeling was in the presence of actin or DNP. When we measured the effect of each ligand on the fluorescence of FHS previously attached to S1, only DNP quenched the emission. Together, the results suggest that the helix-loop-helix region is flexible, it is part of the communication pathway between the ATP- and actin-binding sites of myosin and it is proximal to the region of myosin where DNP binds.

Site-Specific Turn-On Fluorescent Labeling of DNA-Interacting Protein Using Oligodeoxynucleotides That Modify Lysines To Produce 5,6-Dimethoxy 3-Methyleneisoindolin-1-one

“…DNA–protein interaction is also regulated by conformational changes in proteins that cause topological changes in the lysine residue involved in such interactions. − Fluorescent labeling is a promising method for studying the localization, structures, and functions of biomolecules. Fluorescent modification of a lysine residue of interest might afford important information about its position and functions that would help in understanding protein structure, function, and molecular mechanism. − In this study, we developed oligodeoxynucleotides (ODNs) that modify primary amines to produce 5,6-dimethoxy 3-methyleneisoindolin-1-one for fluorescent protein modification at a specific lysine residue. We have previously studied the reactivity of ODNs containing 1,4-dicarbonyls ( 1 ), which are a ring-opened form of the C4′-oxidized abasic site (OAS), to modify the DNA-interacting protein at the target lysine residue. − OAS is an oxidatively damaged DNA lesion, and we have determined in an earlier study that ODNs containing OAS modify the primary amine as a five-membered ring lactam ( 2 ) under mild conditions (Scheme ).…”