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

Archives of biochemistry and biophysics (2010-10-05)
Theresa R Bomfim, Luciana E S F Machado, Luis Mauricio T R Lima, Martha M Sorenson, Verônica P Salerno
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
6-[Fluorescein-5(6)-carboxamido]hexanoic acid, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
6-[Fluorescein-5(6)-carboxamido]hexanoic acid N-hydroxysuccinimide ester, suitable for fluorescence, ≥75% (HPLC)

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