Open AccessActin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1
Author(s) -
Peyser Y. Michael,
Muhlrad Andras
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00530.x
Subject(s) - chemistry , nucleotide , pyrophosphate , quenching (fluorescence) , actin , myosin , reactivity (psychology) , fluorescence , stereochemistry , biochemistry , enzyme , medicine , alternative medicine , pathology , quantum mechanics , gene , physics
Bertrand et al . [Bertrand, R., Derancourt, J. & Kassab, R. (1995) Biochemistry 34 , 9500–9507] reported that 6‐[fluoresceine‐5(and 6)‐carboxamido] hexanoic acid succinimidyl ester (FHS) selectively modifies Lys553, which is part of the strong actin‐binding site of myosin subfragment 1 (S1). We found that the reaction of FHS with Lys533 is accompanied by a decrease in the fluorescence intensity of the reagent. The rate of the FHS reaction increased with increasing pH implying that the unprotonated form of the ε‐amino group of Lys553 reacts with FHS. Addition of 0.4 m KCl reduced the rate of reaction significantly, which indicates ionic strength‐dependent changes in the structure of S1. Limited trypsinolysis of S1 before the FHS reaction also decreased the rate of the reaction showing that the structural integrity of S1 is needed for the reactivity of Lys553. ATP, ADP, ADP·BeF x , ADP·AlF 4 , ADP·V i and pyrophosphate significantly decreased the rate of Lys553 labelling, suggesting nucleotide‐induced conformational changes in the environment of Lys553. The fluorescence emission spectrum of the Lys553‐bound FH moiety and the quenching of its fluorescence by nitromethane was not influenced by nucleotides, implying that the chemical reactivity but not the accessibility of Lys553 was decreased by the nucleotide‐induced conformational change. In the presence of ATP when the M ** ADP·P i state of the ATPase cycle is predominantly populated, the reaction rate decreased more than in the case of the S1·ADP·AlF 4 − and S1·ADP·V i complexes, which are believed to mimic the M ** ADP.P i state. This indicates that the conformation of the S1–ADP·AlF 4 − and S1·ADP·V i complexes in the vicinity of Lys553 does not resemble the structure of the M ** ADP·P i state. The rate of Lys553 labelling decreased strongly in the presence of actin. The nitromethane quenching of the Lys553‐bound FHS was not influenced by actin, which indicates that the reduced reaction rate is not due to steric hindrance caused by the bulky protein but by actin induced conformational changes in the vicinity of Lys553.