TAILIEUCHUNG - Báo cáo Y học: Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N-terminal region of the heavy chain

The thermal unfoldingofmyosinsubfragment 1 (S1) cleaved by trypsin was studied by differential scanning calorimetry. In the absence of nucleotides, trypsin splits the S1 heavy chain into three fragments (25, 50, and 20 kDa). This cleavage has no appreciable influence on the thermal unfolding of S1 examined in the presence of ADP, in the ternary complexes of S1 with ADP and phosphate analogs, such as orthovanadate (Vi) or berylliumfluoride (BeFx), and in the presence of F-actin. | Eur. J. Biochem. 269 5678-5688 2002 FEBS 2002 doi Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N-terminal region of the heavy chain Olga P. Nikolaeva1 Victor N. Orlov1 Andrey A. Bobkov2 and Dmitrii I. Levitsky1 2 1A. N. Belozersky Institute of Physico-Chemical Biology Moscow State University and 2 A. N. Bach Institute of Biochemistry Russian Academy of Sciences Moscow Russia The thermal unfolding of myosin subfragment 1 S1 cleaved by trypsin was studied by differential scanning calorimetry. In the absence of nucleotides trypsin splits the S1 heavy chain into three fragments 25 50 and 20 kDa . This cleavage has no appreciable influence on the thermal unfolding of S1 examined in the presence of ADP in the ternary complexes of S1 with ADP and phosphate analogs such as orthovanadate Vi or beryllium fluoride BeFx and in the presence of F-actin. In the presence of ATP and in the complexes S1-ADP Vi or S1-ADP BeFx trypsin produces two additional cleavages in the S1 heavy chain a faster cleavage in the N-terminal region between Arg23 and Ile24 and a slower cleavage at the 50 kDa fragment. It has been shown that the N-terminal cleavage strongly decreases the thermal stability of S1 by shifting the maximum of its thermal transition by about 7 C to a lower temperature from 50 C to C whereas the cleavage at both these sites causes dramatic destabilization of the S1 molecule leading to total loss of its thermal transition. Our results show that S1 with ATP-induced N-terminal cleavage is able like uncleaved S1 to undergo global structural changes in forming the stable ternary complexes with ADP and Pi analogs Vi BeFx . These changes are reflected in a pronounced increase of S1 thermal stability. However S1 cleaved by trypsin in the N-terminal region is unable unlike S1 to undergo structural changes induced by interaction with F-actin that are expressed in a 4-5 C shift of the S1 thermal .

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