Open AccessDifferential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N‐terminal region of the heavy chain
Author(s) -
Nikolaeva Olga P.,
Orlov Victor N.,
Bobkov Andrey A.,
Levitsky Dmitrii I.
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.03279.x
Subject(s) - cleavage (geology) , chemistry , differential scanning calorimetry , trypsin , myosin , nucleotide , crystallography , stereochemistry , enzyme , biochemistry , biology , paleontology , physics , fracture (geology) , gene , thermodynamics
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 (V i ) or beryllium fluoride (BeF x ), and in the presence of F‐actin. In the presence of ATP and in the complexes S1·ADP·V i or S1·ADP·BeF x , 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 42.4 °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 P i analogs (V i , BeF x ). 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 transition to higher temperature. Thus, the cleavage between Arg23 and Ile24 does not significantly affect nucleotide‐induced structural changes in the S1, but it prevents structural changes that occur when S1 is bound to F‐actin. The results suggest that the N‐terminal region of the S1 heavy chain plays an important role in structural stabilization of the entire motor domain of the myosin head, and a long‐distance communication pathway may exist between this region and the actin‐binding sites.