Location of Hinges in Myosin II Rod
Location of Hinges in Myosin II Rod
Tech Area / Field
3 Approved without Funding
Institute of Molecular Biology and Biophysics, Georgia, Tbilisi
- University of Washington / Department of Bioengineering, USA, WA, Seattle\nUniversity of Nevada / Department of Biochemistry, USA, NV, Reno
Project summaryThe purpose of the proposed work is the investigation of striated muscle, smooth muscle and nonmuscle II myosins in order to get information about the possible number and location of the hinges in the myosin rod and evaluate the conformational abilities of the localized regions.
According to widely spread point of view,in the process of transformation of chemical energy into mechanical work, the conformational transitions, which take place in the myosin head have the leading role and the cross-bridge fibrilar part is passive. With that, according to many other investigators' points of view (including ours), for creating the true model of structural transition in the myosin molecule during the mechano-chemical act, the conformational transitions in the hinge regions of the fibrilar part of the cross-bridge should be considered.
The existence of hinges in the rod of myosins is of no doubt. But their number, location and the type of the possible conformational transitions in them are undetermined, perhaps because of the underestimation of their role. In most cases it refers to the hinges situated in the junctions of S2/LMM and S1/S2. But according to some authors, the number of regions of strieted muscle myosin molecules which are targets of the attack of proteases in the case of limited proteolysis and which correspond to bending points depends on the enviromental conditions and is more than two.The investigation of the dependence of conformational transitions (10S-6S) on enviromental conditions (solvent composition) for smooth muscle and nonmuscle myosins shows that a rod can bend in the several (more than two) points and thus this phenomenon confirms the last assumptions. Our data which are obtained by the computer analysis of the amino acid sequences of the heavy chains of different muscle and nonmuscle myosins also tell us that the number of hinges may be more than two and propose some point of view about their possible location and the type of the possible conformational transitions in them.
For bending of the rod, which has the two-stranded coiled-coil structure, destabilization of the coiled-coil in some regions and transition to another conformation must occur. Consequently,the number of fragments obtained by limited proteolysis of myosin can give us information about the number of bending regions in this conditions (for given solvent composition). Using the obtained fragment's N- and C- terminal amino acid sequence analysis, the location of the bending regions can be carried out. In another composition, the number of the fragments of digestion will be different and we can receive the picture of structural changes in the rod in response to the changes of solvent composition.
The method of limited digestion is used for the investigation of the structural arrangement of many proteins and for separation of their stable blocks (fragments).If amino acid sequence of a protein is known, by the fragment’s N- and C-terminal sequence analysis it is possible to find the order of fragments in the sequence and reveal structurally labile in a given condition regions. In our previous studies we have successfully used triptic digestion and N- and C- terminal sequence analysis of isolated peptides with the aim to investigate substructure of alpha-actinin.
By comparative (computer) analysis of amino acid sequences of the potential bending regions we can reveal common features of these regions and also determine differences among them. On the basis of this information we shall select the model fragments for the future conformational analysis. We have some experience in computer investigation of coiled -coils generally and myosin's rods in particular.
We suppose, that by these works we can localize regions of bending in rods of different muscle and nonmuscle myosins, determine their number and thus obtain information about potential hinges. Besides, we shall verify our point of view about the number and location of the potential hinges in rods of myosins, obtained by computer analysis of amino acid sequences of the myosins.
Location and investigation of hinges apparently helps to understand the role of the conformational transitions along the myosin rod in contractile process, which as we think has been badly neglected. Obtained by the proposed work data will apparently be useful in future studies of the molecular basis of contraction. For instance, we are informed that our collaborator with Sendy Berstein of San Diego State University are planing to modificate genetically hinge region in Drosophila and study mechanics of single thick fillaments and single myofibrils, comparing wild type with mutants. We hope,that our data will be interesting and useful for planning investigations of this kind. Consequently we are going to exchange information and consult with our collaborator constantly.
Thus, participation of our group in carrying out the proposed project will give some of us possibility to reorientate our activity. It helps us to get involved in the international community of science and supports our next investigations.
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