Abstract
BackgroundThermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins (actin and myosin) in their native environment in striated muscle fibres. The interaction of actin with myosin in the highly organized muscle structure is affected by internal forces; therefore their altered conformation and interaction may differ from those obtained in solution. The energetics of long functioning intermediate states of ATP hydrolysis cycle was studied in muscle fibres by differential scanning calorimetry (DSC).ResultsSETARAM Micro DSC-II was used to monitor the thermal denaturation of the fibre system in rigor and in the presence of nucleotide and nucleotide analogues. The AM.ADP.Pi state of the ATP hydrolysis cycle has a very short lifetime therefore, we mimicked the different intermediate states with AMP.PNP and/or inorganic phosphate analogues Vi and AlF4 or BeFx. Studying glycerol-extracted muscle fibres from the rabbit psoas muscle by DSC, three characteristic thermal transitions were detected in rigor. The thermal transitions can be assigned to myosin heads, myosin rods and actin with transition temperatures (Tm) of 52.9 ± 0.7°C, 57.9 ± 0.7°C, 63.7 ± 1.0°C. In different intermediate states of the ATP hydrolysis mimicked by nucleotide analogues a fourth thermal transition was also detected which is very likely connected with nucleotide binding domain of myosin and/or actin filaments. This transition temperature Tm4 depended on the mimicked intermediate states, and varied in the range of 66°C – 77°C.ConclusionAccording to DSC measurements, strongly and weakly binding states of myosin to actin were significantly different. In the presence of ADP only a moderate change of the DSC pattern was detected in comparison with rigor, whereas in ADP.Pi state trapped by Vi, AlF4 or BeFx a remarkable stabilization was detected on the myosin head and actin filament which is reflected in a 3.0 – 10.0°C shift in Tm to higher temperature. A similar effect was observed in the case of the nonhydrolyzable AMP.PNP analogue. Differential DSC measurements suggest that stabilization actin structure in the intermediate states of ATP hydrolysis may play an additional role in actin-myosin interaction.
Highlights
Thermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins in their native environment in striated muscle fibres
In the presence of Ca2+, the energy released from ATP hydrolysis produces conformational changes in myosin and actin, which can be manifested as an internal motion of the myosin head while bound to actin [1,2,3,4,5,6,7,8,9]
The powerful differential scanning calorimetry (DSC) technique allows the derivation of heat capacity of proteins as a function of temperature
Summary
Thermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins (actin and myosin) in their native environment in striated muscle fibres. The interaction of actin with myosin in the highly organized muscle structure is affected by internal forces; their altered conformation and interaction may differ from those obtained in solution. The energetics of long functioning intermediate states of ATP hydrolysis cycle was studied in muscle fibres by differential scanning calorimetry (DSC). Force generation in muscle during contraction arises from direct interaction of the two main protein components of the muscle, myosin and actin. Using the advantage of the SETARAM microcalorimeter in this work, we tried to approach the temperature-induced unfolding processes in different intermediate states of ATP hydrolysis in striated muscle fibres. In the weakly binding state the inorganic phosphate (Pi) was substituted by the phosphate analogue orthovanadate, AlF4 and BeFx
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