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Spin-echo 1H NMR studies of differential mobility in gizzard myosin and its subfragments
L E Sommerville1, G D Henry, B D Sykes
1St. Lawrence University, Canton, New York 13617.
Abstract:
The unexpectedly narrow resonances in the 1H NMR spectra of gizzard myosin, heavy meromyosin, and subfragment 1 were examined by spin-echo NMR spectroscopy. These resonances originated predominantly in the myosin heads, or subfragment 1 units. Smooth muscle myosin undergoes a dramatic change in hydrodynamic properties and can exist either as a folded (10S) or as an extended (6S) species. Factors that influence this transition, namely, ionic strength and phosphorylation (or thiophosphorylation), were varied in the NMR experiments. T2 relaxation experiments on dephosphorylated myosin indicated several components of different relaxation times that were not influenced by changes in ionic strength. Our experiments focused on the components with longer relaxation times, i.e., corresponding to nuclei with more mobility, and these were observed selectively in a spin-echo experiment. With dephosphorylated myosin and HMM, increases in ionic strength caused an increased intensity in several of the narrower resonances. The ionic strength dependence of these changes paralleled that for the 10S to 6S transition. With thiophosphorylated myosin and HMM, changes in ionic strength also influenced the intensities of the narrower resonances, and in addition changes in the 1H NMR spectrum due to thiophosphorylation were observed. The narrow resonances seen with myosin and HMM were observed with S1, but the spin-echo spectra of S1 were not influenced either by changes in ionic strength or by phosphorylation. These results suggest that a fraction of the 1H resonances in smooth muscle myosin and its fragments originates from both aliphatic and aromatic residues of increased mobility compared to the mobility expected from hydrodynamic properties of these proteins. In general, the intensities of these residues increase with increasing ionic strength, and this is consistent with an increase in the percentage of mobile residues during the 10S to 6S transition. Segmental flexibility appeared also to be influenced by phosphorylation within the 6S conformation. These changes were not detected in the isolated myosin heads and thus required a higher order of structure, either the subfragment 2 region or the interaction of myosin heads.
Insights
Spin-echo NMR reveals mobile residues in smooth muscle myosin. Increased ionic strength and phosphorylation influence these mobile protein regions, suggesting a higher-order structure is required for these effects.
Area of Science:
- Biochemistry
- Structural Biology
- Nuclear Magnetic Resonance Spectroscopy
Background:
- Smooth muscle myosin exhibits distinct hydrodynamic states (10S and 6S) influenced by ionic strength and phosphorylation.
- Nuclear Magnetic Resonance (NMR) spectroscopy can probe protein dynamics and structure.
- Previous studies have not fully elucidated the dynamic properties of myosin under varying conditions.
Purpose of the Study:
- To investigate the origin and dynamics of narrow resonances observed in the 1H NMR spectra of gizzard myosin, heavy meromyosin (HMM), and subfragment 1 (S1).
- To determine the influence of ionic strength and phosphorylation/thiophosphorylation on these dynamic regions.
- To understand the structural requirements for observing these mobile resonances.
Main Methods:
- Utilized spin-echo 1H NMR spectroscopy to examine myosin, HMM, and S1.
- Performed T2 relaxation experiments to identify components with different relaxation times.
- Varied ionic strength and phosphorylation/thiophosphorylation states during NMR analysis.
Main Results:
- Observed unexpectedly narrow 1H NMR resonances predominantly in myosin heads (S1), indicating increased mobility of aliphatic and aromatic residues.
- Increased ionic strength enhanced the intensity of these narrow resonances, correlating with the 10S to 6S myosin transition.
- Phosphorylation/thiophosphorylation also influenced resonance intensities, with changes not observed in isolated S1, suggesting a role for higher-order structure.
Conclusions:
- A fraction of 1H resonances in smooth muscle myosin and its fragments originates from highly mobile residues.
- The observed mobility is dependent on protein conformation, ionic strength, and phosphorylation, linked to the 10S-6S transition.
- The requirement of higher-order structure (e.g., subfragment 2 interaction) for observing these dynamic changes highlights the importance of myosin head interactions.