Temperature dependence of myosin-II tail fragment assembly.
Dictyostelium myosin-II bipolar thick filament (BTF) assembly is heavily dependent on ionic strength and temperature and is reversible by the phosphorylation of just three threonines. Truncated tail fragments of Dictyostelium myosin-II are commonly used as models for BTF assembly, as they self-assemble into regular paracrystals that recapitulate the ionic strength and phosphorylation dependence of full-length Dictyostelium myosin-II BTF assembly. Here we show that Dictyostelium myosin-II tail fragment assembly is highly temperature dependent, similar to full-length Dictyostelium myosin-II. Assembly of paracrystals was far more robust at 4 degrees C than at higher temperatures. Pre-assembled paracrystals disassembled completely when shifted to 37 degrees C, indicating that assembly does not greatly improve the thermostability of these tail fragments. The melting temperatures of individual Dictyostelium myosin-II tail coiled-coils under both low and high ionic strength conditions that prohibit paracrystal assembly are extremely low, 21 degrees C and 28 degrees C, respectively. These data are consistent with reversible thermal denaturation of the coiled-coil as the most likely explanation for assembly incompetence under either very low ionic strength or high temperature conditions. Assembled paracrystals of a structurally similar fragment of nonmuscle myosin-IIA were far more thermodynamically stable than their Dictyostelium counterparts at the temperatures examined here.
McMahon
Peggy M
PM
University of Illinois College of Veterinary Medicine, Urbana, IL 61802, USA. pmmcmaho@illinois.edu
Hostetter
Daniel R
DR
Rice
Sarah E
SE
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003461:Crystallography; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D004560:Electricity; D006801:Humans; D046529:Microscopy, Electron, Transmission; D024462:Myosin Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024507:Nonmuscle Myosin Type IIA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D044366:Transition Temperature
D010446:Peptide Fragments; D011994:Recombinant Proteins; D024462:Myosin Type II; D024507:Nonmuscle Myosin Type IIA
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