Effects of ions and pH on the thermal stability of thin and thick filaments of skeletal muscle: high-sensitivity differential scanning calorimetric study.


Abstract

Differential scanning calorimetry (DSC) is unique for studying conformational changes in supramolecular structures because it is immune to interference by the turbidity and other optical artifacts of a sample solution. We have employed DSC to study thermal stability of myosin and actin in their filamentous forms (i.e., thick and thin filaments). The thermal stability of the myosin monomer, as well as polymers, showed remarkable sensitivities to pH and to the ionic strength of the solution. At pH 7.5, the endotherm of myosin filaments was broad and resembled that of the monomer in solution. Reducing the pH to 6.3 split the endotherm of the filament into two major transitions. The first one, with a Tm of 47 degrees C, a delta Hcal of 805 kcal/mol, and a cooperative ratio (CR) of 0.1, was relatively insensitive to the pH changes whereas the second one which represented approximately 80% of the helical structure was pH sensitive. The second transition released 2.17 H+ per mole at 0.17 M KCl and was defined by a Tm of 53.9 degrees C, a delta Hcal of 917 kcal/mol, and a CR of 0.35. The major fragment contributing to the splitting of the endotherm was interpreted to be S-2 because the Tm of purified S-2 in a similar medium also shifted from 39.5 degrees C at pH 7.3 to 49.6 degrees C at pH 6.0. KCl had similar effects on the shape of the endotherm of the thick filament. A decrease of KCl from 0.2 to 0.1 M enhanced the effect of pH on the second transition.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4003, 4004, 4005, 4006, 4007, 4008, 4009, 4010, 4011, 4012, 4013, 10610, 10611, 10612, 10613, 10614, 10615, 10616, 10617, 10618, 10619, 10620, 10621, 10622, 10623, 10624, 10625, 10626, 10627, 10628, 10629, 10630, 10631, 10632, 10633, 10634, 10635, 10636, 10637, 10638, 10639, 10640, 10641, 10642, 10643, 10644, 10645, 10646, 10647, 10648, 10649, 10650, 10651, 10652, 10653, 10654, 10655, 10656, 10657, 10658, 10659, 10660, 10661, 10662, 10663 Extra Details: additive : EDTA(1 mM),the transition is from native to intermediate supramolecular structures ; thick and thin filaments; actin;,myosin ; cooperativity

Submission Details

ID: AjdAxpQE3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:24 p.m.

Version: 1

Publication Details
Bertazzon A;Tsong TY,Biochemistry (1990) Effects of ions and pH on the thermal stability of thin and thick filaments of skeletal muscle: high-sensitivity differential scanning calorimetric study. PMID:2207085
Additional Information

Number of data points 135
Proteins Calmodulin ; Myosin light chain kinase 2, skeletal/cardiac muscle ; Actin, alpha skeletal muscle ; Actin, alpha skeletal muscle
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: dHcal ionic:: , buffers:HEPES: 2 mM, pH:7.9, details:Additives Na-ATP (1.0 mM),, prot_conc:- ; Experimental Assay: Tm pH:7.9, buffers:HEPES: 2 mM, ionic:: , details:Additives Na-ATP (1.0 mM),, prot_conc:- ; Experimental Assay: dHvH ionic:: , details:Additives Na-ATP (1.0 mM),, buffers:HEPES: 2 mM, pH:7.9, prot_conc:- ; Experimental Assay: dHcal details:Additives Na-ATP (1.0 mM),, buffers:HEPES: 2 mM, ionic:: , pH:7.6, prot_conc:- ; Experimental Assay: Tm details:Additives Na-ATP (1.0 mM),, buffers:HEPES: 2 mM, ionic:: , pH:7.6, prot_conc:- ; Experimental Assay: dHvH ionic:: , buffers:HEPES: 2 mM, details:Additives Na-ATP (1.0 mM),, pH:7.6, prot_conc:- ; Experimental Assay: dHcal details:Additives Na-ATP (1.0 mM),, buffers:HEPES: 2 mM, ionic:: , pH:7.3, prot_conc:- ; Experimental Assay: Tm details:Additives Na-ATP (1.0 mM),, buffers:HEPES: 2 mM, ionic:: , pH:7.3, prot_conc:- ; Experimental Assay: dHvH ionic:: , buffers:HEPES: 2 mM, details:Additives Na-ATP (1.0 mM),, pH:7.3, prot_conc:- ; Experimental Assay: dHcal details:Additives Na-ATP (1.0 mM),, buffers:HEPES: 2 mM, ionic:: , pH:6.9, prot_conc:- ; Experimental Assay: Tm details:Additives Na-ATP (1.0 mM),, buffers:HEPES: 2 mM, ionic:: , pH:6.9, prot_conc:- ; Experimental Assay: dHvH ionic:: , buffers:HEPES: 2 mM, details:Additives Na-ATP (1.0 mM),, pH:6.9, prot_conc:- ; Experimental Assay: dHcal ionic:: , buffers:HEPES: 2 mM, details:Additives Na-ATP (1.0 mM),, pH:6.4, prot_conc:- ; Experimental Assay: Tm ionic:: , buffers:HEPES: 2 mM, details:Additives Na-ATP (1.0 mM),, pH:6.4, prot_conc:- ; Experimental Assay: dHvH ionic:: , buffers:HEPES: 2 mM, pH:6.4, details:Additives Na-ATP (1.0 mM),, prot_conc:- ; Experimental Assay: dHcal prot_conc:-, buffers:HEPES: 2 mM, details:Additives Na-ATP (1.0 mM),, pH:5.9, ionic:: 2 mM,50 ; Experimental Assay: Tm prot_conc:-, buffers:HEPES: 2 mM, details:Additives Na-ATP (1.0 mM),, pH:5.9, ionic:: 2 mM,50 mM ; Experimental Assay: dHvH prot_conc:-, buffers:HEPES: 2 mM, details:Additives Na-ATP (1.0 mM),, pH:5.9, ionic:: 2 mM,50 m ; Experimental Assay: dHcal pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, ionic:Ca2+: 8.0 mM, prot_conc ; Experimental Assay: Tm pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, ionic:Ca2+: 8.0 mM, prot_conc:- ; Experimental Assay: dHvH pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, ionic:Ca2+: 8.0 mM, prot_conc: ; Experimental Assay: dHcal ionic:Ca2+: 4.0 mM, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, prot_conc ; Experimental Assay: Tm ionic:Ca2+: 4.0 mM, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, prot_conc:- ; Experimental Assay: dHvH ionic:Ca2+: 4.0 mM, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, prot_conc: ; Experimental Assay: dHcal prot_conc:-, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, ionic:Ca2+: 2.0 ; Experimental Assay: Tm prot_conc:-, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, ionic:Ca2+: 2.0 mM ; Experimental Assay: dHvH prot_conc:-, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, ionic:Ca2+: 2.0 m ; Experimental Assay: dHcal ionic:Ca2+: 1.0 mM, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, prot_conc ; Experimental Assay: Tm ionic:Ca2+: 1.0 mM, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, prot_conc:- ; Experimental Assay: dHvH ionic:Ca2+: 1.0 mM, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, prot_conc: ; Experimental Assay: dHcal ionic:Ca2+: 0.4 mM, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, prot_conc ; Experimental Assay: Tm ionic:Ca2+: 0.4 mM, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, prot_conc:- ; Experimental Assay: dHvH ionic:Ca2+: 0.4 mM, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, prot_conc: ; Experimental Assay: dHcal prot_conc:-, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, ionic:Ca2+: 0.2 ; Experimental Assay: Tm prot_conc:-, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, ionic:Ca2+: 0.2 mM ; Experimental Assay: dHvH prot_conc:-, pH:7.0, details:Additives Na-ATP (0.2 mM),, buffers:HEPES: 2 mM, ionic:Ca2+: 0.2 m ; Experimental Assay: dHcal buffers:potassium phosphate: 20 mM, pH:7.0, prot_conc:-, ionic:KCl: 0.17 M, details:Additives ; Experimental Assay: dHvH buffers:potassium phosphate: 20 mM, pH:7.0, prot_conc:-, ionic:KCl: 0.17 M, details:Additives E ; Experimental Assay: dHcal pH:6.7, buffers:potassium phosphate: 20 mM, prot_conc:-, ionic:KCl: 0.17 M, details:Additives ; Experimental Assay: dHvH pH:6.7, buffers:potassium phosphate: 20 mM, prot_conc:-, ionic:KCl: 0.17 M, details:Additives E ; Experimental Assay: dHcal buffers:potassium phosphate: 20 mM, pH:6.5, prot_conc:-, ionic:KCl: 0.17 M, details:Additives ; Experimental Assay: dHvH buffers:potassium phosphate: 20 mM, pH:6.5, prot_conc:-, ionic:KCl: 0.17 M, details:Additives E ; Experimental Assay: dHcal buffers:potassium phosphate: 20 mM, prot_conc:-, ionic:KCl: 0.17 M, pH:6.3, details:Additives ; Experimental Assay: dHvH buffers:potassium phosphate: 20 mM, prot_conc:-, ionic:KCl: 0.17 M, pH:6.3, details:Additives E ; Experimental Assay: dHcal pH:6.7, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.15 M, details:Additives ; Experimental Assay: dHvH pH:6.7, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.15 M, details:Additives E ; Experimental Assay: dHcal pH:6.5, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.15 M, details:Additives ; Experimental Assay: dHvH pH:6.5, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.15 M, details:Additives E ; Experimental Assay: dHcal prot_conc:-, buffers:potassium phosphate: 20 mM, pH:6.3, ionic:KCl: 0.15 M, details:Additives ; Experimental Assay: dHvH prot_conc:-, buffers:potassium phosphate: 20 mM, pH:6.3, ionic:KCl: 0.15 M, details:Additives E ; Experimental Assay: Tm ionic:KCl: 0.25 M, prot_conc:-, buffers:potassium phosphate: 20 mM, pH:6.3, details:Additives EDT ; Experimental Assay: Tm ionic:KCl: 0.20 M, prot_conc:-, buffers:potassium phosphate: 20 mM, pH:6.3, details:Additives EDT ; Experimental Assay: Tm buffers:potassium phosphate: 20 mM, prot_conc:-, ionic:KCl: 0.17 M, pH:6.3, details:Additives EDT ; Experimental Assay: Tm prot_conc:-, buffers:potassium phosphate: 20 mM, pH:6.3, ionic:KCl: 0.15 M, details:Additives EDT ; Experimental Assay: Tm ionic:KCl: 0.13 M, prot_conc:-, buffers:potassium phosphate: 20 mM, pH:6.3, details:Additives EDT ; Experimental Assay: Tm prot_conc:-, details:Additives EDTA (1 mM),, buffers:potassium phosphate: 20 mM, pH:6.3, ionic:KC ; Experimental Assay: Tm ionic:KCl: 0.25 M, pH:6.5, prot_conc:-, buffers:potassium phosphate: 20 mM, details:Additives EDT ; Experimental Assay: Tm ionic:KCl: 0.20 M, prot_conc:-, buffers:potassium phosphate: 20 mM, pH:6.5, details:Additives EDT ; Experimental Assay: Tm buffers:potassium phosphate: 20 mM, pH:6.5, prot_conc:-, ionic:KCl: 0.17 M, details:Additives EDT ; Experimental Assay: Tm pH:6.5, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.15 M, details:Additives EDT ; Experimental Assay: Tm ionic:KCl: 0.13 M, pH:6.5, prot_conc:-, buffers:potassium phosphate: 20 mM, details:Additives EDT ; Experimental Assay: Tm pH:6.5, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.10 M, details:Additives EDT ; Experimental Assay: Tm prot_conc:-, ionic:KCl: 0.25 M, details:Additives EDTA (1 mM),, buffers:potassium phosphate: 20 m ; Experimental Assay: Tm pH:6.7, ionic:KCl: 0.20 M, prot_conc:-, buffers:potassium phosphate: 20 mM, details:Additives EDT ; Experimental Assay: Tm pH:6.7, buffers:potassium phosphate: 20 mM, prot_conc:-, ionic:KCl: 0.17 M, details:Additives EDT ; Experimental Assay: Tm pH:6.7, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.15 M, details:Additives EDT ; Experimental Assay: Tm ionic:KCl: 0.13 M, pH:6.7, prot_conc:-, buffers:potassium phosphate: 20 mM, details:Additives EDT ; Experimental Assay: Tm pH:6.7, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.10 M, details:Additives EDT ; Experimental Assay: Tm ionic:KCl: 0.25 M, pH:7.0, prot_conc:-, buffers:potassium phosphate: 20 mM, details:Additives EDT ; Experimental Assay: Tm pH:7.0, ionic:KCl: 0.20 M, prot_conc:-, buffers:potassium phosphate: 20 mM, details:Additives EDT ; Experimental Assay: Tm buffers:potassium phosphate: 20 mM, pH:7.0, prot_conc:-, ionic:KCl: 0.17 M, details:Additives EDT ; Experimental Assay: Tm pH:7.0, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.15 M, details:Additives EDT ; Experimental Assay: Tm ionic:KCl: 0.13 M, pH:7.0, prot_conc:-, buffers:potassium phosphate: 20 mM, details:Additives EDT ; Experimental Assay: Tm pH:7.0, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.10 M, details:Additives EDT ; Experimental Assay: Tm pH:7.5, buffers:potassium phosphate: 20 mM, prot_conc:-, ionic:KCl: 0.17 M, details:Additives EDT ; Experimental Assay: Tm pH:7.5, prot_conc:-, buffers:potassium phosphate: 20 mM, ionic:KCl: 0.15 M, details:Additives EDT ; Experimental Assay: Tm pH:7.5, ionic:KCl: 0.13 M, prot_conc:-, buffers:potassium phosphate: 20 mM, details:Additives EDT ; Experimental Assay: Tm buffers:Potassium phosphate: 20 mM, ionic:: , pH:7.3, details:Additives EDTA (1 mM), ; Experimental Assay: Tm pH:6.8, buffers:Potassium phosphate: 20 mM, ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: Tm buffers:Potassium phosphate: 20 mM, ionic:: , pH:5.9, details:Additives EDTA (1 mM), ; Experimental Assay: dHcal prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, pH:7.0, buffers:Potassium phosphate: 20 mM, detail ; Experimental Assay: Tm prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, pH:7.0, buffers:Potassium phosphate: 20 mM, details:A ; Experimental Assay: dHvH prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, pH:7.0, buffers:Potassium phosphate: 20 mM, details ; Experimental Assay: dHcal prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, details:Additives EDTA (1 mM),, buffers:Potassium ; Experimental Assay: Tm prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, details:Additives EDTA (1 mM),, buffers:Potassium pho ; Experimental Assay: dHvH prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, details:Additives EDTA (1 mM),, buffers:Potassium p ; Experimental Assay: dHcal prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, pH:6.5, buffers:Potassium phosphate: 20 mM, detail ; Experimental Assay: Tm prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, pH:6.5, buffers:Potassium phosphate: 20 mM, details:A ; Experimental Assay: dHvH prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, pH:6.5, buffers:Potassium phosphate: 20 mM, details ; Experimental Assay: dHcal prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, buffers:Potassium phosphate: 20 mM, pH:6.3, detail ; Experimental Assay: Tm prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, buffers:Potassium phosphate: 20 mM, pH:6.3, details:A ; Experimental Assay: dHvH prot_conc:1.8-3.5 mg/ml, ionic:KCl: 0.13 M, buffers:Potassium phosphate: 20 mM, pH:6.3, details
Libraries Mutations for sequence A:DEDETTALVCDNGSGLVKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIITNWDDMEKIWHHTFYNELRVAPEEHPTLLTEAPLNPKANREKMTQIMFETFNVPAMYVAIQAVLSLYASGRTTGIVLDSGDGVTHNVPIYEGYALPHAIMRLDLAGRDLTDYLMKILTERGYSFVTTAEREIVRDIKEKLCYVALDFENEMATAASSSSLEKSYELPDGQVITIGNERFRCPETLFQPSFIGMESAGIHETTYNSIMKCDIDIRKDLYANNVMSGGTTMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWITKQEYDEAGPSIVHR/D:LKIAAFNIRTFGETKMSNATLASYIVRIVRRYDIVLIQEVRDSHLVAVGKLLDYLNQDDPNTYHYVVSEPLGRNSYKERYLFLFRPNKVSVLDTYQYDDGCGNCGNDSFSREPAVVKFSSHSTKVKEFAIVALHSAPSDAVAEINSLYDVYLDVQQKWHLNDVMLMGDFNADCSYVTSSQWSSIRLRTSSTFQWLIPDSADTTATSTNCAYDRIVVAGSLLQSSVVPGSAAPFDFQAAYGLSNEMALAISDHYPVEVTLT ; Mutations for sequence A:ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGFISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVTMMTSK/B:KRRWKKNFIAVSAANRFKKISSSGAL
Sequence Assay Result Units