Charge changes in loop 2 affect the thermal unfolding of the myosin motor domain bound to F-actin.
The thermal unfolding of Dictyostelium discoideum myosin head fragments with alterations in the actin-binding surface loop 2 was studied by differential scanning calorimetry. Lengthening of loop 2 without concomitant charge changes led to decreases in the transition temperature of not more than 1.8 degrees C. Insertions with multiple positive or negative charges had a stronger destabilizing effect and led to reductions in the thermal transition temperature of up to 3.7 degrees C. In the presence of nucleotide, most mutants displayed similar or higher transition temperatures than M765. Only constructs M765(11/+6) and M765(20/+12) with long positively charged inserts showed transition temperatures that were more than 2 degrees C below the values measured for M765 in the presence of ADP, ADP-V(i), and ADP-BeF(3). Interaction with F-actin in the presence of ADP shifted the thermal transition of M765 by 6 degrees C, from 49.1 to 55.1 degrees C. The actin-induced increase in thermal stability varied between 1.2 and 9.1 degrees C and showed a strong correlation with the mutant constructs' affinity for actin. Our results show that length and charge changes in loop 2 do not significantly affect nucleotide-induced structural changes in the myosin motor domain, but they affect structural changes that occur when the motor domain is strongly bound to actin and affect the coupling between the actin- and nucleotide-binding sites.
Ponomarev
M A
MA
A.N.Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prosp. 33, Moscow 117071, Russia.
Furch
M
M
Levitsky
D I
DI
Manstein
D J
DJ
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002152:Calorimetry, Differential Scanning; D004023:Dictyostelium; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D016254:Mutagenesis, Insertional / Q000235:genetics; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics
D000199:Actins; D020409:Molecular Motor Proteins; D010446:Peptide Fragments; D010710:Phosphates; D000244:Adenosine Diphosphate; D009218:Myosins