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 wit h alterations in the actin-binding surface loop 2 was studied by differenti al scanning calorimetry. Lengthening of loop 2 without concomitant charge c hanges led to decreases in the transition temperature of not more than 1.8 degrees C. Insertions with multiple positive or negative charges had a stro nger destabilizing effect and led to reductions in the thermal transition t emperature of up to 3.7 degrees C, In the presence of nucleotide, most muta nts displayed similar or higher transition temperatures than M765. Only con structs M765(11/+6) and M765(20/+12) with long positively charged inserts s howed transition temperatures that were more than 2 degrees C below the val ues measured for M765 in the presence of ADP, ADP-V-i, and ADP-BeF3. Intera ction 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 increa se in thermal stability varied between 1.2 and 9.1 degrees C and showed a s trong correlation with the mutant constructs' affinity for actin. Our resul ts show that length and charge changes in loop 2 do not significantly affec t nucleotide-induced structural changes in the myosin motor domain, but the y affect structural changes that occur when the motor domain is strongly bo und to actin and affect the coupling between the actin- and nucleotide-bind ing sites.