Kinetic Resolution of a Conformational Transition and the ATP Hydrolysis Step Using Relaxation Methods with a Dictyostelium Myosin II Mutant Containing a Single Tryptophan Residue

Abstract

The fluorescence emission intensity from a conserved tryptophan residue (W501) located in the relay loop (F466 to L516) of the Dicytostelium discoideum myosin II motor domain is sensitive to ATP binding and hydrolysis. The initial binding process is accompanied by a small quench in fluorescence, and this is followed by a large enhancement that appears coincident with the hydrolysis step. Using temperature and pressure jump methods, we show that the enhancement process is kinetically distinct from but coupled to the hydrolysis step. The fluorescence enhancement corresponds to the open-closed transition (k(obs) approximately 1000 s(-1) at 20 degrees C). From the overall steady-state fluorescence signal and the presence or absence of a relaxation transient, we conclude that the ADP state is largely in the open state, while the ADP.AlF(4) state is largely closed. At 20 degrees C the open-closed equilibria for the AMP.PNP and ADP.BeF(x) complexes are close to unity and are readily perturbed by temperature and pressure. In the case of ATP, the equilibrium of this step slightly favors the open state, but coupling to the subsequent hydrolysis step gives rise to a predominantly closed state in the steady state. Pressure jump during steady-state ATP turnover reveals the distinct transients for the rapid open-closed transition and the slower hydrolysis step.

Myosin cleft movement and its coupling to actomyosin dissociation

“…The N-terminal sequence of Md was modified to MDGTP where P corresponds to P3 of the native sequence. The Cterminal sequence was the same as in the M761 construct and contained a His 8 fusion for ease of purification 26,27 . After cloning, the PCR amplified fragment was sequenced.…”

Dynamic reorganization of the motor domain of myosin subfragment 1 in different nucleotide states

“…The equilibrium between these conformations is controlled by the bound nucleotide and was characterized previously for unlabelled myosins by using temperature and pressure jump experiments [15,16]. In the present study we applied external labels, which probably modified the open-closed equilibrium.…”

“…In the present study we applied external labels, which probably modified the open-closed equilibrium. The tryptophan fluorescence measured for these labelled S1 samples would be informative regarding these undesired effects [15,16]. However, the absorption and emission spectra of tryptophan overlap with those of the fluorophores used, which did not allow us to carry out these control experiments.…”

“…The apo-form and ADP bound form of either a single tryptophan D. discoideum myosin II motor domain construct [15] or skeletal muscle myosin S1 [16] were predominantly in the open conformation, while the ADP.AlF -4 bound forms were predominantly in the closed conformation over the 4-30°C temperature range. The open/closed equilibrium was shifted towards the closed conformation by increased temperature when the motor domain bound ADP.BeF x [15,16].…”

“…Rapid kinetic experiments using tryptophan fluorescence indicated that the delicately poised equilibrium between the closed and open conformations was influenced by temperature changes in a nucleotide dependent manner [14][15][16]. The apo-form and ADP bound form of either a single tryptophan D. discoideum myosin II motor domain construct [15] or skeletal muscle myosin S1 [16] were predominantly in the open conformation, while the ADP.AlF -4 bound forms were predominantly in the closed conformation over the 4-30°C temperature range. The open/closed equilibrium was shifted towards the closed conformation by increased temperature when the motor domain bound ADP.BeF x [15,16].…”