In muscle fibres labelled with
iodoacetamidotetramethylrhodamine at Cys707 of the
myosin heavy chain, the probes have been reported to change orientation when the fibre is activated, relaxed or put into rigor. In order to test whether these motions are indications of the cross-bridge power
stroke, we monitored tension and linear dichroism of the probes in single
glycerol-extracted fibres of rabbit psoas muscle during mechanical transients initiated by
laser pulse photolysis of
caged ATP and
caged ADP. In rigor dichroism is negative, indicating average probe absorption dipole moments oriented more than 54.7 degrees away from the fibre axis. During activation from rigor induced by photoliberation of
ATP from
caged ATP in the presence of
calcium, the dichroism reversed sign promptly (half-time 12.5 ms for 500 microM-
ATP) upon release of
ATP, but then changed only slightly during tension development 20 to 100 milliseconds later. During the onset of rigor following transfer of the fibre from an
ATP-containing relaxing
solution to a rigor medium lacking
ATP, force generation preceded the change in dichroism. The dichroism change occurred slowly (half-time 47 s), because binding of
ADP to sites within the muscle fibre limited its rate of diffusion out of the fibre. When
ADP was introduced or removed, the dichroism transient was similar in time course and magnitude to that obtained after the introduction or removal of
ATP. Neither adding nor removing
ADP produced substantial changes in force. These results demonstrate that orientation of the
rhodamine probes on the
myosin head reflects mainly structural changes linked to
nucleotide binding and release, rather than rotation of the cross-bridge during force generation.