Voltage-gated ion channels are transmembrane proteins that are essential for nerve impulses and regulate ion flow across cell membranes in response to changes in membrane potential. They are made up of four homologous domains or subunits, each of which contains six transmembrane segments. Studies of potassium channels have shown that the second (S2) and fourth (S4) segments contain several charged residues, which sense changes in voltage and form part of the voltage sensor. Although these regions clearly undergo conformational changes in response to voltage, little is known about the nature of these changes because voltage-dependent distance changes have not been measured. Here we use lanthanide-based resonance energy transfer to measure distances between Shaker potassium channel subunits at specific residues. Voltage-dependent distance changes of up to 3.2 A were measured at several sites near the S4 segment. These movements directly correlated with electrical measurements of the voltage sensor, establishing the link between physical changes and electrical charge movement. Measured distance changes suggest that the region associated with the S4 segment undergoes a rotation and possible tilt, rather than a large transmembrane movement, in response to voltage. These results demonstrate the first in situ measurement of atomic scale movement in a trans-membrane protein.

Myosin V is a homodimeric motor protein involved in trafficking of vesicles in the cell. It walks bipedally along actin filaments, moving cargo approximately 37 nm per step. We have measured the step size of individual myosin heads by fusing an enhanced green fluorescent protein (eGFP) to the N-terminus of one head of the myosin dimer and following the motion with nanometer precision and subsecond resolution. We find the average step size to be 74.1 nm with 9.4 nm (SD) and 0.3 nm (SE). Our measurements demonstrate nanometer localization of single eGFPs, confirm the hand-over-hand model of myosin V procession, and when combined with previous data, suggest that there is a kink in the leading lever arm in the waiting state of myosin V. This kink, or "telemark skier" configuration, may cause strain, which, when released, leads to the powerstroke of myosin, throwing the rear head forward and leading to unidirectional motion.

We studied the structural dynamics of chicken myosin V by combining the localization power of fluorescent imaging with one nanometer accuracy (FIONA) with the ability to detect angular changes of a fluorescent probe. The myosin V was labeled with bifunctional rhodamine on one of its calmodulin light chains. For every 74 nm translocation, the probe exhibited two reorientational motions, associated with alternating smaller and larger translational steps. Molecules previously identified as stepping alternatively 74-0 nm were found to actually step 64-10 nm. Additional tilting often occurred without full steps, possibly indicating flexibility of the attached myosin heads or probing of their vicinity. Processive myosin V molecules sometimes shifted from the top to the side of actin, possibly to avoid an obstacle. The data indicate marked adaptability of this molecular motor to a nonuniform local environment and provide strong support for a straight-neck model of myosin V in which the lever arm of the leading head is tilted backwards at the prepowerstoke angle.

Myosin is thought to generate movement of actin filaments via a conformational change between its lightchain domain and its catalytic domain that is driven by the binding of nucleotides and actin. To monitor this change, we have measured distances between a gizzard regulatory light chain (Cys 108) and the active site (near or at Trp 130) of skeletal myosin subfragment 1 (S1) by using luminescence resonance energy transfer and a photoaffinity ATP-lanthanide analog. The technique allows relatively long distances to be measured, and the label enables site-specific attachment at the active-site with only modest affect on myosin's enzymology. The distance between these sites is 66.8 ؎ 2.3 Å when the nucleotide is ADP and is unchanged on binding to actin. The distance decreases slightly with ADP-BeF 3 , (؊1.6 ؎ 0.3 Å) and more significantly with ADP-AlF 4 (؊4.6 ؎ 0.2 Å). During steady-state hydrolysis of ATP, the distance is temperature-dependent, becoming shorter as temperature increases and the complex with ADP⅐P i is favored over that with ATP. We conclude that the distance between the active site and the light chain varies as Acto-S1-ADP Ϸ S1-ADP > S1-ADP-BeF 3 > S1-ADP-AlF 4 Ϸ S1-ADP-P i and that S1-ATP > S1-ADP-P i . The changes in distance are consistent with a substantial rotation of the light-chain binding domain of skeletal S1 between the prepowerstroke state, simulated by S1-ADP-AlF 4 , and the post-powerstroke state, simulated by acto-S1-ADP.Muscle contraction is thought to occur when the myosin head (subfragment 1 or S1) undergoes an ATP-dependent conformational change that translates the actin filament. More specifically, the long ␣-helix tail of S1, bound to two calmodulin-like proteins known as the regulatory light chain (RLC) and essential light chain, is believed to act as a lever arm by rotating Ϸ45-70 degrees during the powerstroke, thereby translating the actin filament 5-15 nm (reviewed in refs. 1-3; see also ref. 4 and Fig. 1). Considerable evidence supports this model, although, particularly with skeletal myosin, a significant rotation of the light-chain (LC) domain has been difficult to detect directly. Here, we have measured the distance between the catalytic domain and the RLC, a distance that is expected to change if the LC domain undergoes a rotation. The measurement is possible because of two advances: (i) the use of luminescence resonance energy transfer (LRET), a modification of the conventional fluorescence resonance energy transfer, in which longer distances with greater accuracy can be measured, including the relatively large distances from the catalytic domain to the RLC within myosin (5-7); and (ii) the use of newly synthesized active-site labels containing a luminescent lanthanide chelate that covalently attach near the active site and yet alter the enzymology only modestly (H.L., J. Grammar, M.X., P.R.S., and R.G.Y., unpublished work).Specifically, we have measured energy transfer from a terbiumdonor photo-incorporated at an active-site residue of the catalytic domain (li...