High-Field High-Repetition-Rate Sources for the Coherent THz Control of Matter.

Ultrashort flashes of THz light with low photon energies of a few meV, but strong electric or magnetic field transients have recently been employed to prepare various fascinating nonequilibrium states in matter. Here we present a new class of sources based on superradiant enhancement of radiation from relativistic electron bunches in a compact electron accelerator that we believe will revolutionize experiments in this field. Our prototype source generates high-field THz pulses at unprecedented quasi-continuous-wave repetition rates up to the MHz regime. We demonstrate parameters that exceed state-of-the-art laser-based sources by more than 2 orders of magnitude. The peak fields and the repetition rates are highly scalable and once fully operational this type of sources will routinely provide 1 MV/cm electric fields and 0.3 T magnetic fields at repetition rates of few 100 kHz. We benchmark the unique properties by performing a resonant coherent THz control experiment with few 10 fs resolution.

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.

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.

The dynamics of the relay loop tryptophan residue in the Dictyostelium myosin motor domain and the origin of spectroscopic signals.

Steady-state and time-resolved fluorescence measurements were performed on a Dictyostelium discoideum myosin II motor domain construct retaining a single tryptophan residue at position 501, located on the relay loop. Other tryptophan residues were mutated to phenylalanine. The Trp-501 residue showed a large enhancement in fluorescence in the presence of ATP and a small quench in the presence of ADP as a result of perturbing both the ground and excited state processes. Fluorescence lifetime and quantum yield measurements indicated that at least three microstates of Trp-501 were present in all nucleotide states examined, and these could not be assigned to a particular gross conformation of the motor domain. Enhancement in emission intensity was associated with a reduction of the contribution from a statically quenched component and an increase in a component with a 5-ns lifetime, with little change in the contribution from a 1-ns lifetime component. Anisotropy measurements indicated that the Trp-501 side chain was relatively immobile in all nucleotide states, and the fluorescence was effectively depolarized by rotation of the whole motor domain with a correlation time on 50-70 ns. Overall these data suggest that the backbone of the relay loop remains structured throughout the myosin ATPase cycle but that the Trp-501 side chain experiences a different weighting in local environments provided by surrounding residues as the adjacent converter domain rolls around the relay loop.

Resolution of conformational states of Dictyostelium myosin II motor domain using tryptophan (W501) mutants: implications for the open-closed transition identified by crystallography.

When myosin interacts with ATP there is a characteristic enhancement in tryptophan fluorescence which has been widely exploited in kinetic studies. Using Dictyostelium motor domain mutants, we show that W501, located at the end of the relay helix close to the converter region, responds to two independent conformational events on nucleotide binding. First, a rapid isomerization gives a small fluorescence quench and then a slower reversible step which controls the hydrolysis rate (and corresponds to the open-closed transition identified by crystallography) gives a large enhancement. A mutant lacking W501 shows no ATP-induced enhancement in the fluorescence, yet quenched-flow measurements demonstrate that ATP is rapidly hydrolyzed to give a products complex as in the wild-type. The nucleotide-free, open and closed states of a single tryptophan-containing construct, W501+, show distinct fluorescence spectra and susceptibilities to acrylamide quenching which indicate that W501 becomes internalized in the closed state. The open-closed transition does not require hydrolysis per se and can be induced by a nonhydrolyzable analogue. At 20 degrees C, the equilibrium may favor the open state, but with ATP as substrate, the subsequent hydrolysis step pulls the equilibrium toward the closed state such that a tryptophan mutant containing only W501 yields an overall 80% enhancement. These studies allow solution-based assays to be rationalized with the crystal structures of the myosin motor domain and show that three different states can be distinguished at the interface of the relay and converter regions.

Fluorescence measurements detect changes in scallop myosin regulatory domain.

Ca2+-induced conformational changes of scallop myosin regulatory domain (RD) were studied using intrinsic fluorescence. Both the intensity and anisotropy of tryptophan fluorescence decreased significantly upon removal of Ca2+. By making a mutant RD we found that the Ca2+-induced fluorescence change is due mainly to Trp21 of the essential light chain which is located at the unusual Ca2+-binding EF-hand motif of the first domain. This result suggests that Trp21 is in a less hydrophobic and more flexible environment in the Ca2+-free state, supporting a model for regulation based on the 2 A resolution structure of scallop RD with bound Ca2+ [Houdusse A. and Cohen C. (1996) Structure 4, 21-32]. Binding of the fluorescent probe, 8-anilinonaphthalene-1-sulphonate (ANS) to the RD senses the dissociation of the regulatory light chain (RLC) in the presence of EDTA, by energy transfer from a tryptophan cluster (Trp818, 824, 826, 827) on the heavy chain (HC). We identified a hydrophobic pentapeptide (Leu836-Ala840) at the head-rod junction which is required for the effective energy transfer and conceivably is part of the ANS-binding site. Extension of the HC component of RD towards the rod region results in a larger ANS response, presumably indicating changes in HC-RLC interactions, which might be crucial for the regulatory function of scallop myosin.

Dimerization of the head-rod junction of scallop myosin.

We have compared the dimerization properties and coiled-coil stability of various recombinant fragments of scallop myosin around the head-rod junction. The heavy-chain peptide of the regulatory domain and its various extensions toward the alpha-helical rod region were expressed in Escherichia coli, purified, and reconstituted with the light chains. Rod fragments of the same length but without the light-chain binding domain were also expressed. Electron micrographs show that the regulatory domain complex containing 340 residues of the rod forms dimers with two knobs (two regulatory domains) at one end attached to an approximately 50-nm coiled coil. These parallel dimers are in equilibrium with monomers (Kd = 10.6 microM). By contrast, complexes with shorter rod extensions remain predominantly monomeric. Dimers are present, accounting for ca. 5% of the molecules containing a rod fragment of 87 residues and ca. 30% of those with a 180-residue peptide. These dimers appear to be antiparallel coiled coils, as judged by their length and the knobs observed at the two ends. The rod fragments alone do not dimerize and form a coiled-coil structure unless covalently linked by disulfide bridges. Our results suggest that the N-terminal end of the coiled-coil rod is stabilized by interactions with the regulatory domain, most likely with residues of the regulatory light chain. This labile nature of the coiled coil at the head-rod junction might be a structural prerequisite for regulation of scallop myosin by Ca2+-ions.