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Pulsed dye laser (PDL) is an effective treatment option for erythematotelangiectatic rosacea. The use of a test spot allows patients to experience the procedure on a small area prior to further treatment. The purpose of this study was to elucidate whether the use of a no charge test spot influenced return rates for further PDL treatment. Data were obtained retrospectively using International Classification of Diseases (ICD)-10 codes for rosacea. Sixty charts were identified: 26 patients initially received a PDL test area free of charge, whereas 34 patients initially underwent full PDL treatment. Patients who experienced the test spot laser treatment had a lower return rate compared to those that directly underwent full PDL treatment. However, this difference was not statistically significant (Fisher's exact test p = 0.2883). Future studies evaluating and identifying factors that influence PDL return rates are needed. Abbreviations: ETR: Erythematotelangiectatic rosacea; PDL: pulsed dye laser; ICD: International classification of diseases.

Gorlin–Goltz syndrome (GGS) is a rare genetic syndrome with variable expressivity and autosomal dominant inheritance. The major features of GGS include numerous basal cell carcinomas (BCCs), keratocysts of the jaw, palmar/plantar pits and calcification of the falx cerebri. Authors report the case of a 51-year-old male with a 19-year history of GGS and multiple BCCs of the head and neck. He presented with a large ulcerating lesion on the right side of his face involving cutaneous, subcutaneous and muscular tissues of the temporal and orbital region. Additionally, magnetic resonance imaging revealed involvement of the right zygomatic bone, infratemporal fossa and mandible. This case is notable in that BCC invasion of the facial bones is rare. Extensive resection and reconstruction with a latissimus dorsi microvascular free muscle flap was performed. The success of this challenging case exemplifies the need for a multidisciplinary team that included dermatology, plastic surgery, oculoplastics and otolaryngology.

We present a method for obtaining high-resolution information on protein backbone structure and dynamics using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) and molecular modeling. Two complementary EPR techniques were employed to measure protein orientation (conventional EPR) and intra-protein distances (dipolar electron-electron resonance, DEER). BSL attaches at Cys positions i and i+4 on a helix, greatly reducing probe mobility relative to the peptide backbone, compared to monofunctional labels. Accurate modeling of BSL provides the coordinates required to directly relate spectroscopic data to backbone structure (both orientation and distance), and dynamics (rotational motion). In the current work, the motor protein Dictyostelium myosin II was used to demonstrate this approach. We measured nucleotide-dependent structural transitions of two key helices within the myosin catalytic domain (CD). Two double-Cys sites were engineered, with one Cys pair located on the relay helix, and the other on a stable helix in the upper 50kD domain. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin. BSL on a construct with two pairs was used to measure interprobe distances. The effect of ADP binding on both orientation and distance was clearly detected with BSL, but not with a monofunctional label. The significance of this work is twofold: (1) A structural transition in the relay helix upon ADP binding was clearly defined with high resolution. (2) BSL spectra demonstrate superior resolution, compared to monofunctional spin labels, making it possible to directly translate spectroscopic data to protein structure and dynamics.

We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M′.D.P, we used pPDM to cross-link SH1 (Cys707) to SH2 (Cys697) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M′.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M′D.P analog on the force generation pathway.

The use of site-directed spectroscopic probes in elucidating the molecular mechanism of force generation in muscle is reviewed, focusing on recent technical and conceptual advances. High-resolution crystal structures of actin and myosin suggest mechanistic models for force generation, but spectroscopic probe experiments under functional conditions are required to test and refine these models. An array of complementary spectroscopic techniques has been developed that can detect disorder and dynamics directly, resolve multiple conformational states, and can be applied to complex protein assemblies, taking advantage of the beautiful symmetry of the myofibrillar lattice. Recently developed instrumentation has the capacity to resolve simultaneously both biochemical and structural kinetics of myosin and/or actin. These new insights support a model in which structural and biochemical transitions are not tightly coupled, and disorder-to-order transitions play central roles in both force generation and its regulation.

Crosslinking the two most reactive Cys (SH1 and SH2) of the myosin catalytic domain (CD) inhibits force production and ATP hydrolysis and locks myosin in a weak actin-binding conformation with the CD immobilized and orientationally disordered. These results suggest that crosslinking traps a state in which the myosin head is on the cusp of force generation. In the present study, we measured the structural dynamics of myosin's light chain domain (LCD) in skeletal muscle fibers during rigor, relaxation, and with SH1 and SH2 crosslinked. To measure LCD structural dynamics, we exchanged spin labeled RLC for native RLC in permeabilized muscle fibers, with retention of function, then used EPR spectroscopy to measure structural dynamics. EPR spectra indicate when SH1 and SH2 are crosslinked, the LCD is in an orientation intermediate between relaxation and rigor, indicative of a state beginning to generate force. The saturation transfer EPR (STEPR) spectrum from these fibers does not change with crosslinking, demonstrating that that the LCD undergoes very slow dynamics, as in rigor, and is less dynamic than relaxation. In order to relate LCD structural dynamics with those of the CD, we measured CD structural dynamics in fibers by directly crosslinking SH1 and SH2 with BSL. EPR spectra from these fibers reveal that the CD is highly disordered, with dynamics ten times slower than in relaxation. Thus when SH1 and SH2 are crosslinked, both domains exhibit structural dynamics intermediate between relaxation (pre-power stroke) and rigor (post-power stroke). This supports the conclusion that SH1-SH2 crosslinking traps a state analogous to an initial force-generating state. We propose that this state is the missing link needed to explain how myosin undergoes a transition from dynamic disorder to order as it converts chemical energy to mechanical work.

We have used electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) to measure structural transitions of the catalytic domain (CD) in Dictyostelium myosin II. The use of BSL is a critical feature in this work. The bifunctional attachment of BSL eliminates most of the nanosecond motions characteristic of monofunctional labels, making it possible to measure protein structural transitions with a precision not previously achievable. Two double-Cys constructs were engineered with Cys residues at helical locations i and i+4 (494.498 and 639.643). Residues 494.498 are located on the relay helix and residues 639.643 are located on a helix adjacent to the relay helix. After BSL labeling, myosin was bound to actin in oriented muscle fibers, making it possible to measure helix orientation relative to the fiber axis. Spectra were acquired in APO and ADP states. Simulation of the 494.498.BSL spectra demonstrates that in APO and ADP states there are two highly ordered populations of the relay helix. APO and ADP spectra contain the same two spectral components, but the distribution of these two components differs between states. Similarly, spectra from the 639.643 construct reveal an ADP-induced structural transition, but the difference between APO and ADP spectra demonstrates a 3o rotation of the 639.643 helix. Our results demonstrate structural transitions of two helixes within the CD of actin-bound myosin associated with nucleotide binding. Measuring these transitions is essential to understanding the molecular mechanism of force generation. This is particularly true for the relay helix, which plays a key role in the coupling of myosin ATPase and motor function. Additionally, these results demonstrate the utility of BSL for measuring transitions in protein orientation, order, and dynamics.

We present a method for obtaining high-resolution information on protein backbone structure and dynamics using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) and molecular modeling. Two complementary EPR techniques were employed to measure protein orientation (conventional EPR) and intra-protein distances (dipolar electron-electron resonance, DEER). BSL attaches at Cys positions i and i+4 on a helix, greatly reducing probe mobility relative to the peptide backbone, compared to monofunctional labels. Accurate modeling of BSL provides the coordinates required to directly relate spectroscopic data to backbone structure (both orientation and distance), and dynamics (rotational motion). In the current work, the motor protein Dictyostelium myosin II was used to demonstrate this approach. We measured nucleotide-dependent structural transitions of two key helices within the myosin catalytic domain (CD). Two double-Cys sites were engineered, with one Cys pair located on the relay helix, and the other on a stable helix in the upper 50kD domain. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin. BSL on a construct with two pairs was used to measure interprobe distances. The effect of ADP binding on both orientation and distance was clearly detected with BSL, but not with a monofunctional label. The significance of this work is twofold: (1) A structural transition in the relay helix upon ADP binding was clearly defined with high resolution. (2) BSL spectra demonstrate superior resolution, compared to monofunctional spin labels, making it possible to directly translate spectroscopic data to protein structure and dynamics. This work was funded by grants from NIH (R01 AR32961, T32 AR07612, P30 AR0507220).

We are using electron paramagnetic resonance and site-directed spin labeling to measure the orientation of the light chain domain of skeletal muscle myosin in pre-power stroke states trapped by the phosphate analog AlF4. Previous work (Kraft et al. (2005) Proc. Natl. Acad. Sci. USA 102:13861-66) has shown via single-fiber X-ray diffraction that AlF4 traps two distinct pre-powerstroke myosin states in activated muscle. The first state (ADP.AlF4-I) produces a weak actin binding, disordered myosin whereas the second state (ADP.AlF4-II) produces a strong binding, stereospecific actomyosin complex. This weak-to-strong transition is a clear indication of the initiation of the power stroke before phosphate release, but it does not reveal the state of the light chain domain (LCD), which undergoes a rotation during the power stroke. We measured the orientation of the light chain domain by exchanging the native regulatory light chain (RLC) of skinned rabbit psoas muscle fiber bundles with a Di-Cys mutant RLC labeled with a bifunctional spin label. Our group has shown previously (Thompson and Naber et al. 2008 Biophys J, in press) that a bifunctional methanethiosulfonate spin label binds rigidly to myosin and reports protein orientation accurately. EPR spectra of oriented fibers show that the LCD produces a distinct orientation from rigor and relaxation in the ADP.AlF4-II state whereas the ADP.AlF4-I state is indistinguishable from relaxed muscle, supporting the hypothesis that the II state represents a state early in the power stroke with a distinct LCD orientation. This work was supported by NIH (AR32961, AR007612). We thank Bernhard Brenner and Theresia Kraft for guidance.