Ryan Mello

Measuring Myosin Light Chain Domain Orientation in the Pre-Power Stroke AlF4 States with a Bifunctional Spin Label in Skinned Muscle Fibers

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.

To test or not to test: A study examining the return rates of rosacea patients treated with a pulsed dye laser

ABSTRACT 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

A multidisciplinary approach to the successful management of Gorlin syndrome

Abstract 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.

High-Resolution EPR of a Bifunctional Spin Label Reveals an ADP Induced Structural Rearrangement of the Actin-Bound Myosin Catalytic Domain

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.

Myosin Crosslinking and EPR Capture the Start of Force Generation in Muscle Fibers

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 myosins 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.