Resolved Structural States of Calmodulin in Regulation of Skeletal Muscle Calcium Release.

Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca2+, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca2+, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca2+, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca2+ stabilized the closed conformation by a factor of two. We conclude that the Ca2+-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca2+-dependent structural dynamics of bound CaM.

Myosin lever arm orientation in muscle determined with high angular resolution using bifunctional spin labels.

Despite advances in x-ray crystallography, cryo-electron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide high-resolution structural information about the myosin light chain domain (LCD; lever arm) under ambient conditions in vertebrate muscle. Here, we measure the orientation of LCD elements in demembranated muscle fibers by electron paramagnetic resonance (EPR) using a bifunctional spin label (BSL) with an angular resolution of 4°. To achieve stereoselective site-directed labeling with BSL, we engineered a pair of cysteines in the myosin regulatory light chain (RLC), either on helix E or helix B, which are roughly parallel or perpendicular to the myosin lever arm, respectively. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtain EPR spectra from which the angular distributions of BSL, and thus the lever arm, can be determined with high resolution relative to the muscle fiber axis. In the absence of ATP (rigor), each of the two labeled helices exhibits both ordered (σ ∼9-11°) and disordered (σ > 38°) populations. Using these angles to determine the orientation of the lever arm (LCD combined with converter subdomain), we observe that the oriented population corresponds to a lever arm that is perpendicular to the muscle fiber axis and that the addition of ATP in the absence of Ca2+ (inducing relaxation) shifts the orientation to a much more disordered orientational distribution. Although the detected orientation of the myosin light chain lever arm is ∼33° different than predicted from a standard "lever arm down" model based on cryo-EM of actin decorated with isolated myosin heads, it is compatible with, and thus augments and clarifies, fluorescence polarization, x-ray interference, and EM data obtained from muscle fibers. These results establish feasibility for high-resolution detection of myosin LCD rotation during muscle contraction.

Adaptation of cardiovascular system stent implants.

Time-consuming design and manufacturing processes are a serious disadvantage when adapting human cardiovascular implants as they cause unacceptable delays after the decision to intervene surgically has been made. An ideal cardiovascular implant should have a broad range of characteristics such as strength, viscoelasticity and blood compatibility. The present research proposes the sequence of the geometrical adaptation procedures and presents their results. The adaptation starts from the identification of a person's current health status while performing abdominal aortic aneurysm (AAA) imaging, which is a point of departure for the mathematical model of a cardiovascular implant. The computerized tomography scan shows the patient-specific geometry parameters of AAA and helps to create a model using COMSOL Multiphysics software. The initial parameters for flow simulation are taken from the results of a patient survey. The simulation results allow choosing the available shape of an implant which ensures a non-turbulent flow. These parameters are essential for the design and manufacturing of an implant prototype which should be tested experimentally for the assurance that the mathematical model is adequate to a physical one. The article gives a focused description of competences and means that are necessary to achieve the shortest possible preparation of the adapted cardiovascular implant for the surgery.