Nanothermometry Reveals Calcium-Induced Remodeling of Myosin

Abstract

Ions greatly influence protein structure–function and are critical to health and disease. A 10, 000-fold higher calcium in the sarcoplasmic reticulum (SR) of muscle suggests elevated calcium levels near active calcium channels at the SR membrane and the impact of localized high calcium on the structure–function of the motor protein myosin. In the current study, combined quantum dot (QD)-based nanothermometry and circular dichroism (CD) spectroscopy enabled detection of previously unknown enthalpy changes and associated structural remodeling of myosin, impacting its function following exposure to elevated calcium. Cadmium telluride QDs adhere to myosin, function as thermal sensors, and reveal that exposure of myosin to calcium is exothermic, resulting in lowering of enthalpy, a decrease in alpha helical content measured using CD spectroscopy, and the consequent increase in motor efficiency. Isolated muscle fibers subjected to elevated levels of calcium further demonstrate fiber lengthening and decreased motility of actin filaments on myosin-functionalized substrates. Our results, in addition to providing new insights into our understanding of muscle structure–function, establish a novel approach to understand the enthalpy of protein–ion interactions and the accompanying structural changes that may occur within the protein molecule.

Secondary Structure of the Novel Myosin Binding Domain WYR and Implications within Myosin Structure

“…Previous studies have shown Myosin, LMM, and segments of the LMM to exhibit traditional helical character in which a maximum is present at~195 nm and two minima exist at 208 nm and 222 nm [27,28,[54][55][56][57]. We obtained similar results over a five-fold range of LMM concentrations (2 µM to 10 µM) (Figure S9).…”

The Optimized Quantum Dots Mediated Thermometry Reveals the Efficiency of Myosin Extracted from Muscle Mini Bundles

“…However, our understanding of myosin ATPase properties at the muscle ber level remains incomplete. As a semiconductor nanocrystal, quantum dots were selected as a nanoscale thermometer [12,17] because of their superior uorophores and thermal sensitivity compared to organic dyes or other uorescent proteins. Furthermore, QD have been investigated to measure altered temperature of the ATPase reaction by puri ed myosin to de ne myosin e ciency [12,17].…”

“…As a semiconductor nanocrystal, quantum dots were selected as a nanoscale thermometer [12,17] because of their superior uorophores and thermal sensitivity compared to organic dyes or other uorescent proteins. Furthermore, QD have been investigated to measure altered temperature of the ATPase reaction by puri ed myosin to de ne myosin e ciency [12,17]. The decreased uorescence intensity of QD re ects higher amount of heat production in the process of ATP hydrolysis, signifying lower work e ciency of the myosin motor protein [12,17,23].…”

“…So far, several miniature approaches, such as single bre or myo bril contractile measurement or single bre in vitro motility assays, have been applied to evaluate muscle function at cellular, subcellular or molecular levels [9,15,16]. However, the biochemical properties of myosin from these preparations remain incomplete and the introduction of cadmium telluride quantum dots (CdTe QD) mediated thermometry to measure myosin e ciency at the protein level has complemented the understanding of the myosin function in vitro [12,17]. The underlying mechanism of the approach is that the heat loss during ATP hydrolysis by myosin ATPase enzymatic reaction will increase the temperature in the microenvironment, and QD uorescence intensity will accordingly decrease with thermal resolution of ~ 1 mK.…”

Erbium Single‐Band Nanothermometry in the Third Biological Imaging Window: Potential and Limitations

“…In contrast to traditional thermometers, temperature‐sensitive photoluminescent nanoprobes, typically referred to as nanothermometers, allow for contactless, fast, and accurate temperature measurement at the sub‐micrometer/nanoscale: [ 5,6 ] pertinent in the development of state‐of‐the‐art electronics, in situ characterization of materials, unraveling of previously unexplored physical phenomena, and most of all biomedicine. [ 7–13 ] In biomedicine, there is a niche for photoluminescent nanothermometers operating in the near‐infrared (NIR) spectral region, within the biological imaging windows (BW‐I: 750–950 nm, BW‐II: 1000–1350 nm, and BW‐III: 1500–1800 nm). [ 14,15 ] Biological tissues are partially translucent to NIR light, due to reduced optical scattering and absorption, thus leading to more accurate target visualization in the NIR.…”