Characterizing polymerization dynamics using fluorescent molecular rotors and magnetoelastic sensors

Abstract only availableThe dynamics of polymerization are critical in many medical applications; for instance, polymers used to fill aneurysms must be timed accurately. Two distinct methods were examined for their ability to probe the polymerization kinetics of different polymers and to predict the onset of polymerization. Molecular rotors are a class of fluorophores with two de-excitation pathways: fluorescence emission and intramolecular rotation. Highly viscous solvents provide a constrained environment in which intramolecular rotation is inhibited, and radiation is the preferred pathway. It is hypothesized that during polymerization steric hindrance of the intramolecular rotation leads to increased fluorescence emission intensity. Magnetoelastic (ME) sensors have been used to measure fluid viscosity. In a time varying magnetic field, a magnetoelastic strip oscillates at its viscosity-dependent resonant frequency creating a magnetic flux that is detected. Subsequently, viscosity can be analyzed by measuring quantities such as resonance frequency, signal voltage, and Q-factor. In this study, fluorescent molecular rotors and magnetoelastic sensors were evaluated for their efficacy in monitoring the polymerization dynamics of acrylamide gels, collagen, and sol-gels. The ME sensor was effective in characterizing the polymerization dynamics of acrylamide and sol-gel, where a reduced Q-factor indicated mechanical dampening of the oscillation in the polymerized state. For unknown reasons, the ME sensor was unable to characterize the polymerization of collagen. However, the molecular rotors sensed the polymerization of collagen and sol-gel though a marked increase of emission intensity. Molecular rotors deteriorate from ammonium persulfate (APS), a strong oxidant and catalyst for cross-linking in the acrylamide system. While ME sensors are effective in characterizing several polymerization reactions, molecular rotors are more effective in monitoring the polymerization of proteins such as collagen. The results also demonstrate the possibility of using molecular rotors as novel probes capable of characterizing the polymerization dynamics of various biopolymers significant to medicine.NSF-REU Program in Biosystems Modeling and Analysi

Calcineurin determines toxic versus beneficial responses to α-synuclein

Calcineurin (CN) is a highly conserved Ca[superscript 2+]–calmodulin (CaM)-dependent phosphatase that senses Ca[superscript 2+] concentrations and transduces that information into cellular responses. Ca[superscript 2+] homeostasis is disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca[superscript 2+], thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN's spectrum of substrates toward protective pathways. Modulating CN or CN's substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase’s activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.Jeffry M. and Barbara Picower FoundationJPB FoundationHoward Hughes Medical Institute (Collaborative Innovation Award)Eleanor Schwartz Charitable Foundatio

Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms

Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and bio-physically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired cross-reactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ~ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.Fil: Srinivasan, Lakshmi. University Johns Hopkins; Estados UnidosFil: Alzogaray, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Selvakumar, Dakshnamurthy. Fortébio; Estados UnidosFil: Nathan, Sara. University Johns Hopkins; Estados UnidosFil: Yoder, Jesse B.. University Johns Hopkins; Estados UnidosFil: Wright, Katharine M.. University Johns Hopkins; Estados UnidosFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Nwafor, Justin N.. University Johns Hopkins; Estados UnidosFil: Labanda, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Schön, Arne. University Johns Hopkins; Estados UnidosFil: Freire, Ernesto. University Johns Hopkins; Estados UnidosFil: Tomaselli, Gordon F.. University Johns Hopkins; Estados UnidosFil: Amzel, León Mario. University Johns Hopkins; Estados UnidosFil: Ben-Johny, Manu. Columbia University; Estados UnidosFil: Gabelli, Sandra. University Johns Hopkins; Estados Unido

Molecular endpoints of Ca2+/calmodulin- and voltage-dependent inactivation of Cav1.3 channels

Ca2+/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca2+ channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation in distinctive ways, depending on which hypothesis holds true. Thus, we systematically mutate the activation gate, formed by all S6 segments within CaV1.3. These channels feature robust baseline CDI, and the resulting mutant library exhibits significant diversity of activation, CDI, and VDI. For CDI, a clear and previously unreported pattern emerges: activation-enhancing mutations proportionately weaken inactivation. This outcome substantiates an allosteric CDI mechanism. For VDI, the data implicate a “hinged lid–shield” mechanism, similar to a hinged-lid process, with a previously unrecognized feature. Namely, we detect a “shield” in CaV1.3 channels that is specialized to repel lid closure. These findings reveal long-sought downstream mechanisms of inactivation and may furnish a framework for the understanding of Ca2+ channelopathies involving S6 mutations

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Conservation of Calcium Regulation Across Voltage-gated Calcium and Sodium Channels

The voltage-gated Ca2+ and Na channels represent two major ion-channel superfamilies with distinct biophysical properties that support diverse and vital physiological functions. Accordingly, these superfamilies have long been studied as distinct entities. That said, the carboxyl tail of these channels exhibit remarkable homology, hinting at a purposeful module long-shared amongst these molecules. If these homologous tail domains elaborated functions of like correspondence, a common origin of such a module might be suggested. For Ca2+ channels, the interaction of CaM with their carboxy-terminus evokes robust and recognizably similar forms of Ca2+ regulation. By contrast, over a decade of research has revealed subtle and variable Ca2+ effects on Na channels, calling into question the very existence of a shared module. Here, using Ca2+ photouncaging, we find that these dissimilarities in Na channels are only apparent, and that Ca2+ regulatory function and mechanism are fundamentally conserved. To identify the molecular states underlying channel regulation, we develop a structure-function approach relating the strength of regulation to the affinity of underlying calmodulin/channel interactions, by a Langmuir relation. By application of this theoretical framework to Ca2+ channels, we uncovered an unprecedented switching of CaM interaction on the channel carboxy-terminus. This system of structural plasticity furnishes a unified mechanistic framework to understand Ca2+ and Na channel regulation and offers shared principles to approach related channelopathic diseases. In all, these results help substantiate the persistence of an ancient Ca2+ regulatory design across channel superfamilies – a relic that has been preserved for much of living history