Heng Huang

Remote control of ion channels and neurons through magnetic-field heating of nanoparticles

Recently, optical stimulation has begun to unravel the neuronal processing that controls certain animal behaviours. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Here, we show an approach based on radio-frequency magnetic-field heating of nanoparticles to remotely activate temperature-sensitive cation channels in cells. Superparamagnetic ferrite nanoparticles were targeted to specific proteins on the plasma membrane of cells expressing TRPV1, and heated by a radio-frequency magnetic field. Using fluorophores as molecular thermometers, we show that the induced temperature increase is highly localized. Thermal activation of the channels triggers action potentials in cultured neurons without observable toxic effects. This approach can be adapted to stimulate other cell types and, moreover, may be used to remotely manipulate other cellular machinery for novel therapeutics.

Stable, high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (Kd = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (Tm = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67.

Engineered Streptavidin Monomer and Dimer with Improved Stability and Function

Although streptavidins high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult. In this study, we describe the design of a stable monomer that binds biotin both in vitro and in vivo. To this end, we constructed and characterized monomers containing rationally designed mutations. The mutations improved the stability of the monomer (increase in T(m) from 31 to 47 °C) as well as its affinity (increase in K(d) from 123 to 38 nM). We also used the stability-improved monomer to construct a dimer consisting of two streptavidin subunits that interact across the dimer-dimer interface, which we call the A/D dimer. The biotin binding pocket is conserved between the tetramer and the A/D dimer, and therefore, the dimer is expected to have a significantly higher affinity than the monomer. The affinity of the dimer (K(d) = 17 nM) is higher than that of the monomer but is still many orders of magnitude lower than that of the wild-type tetramer, which suggests there are other factors important for high-affinity biotin binding. We show that the engineered streptavidin monomer and dimer can selectively bind biotinylated targets in vivo by labeling the cells displaying biotinylated receptors. Therefore, the designed mutants may be useful in novel applications as well as in future studies in elucidating the role of oligomerization in streptavidin function.

Optimizing of Local Nano-Particle Heating for Thermo-Magnetic Stimulation of Cells

We have developed thermo-magnetic stimulation of cells by coupling radio-frequency magnetic field heating of supraparamagnetic nanoparticles to temperature sensitive ion channels. Optimizing the thermomagnetic heating requires nanoparticles with increased heating power, high, short field pulses, and better understanding of the molecular scale heat transfer from nanoparticles to the surrounding fluid. We are developing supraparamagnetic nanoparticles with increased magnetic moment and heating capacity. Several sizes, materials and core-shell geometries were synthesized. The nanoparticles were made water-soluble by either encasing them in silica or by polymer coating. To optimize the efficiency of these nanoparticles in activating the TRPV1 channels, we label channel-expressing cells with nanoparticles and compare the efficiency of various field frequencies and strengths, as well as pulse durations. To study the molecular scale heat transfer from nanoparticles to the surrounding fluid, we use fluorescence to measure the local temperature in the nanometer space around these nanoparticles. We compare isolated nanoparticles to particles arranged on a surface or in bulk, while also recording the global temperature. The obtained spatio-temporal temperature profiles are modeled using finite element software (COMSOL).

Monitoring Association of Membrane Proteins with Micro-Domains and Cytoskeleton in Live Cells During Signaling and Perturbation

A central challenge to understanding cell signaling is to quantify how the membrane ultra-structure modulates signaling dynamics and how vice-versa signaling influences the membrane ultra-structure. Spatial membrane domains, such as created by lipid rafts and the membrane cytoskeleton, influence membrane protein mobility and hence membrane bound processes. Not only are these domains modified in a range of pathologies but various drugs are also thought to influence them. However, direct visualization of these lipid domains in intact cells is challenging because of their small dimension and dynamics.We present a non-destructive a camera based FCS method for quantifying the membrane protein association with lipid domains in intact cells and with membrane cytoskeleton domains. Most importantly, our method provides continuous monitoring of changes in the protein domain interactions over time. We investigated the modulation of GPI-anchored GFP with lipid domains over time in response to cross linking, Ethanol, Cholesterol Sulfate or Cholera toxin addition, as well as cholesterol extraction. As an example for signaling induced changes, we studied the changes in the interaction of temperature sensitive ion channel, TRPV2, with lipid domains and membrane cytoskeleton upon activation by 2-APB.