Nikhil Malvankar1
Yale University1
Electrogenetics, which uses electric fields to control gene expression and cell function, has applications ranging from production and delivery of bioproducts and biomanufacturing of functional materials. We plan to design and synthesize next generation of electronic biomaterials by combining electrogenetics with synthetic and systems biology. We have found that deep in the ocean or underground, where there is no oxygen, <i>Geobacter </i>bacteria “breathe” by projecting tiny protein filaments called "nanowires" into the soil, to dispose of excess electrons resulting from the conversion of nutrients to energy (<i>Cell</i> 2019).<br/>Using electric fields, we have induced the overexpression of cytochrome OmcZ nanowires that show 1000-fold more electron conductivity than the OmcS nanowires important in natural soil environments (<i>Nature Chem.Bio.</i> 2020). Nanowire-based electrogenetics platform responds rapidly to environmental changes to adapt to signals and stresses for stimuli-responsive and pH-responsive systems. In contrast to optogenetics, which requires specifically designed devices and whose signals are attenuated in high-density cells, these nanowires efficiently control bacterial growth, adhesion and communication.<br/>I will present our recent studies revealed a surprise that these protein nanowires have a core of metal-containing molecules called hemes. Previously it was not suspected. Using high-resolution cryo-electron microscopy, we were able to see the nanowire’s atomic structure and discover that hemes line up to create a continuous path along which electrons travel. I will present our recent experimental and computational studies to identify the nanowire composition, structure and mechanism of nanowire assembly and high electron conductivity by measuring their DC and THz conductivity as a function of nanowire length, temperature, frequency, pH and heme stacking.<br/>These studies solve a longstanding mystery of how microbial protein nanowires move electrons over micrometer distances and enable the bacteria to perform environmentally important functions such as cleaning up radioactive sites, generating biofuels and electricity (<i>Nature Nano</i><i>. </i>2011), or exchange electrons with other bacterial species to share nutrients and resources (<i>Science</i> 2010). This electron exchange process is important in microbial communities found in diverse environments. Nanowires confer novel properties to microbial consortia such as living transistors (<i>Nature Nano</i><i>. </i>2011) and living supercapacitors (<i>ChemPhysChem </i>2012)<br/>We have further found that the nanowires are translocated to the bacterial surface using unique heterodimeric pili that likely serve as a piston to secrete cytochromes, rather than functioning as nanowires as previously thought (<i>Nature</i> 2021.) These previously hidden bacterial hairs serve as a molecular switch for controlling the release of cytochrome nanowires. As all major phyla of prokaryotes use systems similar to these pili, this nanowire translocation machinery may have a widespread effect in identifying the evolution and prevalence of diverse electron-transferring microorganisms and in determining nanowire assembly architecture for designing synthetic protein nanowires.<br/>I will also present our studies on bioinspired synthetic protein nanowires and new methodology to set standards for reporting protein conductivity and underlying mechanisms. Although most protein conductivity studies remain focused on electrons, protons play a very important role, not only in energy generation, but also in the electronic conductivity of proteins. Through measurements of the intrinsic electron transfer rate, we recently showed that both the energetics of the proton acceptor, a neighboring glutamine, and its proximity to tyrosine, regulate the hole transport over micrometers in amyloids through a proton rocking mechanism (<i>PNAS</i> 2021). I will present strategies to couple electron and proton transfer for the development of electronically conductive protein-based biomaterials.