Thrust 1: Protein Metamorphosis and Responsive Nanodevices (de Alba lead).
This Thrust targets the interactions, self-organization, and operation of biological macromolecules, and their interplay to form the assemblies that perform cellular functions. Phase II research focuses on the emerging theme of protein metamorphosis as mechanism to enable natural and synthetic controllable biological nanodevices, organized in two broad areas: 1) the functional roles of gradually morphing proteins; 2) engineering of control systems of the assembly-disassembly of biological macromolecular assemblies.
1.1. Circadian molecular clocks synchronize rhythms of rest and activity to Earth’s rotation. The cyanobacterial oscillator is composed of just three proteins (KaiA, KaiB, and KaiC) that autonomously keep time [6] using a molecular mechanism that remains unknown. We reported that KaiB switches folds between daytime-inactive and nighttime-active states [7] and determined the structures of the clock complexes [8]. For Phase II we target the KaiB fold metamorphosis as the potential ticking mechanism.
1.2. Advanced conformational fluorescence biosensors based on morphing proteins promise real-time monitoring inside living cells at nanoscale resolution [9]. In Phase I we developed the morphing-uponbinding transducer technology and engineered proof of concept fluorescence sensors for pH, ionic strength and [Ca2+]. For Phase II, we will integrate these sensors into a designer plug and play platform to convert them into fully recombinant fluorescence biosensors for real time in vivo imaging. We will also develop first-time fluorescence sensors for monitoring of ATP/ADP and cAMP concentration.
1.3. Gene tracking and cooperative DNA sequence readout by eukaryotic transcription factors (TFs). Morphing proteins can explain key puzzles in eukaryotic gene expression, such as how TFs track target genes ignoring myriads of false positives [10] and are able to coalesce to the transcription machinery [11]. An emerging mechanism for boosting binding specificity is the cooperative sequence readout of TF complexes that are flexible structurally (morphing domains [12]), in composition, and sequence readout [13]. We will test this mechanism combining: 1) single-molecule fluorescence/force spectroscopy to track TFs as they search along DNA molecules; 2) high-throughput SELEX-seq to map out DNA binding profiles; 3) High-throughput profiling of hundreds of “cellular” conditions of TF binding and coalescence.
1.4. Peptide-based design of inflammasome inhibitors by targeting the inflammasome adapter ASC. The inflammatory process is a first defense mechanism, but oftentimes the inflammatory response chronifies leading to disease [14]. Inflammation is controlled by the (dis)assembly of a multiprotein complex known as the inflammasome [15]. Centerstage in the inflammasome is the adapter ASC, a protein with two sticky domains connected by a flexible linker that mediates assembly [15]. We aim at blocking inflammasome assembly via peptides designed de novo to inhibit ASC self-association.
1.5. Engineering controllable protein (dis)assembly for responsive bio-nanodevices and biomaterials. This project aims at developing variants of bacterial microcompartment shell proteins [16] engineered to introduce assembly-disassembly control systems such as redox-controllable hydrogels or allosteric mesoscale protein-based containers that can respond to multiple cues [17].
Crosscutting Thrusts 1 and 2. Synthetic biomimetic membrane-based compartments aim to reproduce the micro-environments found in living cells, including exchange of content and information. A Phase I thrust 1-2 collaboration led to the effective encapsulation in giant lipid vesicles of the cyanobacterial oscillator in its functional form and including fluorescence reporters. Phase II aims to extend the methodology to other soluble protein systems being developed in thrust 1 (fluorescence sensors, sets of transcription factors and DNA, and inflammasome assemblies). The technology will serve as a platform to monitor the functioning of macromolecular assemblies in biomimetic controllable environments.
