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Thrust 1 - Protein Metamorphosis and Responsive Nanodevices

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.

Project Lead

Associate Professor of Bioengineering Eva de Alba
Associate Professor of Bioengineering, Thrust 1 Lead
Biomedical Sciences and Physics Building
Professor of Chemistry and Chemical Biology Mike Colvin
Professor of Chemistry and Chemical Biology, Thrust 1
COB, Room 361
Roberto C. Andresen Eguiluz
Assistant Professor of Materials Science and Engineering, Thrust 1&3, Undergraduate Lead
Assistant Professor of Molecular and Cell Biology Aaron Hernday
Assistant Professor of Molecular and Cell Biology, Thrust 1
SE1, Room 314
Professor of Molecular Cell Biology Patricia LiWang
Professor of Molecular Cell Biology, Thrust 1
Castle, Room 95
Professor, Chemistry and Chemical Biology Andy LiWang
Professor, Chemistry and Chemical Biology, Thrust 1
Castle, Building 1200, Room 36
Assistant Professor of Chemistry & Biochemistry Andrea Merg
Assistant Professor, Chemistry and Biochemistry, Thrust 1
BSP, Room 167
CCBM Co-Director, Professor of Bioengineering Victor Muñoz
CCBM Co-Director & PI, Professor of Bioengineering, Thrust 1
Biomedical Sciences and Physics Building 261
Tomas Rube
Assistant Project Scientist, Thrust 1
Assistant Professor of Bioengineering Anand Bala Subramaniam
Assistant Professor, Bioengineering, Thrust 1
SE2, Room 291
Assistant Professor of Chemistry and Chemical Biology, Thrust 1
Castle Commerce Center
Assistant Professor of Chemistry and Biochemistry Michael Thompson
Assistant Professor of Chemistry and Biochemistry, Thrust 1
Castle, Bldg 1200, Room 96