About our work
The goal of the Savage Lab is to understand how compartmentalization facilitates the molecular physiology of the cell. Specifically, we are interested in the assimilation of carbon dioxide and the strategies used by microbes to optimize this metabolism. Our model system is the cyanobacterium Synechococcus elongatus PCC7942, which uses the multi-scale cooperation of many different protein components to increase the fidelity and rate of the critical enzyme ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) in a process known as the Carbon dioxide Concentrating Mechanism (CCM). We use the tools of biochemistry, molecular biology, and synthetic biology to identify and interrogate the key players and mechanistic principles underlying CCM function. Ultimately, we hope to develop a total cell biological understanding of how CCM function emerges from individual components and to use this understanding for the improvement of carbon dioxide assimilation in plants. Our specific areas of research are as follows:
The role of compartmentalization in catalyzing metabolism
Cyanobacteria carry out photosynthetic carbon dioxide assimilation by controlling when and where biochemical reactions occur. Perhaps most amazing is the use of a protein organelle, the carboxysome, to achieve improved activity of RuBisCO. RuBisCO is a notoriously bad enzyme, and it is thought the inside of the carboxysome provides a microenvironment within the cell of high carbon dioxide (and possibly low oxygen) as a means of improving rate and specificity. Despite numerous structural studies of carboxysome components, however, there are many open questions related to how this massive complex self-assembles and functions in the context of the cellular milieu. To this end, we are investigating the following areas using a combination of molecular biology, biochemistry, and biophysics:
Assembly of the carboxysome. The carboxysome is a 250 MDa+ complex formed from thousands of proteins, yet it self-assembles in the bacterial cytosol into monodisperse particles. How does this amazing process occur? In previous work, we have used microscopy to demonstrate the importance of carboxysome partitioning during cell division and the surprising connection between this process and the bacterial cytoskeleton (Savage et al. 2010; Yokoo et al. 2014). More recently we have focused on assembly directly and have demonstrated the minimal set of proteins sufficient to produce the carboxysome in a heterologous host (Bonacci et al. 2012), and honed in on one of these proteins, CsoS2, as particularly critical to the process (Chaijarasphong et al. 2016). We are now currently building on these advances to understand how specific protein-protein interactions direct the assembly process and are attempting to elucidate the fundamental biophysical principles leading to high fidelity assembly.
Unraveling the effect of compartmentalization on protein function. The concentration of protein inside the carboxysome is 500 – 600 mg/ml, roughly twice that of the bacterial cytoplasm! Molecular crowding and confinement are known to dramatically affect protein function thus raising a general question of the importance of compartmentalization into protein compartments such as the carboxysome. At the same time, selectivity of the protein shell likely influences the function of enzymatic cargo. To explore this effect, we have developed a synthetic system using the capsid-forming encapsulin protein to probe the interplay between compartmentalization and protein activity and stability.
Tools for studying and perturbing cellular physiology
The importance of metabolism spans the biological and chemical sciences, and it is now appreciated that a deeper understanding of metabolic function is critical to the treatment of many diseases and for the metabolic engineering of organisms for sustainable chemical production. However, studies on metabolism are often limited by the difficulty in rapidly and noninvasively probing small molecules within the cell. For example, it is typically impractical to perform simple genetic screens involving a direct assay of metabolism. To this end, work in our group also aims to develop biosensor-based approaches for rapidly measuring cellular metabolite levels involved in central metabolism and carbon dioxide assimilation. At the same time, we are interested in creating novel genome editing tools to facilitate the screening process.
Rapid construction of metabolite biosensors. A major effort underway is to develop and refine techniques for the engineering of genetically-encoded fluorescent protein biosensors that can sense and report on the intracellular concentration of metabolites in living cells (Nadler et al. 2016). Although we are currently focused on the protein engineering methods of this process, we ultimately envision applying biosensors in high-throughput metabolic engineering screens, such as to improve ribulose-1,5-bisphosphate supply, a known limitation in the CBB cycle.
Cas9 engineering. We are also applying our biosensor protein engineering methods, such as domain insertion profiling, to the programmable endonuclease Cas9 in order to create single proteins that can both sense and actuate a cellular response including genome cleavage, gene repression and/or activation. This work aims to: i) enable more precise control of CRISPR-Cas9 nuclease activity, ii) create molecular scaffolds for the target recruitment of specific proteins to precise DNA sequences, iii) generate robust Cas9-based transcriptional activation and repression platforms for synthetic biology (Oakes et al. 2014, Oakes et al. 2016). Moreover our work reveals the constraints on the engineering of CRISPR proteins and provides a foundational platform from which we can expand the functional repertoire of such genome engineering proteins.
The Savage Lab is a member of the Departments of Molecular & Cell Biology and Chemistry at the University of California, Berkeley. UC Berkeley, also affectionately known as Cal, is a public research university dedicated to both education and research and has made such varied contributions to society as the discovery of six chemical elements and the birth of the Free Speech Movement. Cal is located near downtown Berkeley, California and is rooted in the vibrant intellectual, cultural, environmental, culinary, and entrepreneurial communities of the San Francisco Bay area.
The Savage Lab is also a member of the Energy Biosciences Institute (aka the EBI), a collaboration between UC Berkeley, Lawrence Berkeley National Laboratory, the University of Illinois at Urbana-Champaign, and British Petroleum. This collaboration of diverse expertise seeks to advance our understanding of biological research with the hope of informing and improving the decisions and technologies of the energy sector. The EBI is located in the Energy Biosciences Building (EBB), a 113,000 square foot research building on the northwest corner of campus adjacent to downtown Berkeley. The EBB houses laboratories from numerous departments on campus and possesses state-of the art research facilities.