Research in the Robinson group centers on three areas in biochemical engineering: understanding and controlling protein aggregation; cellular mechanisms controlling protein quality and human disease; and overcoming obstacles to expression and characterization of G-protein coupled receptors. In each of these areas, we use the tools of molecular and cellular biology, biochemistry, and biophysics, combined with systems biology, mathematical modeling, and engineering analysis, to develop an improved understanding of biological systems. Using this knowledge, we carry out molecular and cellular engineering to develop improved methods, products, and tools for biotechnology, medical, and research applications.
Aggregation is a long-standing in vitro and in vivo obstacle for studying proteins; it serves as an irreversible, off-pathway process during protein folding, and it is a ubiquitous problem throughout commercial manufacture of protein-based biotechnology products. This is a potentially debilitating setback from a structural biology and protein design perspective, as it can limit the ability of scientists to produce sufficiently high quantities of purified material that are required for subsequent sample preparation and for biophysical characterization. It also greatly limits biotechnology product discovery and development. From a discovery perspective, only those candidate molecules that can be readily expressed and (re)folded to active forms can be included in screens for improved or novel structure and function. Even once viable product candidates are identified, elimination of aggregates and prevention of aggregation is one of the most common technical hurdles for product manufacture, formulation, and final product viability.
In collaborative research with the Roberts group, we seek to
Recent and Representative Publications:
MedImmune (w/ CJR)
Cells are inherently robust to stochastic perturbations, and have evolved to recover readily from short-term exposure to heat, pH changes, and nutrient deprivation during times of stress. This process, termed the stress response, is important in a wide range of basic research and commercial applications since cell growth rates, production of metabolites, and protein expression are all affected by the stress response. Moreover, accumulation of toxic metabolic products or unfolded proteins can in turn induce the stress response, implicated in a number of human diseases. Our two areas of research are:
During protein expression, the stress response to unfolded protein accumulation, termed the unfolded protein response (UPR), resulting in low protein yields during heterologous protein expression. A systematic approach to improving protein production involves understanding on a molecular level how cell regulation mechanisms respond to heterologous protein expression. Key issues include how to maintain reasonable cell health, yet obtain high protein yields and what interactions promote or stabilize formation of active protein with correct post-translation modifications.
Hallmarks of the disease state in the Alzheimer brain, and of several other neurodegenerative diseases including corticobasal degeneration, frontotemporal dementia, and parkinsonism linked to chromosome 17, are the hyperphosphorylation of tau and subsequent formation of insoluble tau aggregates (neurofibrillary tangles or paired helical filaments). In healthy cells, the tau protein binds to and stabilizes microtubules, and is abundant. It is not yet clear whether the problem in these diseases is a loss of tau function (e.g. loss of microtubule stability), or an inherent toxicity of the tau tangles. Major research questions include determining the biochemical and cellular pathways that drive tau homeostasis, including degradation and refolding pathways, and which steps in the tau pathways are the best targets for therapeutic intervention.
NIH R01-GM 075297
Recent and Representative Papers
Membrane proteins are critical for cell-cell recognition, inter and intracellular signaling, and cell homeostasis, and are of great interest in understanding human disease and as drug targets. However, the majority of membrane protein structures have been obtained only from proteins available in natural abundance, due to the major difficulties with heterologous expression to date. Of all unique membrane crystal structures to date, about 10% have been expressed in heterologous systems, and fewer than 5% are of eukaryotic proteins (15 of which are transmembrane proteins: 4 GPCRs, 5 ion channels, 6 aquaporins and other transporters).
G-protein coupled receptors (GPCRs) are membrane proteins expressed in virtually all human tissues, and they transmit a wide variety of signals in response to diverse stimuli (including light, hormones, injury, and inflammation). Although cloning of a mammalian GPCR (β2 adrenergic receptor) was first achieved in the mid-1980s, the first non-rhodopsin GPCR structure only became available almost 20 years later (2007) . There are approximately 360 non-sensory human GPCRs (of ~800-900 total predicted GPCRs), with ligands identified or predicted for only ~2/3 of the non-sensory proteins. Many acute and chronic disease states are linked to GPCR function or malfunction, and 30-60% of commercially available drugs interact with a GPCR, making them targets for nearly 40% of drug discovery efforts worldwide, yet these drugs target less than 10% of all GPCRs. However, efforts to better understand GPCR ligand specificity, structure, stability, and assembly are hampered by the difficulties associated with producing these integral membrane proteins.
Over the last eight years, our laboratory has expressed several GPCRs and other complex proteins in order to determine their relative expressability and to investigate cellular limitations to folding and trafficking to give active protein both from Saccharomyces cerevisiae and E. coli. Our overall goals are to overcome the limitations to expression in S. cerevisiae, identify the mechanisms for membrane insertion and proper trafficking, and biophysical and stability characterization of purified receptors.
Recent and Representative Publications:
NIH COBRE: P20 RR15588 “Membrane Protein Production and Characterization” Sub-project: "Determinants of GPCR Expression in E. coli and Yeast"