Introduction

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.

Identifying and Overcoming Obstacles to GPCR Expression and Characterization

Human G-protein coupled receptors (GPCRs) represent the largest family of integral membrane receptors that are involved in intercellular communication in response to diverse external stimuli.  Upon ligand binding to GPCRs, a signal is initiated intracellularly via interaction with the GTP-binding protein (G protein) to further transmit cellular responses. GPCRs play a myriad number of physiological roles in humans, leading to a substantial pharmacological interest in modulating this signaling activity via novel pharmaceutically active molecules. Indeed, 30-50% of currently marketed drugs are thought to be act through the direct and indirect modulation of GPCR activity.

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.

Our research in this area includes re-engineering of the recognition sequence of GPCRs to serve as cellular sensors, improving expression of GPCRs for drug screening and crystallization, and biophysical characterization of ligand-receptor and membrane-receptor interactions.

Recent and Representative Publications:

1. Naranjo, AN, A Chevalier, GD Cousins, E Ayettey, EC McCusker, C Wenk, AS Robinson (2015) Conserved disulfide bond is not essential for the adenosine A2A receptor: extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition, BBA Biomembranes, 1848: 603-614 Available on-line Dec 5 2014 DOI: 10.1016/j.bbamem.2014.11.010
2. Blocker, KM, ZT Britton, AN Naranjo, PM McNeely, CL Young, AS Robinson (2015) Recombinant G protein-coupled receptor expression in Saccharomyces cerevisiae for protein characterization, in “Membrane Proteins – Production and Function Characterization”, Methods Enzymol., 556:165-83. doi: 10.1016/bs.mie.2014.12.025
3. McNeely, P.M., Naranjo, A.N., and A.S. Robinson (2012) Structure-function studies with G-protein coupled receptors as a paradigm for improving drug discovery and therapeutic development, Biotechnology Journal, 7(12): 1451-1461. DOI: 0.1002/biot.201200076
4. O’Malley, Michelle A., Matthew E. Helgeson, Norman J. Wagner, Anne S. Robinson (2011) “Toward Rational Design of Protein Detergent Complexes: Determinants of Mixed Micelles that are Critical for the in vitro Stabilization of a G-protein Coupled Receptor”, Biophysical Journal, 101 (8): 1938-1948.   DOI: 10.1016/j.bpj.2011.09.018  PMID: 22004748

Collaborators:

Bramie Lenhoff
Department of Chemical Engineering
University of Delaware 
http://www.che.udel.edu/directory/facultyprofile.html?id=252

Wilfred Chen

Department of Chemical Engineering
University of Delaware

http://www.che.udel.edu/directory/facultyprofile.html?id=25009

Funding:

National Science Foundation 1263768 (PI: W. Chen)

Collaborative Proposal: Exploiting synthetic GPCRs and mating factors as extracellular sensors for substrate-dependent assembly of complex cellulosomes

Understanding and Controlling Protein Aggregation

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. 

As these stresses cannot be avoided altogether practically, an attractive alternative strategy is to engineer protein sequence/structure to be less susceptible to aggregation.  If this can be done within a mechanistic context of protein aggregation, then it may be more easily generalized beyond model systems. Therefore, an overall motivation for the collaborative research with the Roberts lab is to provide mechanistic yet practical computational tools and design paradigms for rational protein design to impart resistance to aggregation, and in the longer term to combine them with algorithms focused on maintaining or imbuing new protein function.

(a) Summary of designed point-mutants for gDCrys based on category of design strategy. (b)Three dimensional structure of gDCrys (PDB file: 1HK0) illustrating each variant site; H22 (yellow), C41 (green), L53 (red), M69 (orange), and S130 (blue). From Sahin, E et al. (2011) Computational Design and Biophysical Characterization of Aggregation-Resistant Point Mutations for gD Crystallin Illustrate a Balance of Conformational Stability and Intrinsic Aggregation Propensity, Biochemistry 50, 628-639.

Recent and Representative Publications:

1.  Wu, H, K Truncali, J Ritchie, R Kroe-Barrett, S Singh, AS Robinson, and CJ Roberts (2015) Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1, mAbs, in press
2.  Wu, H., Rachel Kroe-Barrett, Sanjaya Singh, Anne S. Robinson, Christopher J. Roberts (2014) Competing aggregation pathways for monoclonal antibodies, FEBS Letters 588(6): 936-941. http://dx.doi.org/10.1016/j.febslet.2014.01.051
3. JA Costanzo, CJ O’Brien, K Tiller^†, E Tamargo^†, AS Robinson, CJ Roberts, and EJ Fernandez (2013) Computational Design to Control Protein Aggregation Rates Through Conformational Stability, Protein Eng, Des, & Sel, 27 (5): 157-167 10.1093/protein/gzu008  

Collaborator:

Chris Roberts
Department of Chemical Engineering
University of Delaware
http://www.che.udel.edu/directory/facultyprofile.html?id=12564

Funding:

NSF 1264554 (PI: Roberts)
GOALI: Collaborative Proposal: Mechanistic Design of Aggregation Resistance in Multi-Domain Proteins

Cellular Mechanisms Controlling Protein Quality and Human Disease

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:

1. Understanding the effects of these cellular control mechanisms of protein expression levels and protein quality (activity, post-translational modifications).
2. Determining how loss of cellular control may lead to a disease state

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.

Recent and Representative Publications:

1. Young, CL and AS Robinson^* (2014) Protein Folding and Secretion: Mechanistic Insights Advancing Recombinant Protein Production in S. cerevisiae, Current Opinion in Biotechnology 30: 168-177. DOI: 10.1016/j.copbio.2014.06.018
2. Spatara, ML and Robinson, AS* (2010) “Transgenic mouse and cell culture models demonstrate a lack of mechanistic connection between endoplasmic reticulum stress and tau dysfunction” Journal of Neuroscience Research, 88(9):1951-61. PMID: 20143409