Membrane proteins confound anything less than exceptionally heroic attempts aimed at solving their structures. The conventional approaches to membrane protein overexpression, purification, and crystallization typically fail due to problems with insolubility and folding. This project leverages large libraries of soluble and highly crystallizable proteins to identify binding partners for membrane proteins. Selectants from these libraries will provide affinity reagents for membrane protein co-expression, affinity purification and co-crystallization. Co-expression with the binding partner could help avoid membrane protein aggregation, and allow protein folding to take place. Affinity chromatography with the binding partner is aimed at assisting membrane protein purification, and co-crystallization aims to slow protein aggregation and precipitation during formation of crystals. The first specific aim focuses on design and construction of phage-displayed protein libraries for high affinity binding to membrane proteins. Strategic choice of proteins for library formation, such as the highly crystallizable protein lysozyme and the exceptionally soluble protein S-crystallin, for phage display will help insure the success of the project;additional libraries specifically tailored forG-protein coupled receptors (GPCRs) include variants of G- proteins and GPCR ligands. To obtain high affinity binding, thermal stability, solubility, and other properties, the second specific aim features a flow path of selections and screens. In the third specific aim, the affinity reagents from phage display are applied to the production of membrane proteins and their crystallization. By binding to and essentially freezing specific conformations of the membrane protein, the affinity reagents could offer powerful tools both for structural biology, but also other structure-function studies of membrane proteins. In summary, this proposal will define new approaches to protein engineering and molecular recognition, through development of new fusion proteins and their use in the recognition of membrane proteins.

Public Health Relevance

Membrane proteins represent an extraordinarily important class of proteins associated with many diseases and targeted by roughly 40% of therapeutics. Yet, despite their biomedical importance, structures of membrane proteins are relatively rare due to problems with their insolubility and crystallizability. This proposal harnesses classes of proteins known to exhibit great solubility and crystallizability as reagents designed to bind, stabilize, and expedite determination of membrane protein structures.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Chin, Jean
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California Irvine
Schools of Arts and Sciences
United States
Zip Code
Smith, Joshua N; Edgar, Joshua M; Balk, J Mark et al. (2018) Directed evolution and biophysical characterization of a full-length, soluble, human caveolin-1 variant. Biochim Biophys Acta Proteins Proteom 1866:963-972
Britton, Joshua; Majumdar, Sudipta; Weiss, Gregory A (2018) Continuous flow biocatalysis. Chem Soc Rev 47:5891-5918
Meneghini, Luz M; Tripathi, Sarvind; Woodworth, Marcus A et al. (2017) Dissecting binding of a ?-barrel membrane protein by phage display. Mol Biosyst 13:1438-1447
Britton, Joshua; Stubbs, Keith A; Weiss, Gregory A et al. (2017) Vortex Fluidic Chemical Transformations. Chemistry 23:13270-13278
Britton, Joshua; Dyer, Rebekah P; Majumdar, Sudipta et al. (2017) Ten-Minute Protein Purification and Surface Tethering for Continuous-Flow Biocatalysis. Angew Chem Int Ed Engl 56:2296-2301
Britton, Joshua; Smith, Joshua N; Raston, Colin L et al. (2017) Protein Folding Using a Vortex Fluidic Device. Methods Mol Biol 1586:211-220
Britton, Joshua; Meneghini, Luz M; Raston, Colin L et al. (2016) Accelerating Enzymatic Catalysis Using Vortex Fluidics. Angew Chem Int Ed Engl 55:11387-91
Britton, Joshua; Raston, Colin L; Weiss, Gregory A (2016) Rapid protein immobilization for thin film continuous flow biocatalysis. Chem Commun (Camb) 52:10159-62
Gilliam, Amanda J H; Smith, Joshua N; Flather, Dylan et al. (2016) Affinity-Guided Design of Caveolin-1 Ligands for Deoligomerization. J Med Chem 59:4019-25
Mohan, Kritika; Weiss, Gregory A (2016) Chemically Modifying Viruses for Diverse Applications. ACS Chem Biol 11:1167-79

Showing the most recent 10 out of 16 publications