Protein engineering by directed evolution can alter proteins' structures, properties, and functions. However, membrane proteins, despite their importance to living organisms, remain relatively unexplored as targets for protein engineering and directed evolution. This gap in capabilities likely results from the tendency of membrane proteins to aggregate and fail to overexpress in bacteria cells. For example, the membrane protein caveolin-1 has been implicated in many cell signaling pathways and diseases, yet the full-length protein is too aggregation-prone for detailed mutagenesis, directed evolution, and biophysical characterization. Using a phage-displayed library of full-length caveolin-1 variants, directed evolution with alternating subtractive and functional selections isolated a full-length, soluble variant, termed cavsol, for expression in E. coli. Cavsol folds correctly and binds to its known protein ligands HIV gp41, the catalytic domain of cAMP-dependent protein kinase A, and the polymerase I and transcript release factor. As expected, cavsol does not bind off-target proteins. Cellular studies show that cavsol retains the parent protein's ability to localize at the cellular membrane. Unlike truncated versions of caveolin, cavsol forms large, oligomeric complexes consisting of approximately >50 monomeric units without requiring additional cellular components. Cavsol's secondary structure is a mixture of α-helices and β-strands. Isothermal titration calorimetry experiments reveal that cavsol binds to gp41 and PKA with low micromolar binding affinity (KD). In addition to the insights into caveolin structure and function, the approach applied here could be generalized to other membrane proteins.
Keywords: Biophysics; Caveolae; Caveolin; Membrane proteins; Oligomerization; Phage display.
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