Directed evolution and biophysical characterization of a full-length, soluble, human caveolin-1 variant

Abstract

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 cav, for expression in E. coli. Cav 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, cav does not bind off-target proteins. Cellular studies show that cav retains the parent protein's ability to localize at the cellular membrane. Unlike truncated versions of caveolin, cav forms large, oligomeric complexes consisting of approximately >50 monomeric units without requiring additional cellular components. Cav's secondary structure is a mixture of α-helices and β-strands. Isothermal titration calorimetry experiments reveal that cav binds to gp41 and PKA with low micromolar binding affinity (K). In addition to the insights into caveolin structure and function, the approach applied here could be generalized to other membrane proteins.

Reconstitution of Caveolin-1 into Artificial Lipid Membrane: Characterization by Transmission Electron Microscopy and Solid-State Nuclear Magnetic Resonance

“…Recombinant caveolins have been previously prepared, however not necessarily with a focus on homogeneous, milligram-scale levels required for biophysical studies [50]. Alternatively, protein engineering was employed to increase caveolin solubility and bacterial expression levels at the expense of severely modifying the interaction of caveolin with membranes [30]. The massive generation of caveolae-like vesicles in E. coli by CAV1 over-expression strongly indicates that the protein properly folds when expressed in the bacteria and that post-translational modifications that normally occur in mammalian cells are not essential for the membrane scaffolding function of caveolins [51].…”