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. 2018 Sep;1866(9):963-972.
doi: 10.1016/j.bbapap.2018.05.014. Epub 2018 May 29.

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

Joshua N Smith  1 , Joshua M Edgar  2 , J Mark Balk  2 , Mariam Iftikhar  2 , Jessica C Fong  3 , Tivoli J Olsen  2 , Dmitry A Fishman  2 , Sudipta Majumdar  2 , Gregory A Weiss  4
Affiliations

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

Joshua N Smith et al. Biochim Biophys Acta Proteins Proteom. 2018 Sep.

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 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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Figures

Fig. 1
Fig. 1
Selections for soluble and functional cav variants. (A) Schematic diagram detailing the selection and screening process. (i.) Negative selections removed hydrophobic, aggregation-prone variants. In the same round, (ii.) a positive selection tested binding to gp41. (iii.) After four rounds of selections, the DNA from remaining variants was transformed into E. coli and (iv.) screened for protein overexpression. This schematic diagram is not drawn to scale. (B) Conditions used for increasing stringency in each round of selection. Higher ionic strength buffer and additional washes increased the stringency of selection conditions for each round. To discourage nonspecific and off-target binding, the blocking agent was varied during rounds of selections.
Fig. 2
Fig. 2
Protein-based ELISAs investigating specific binding of cavsol with its binding partners. Dose-dependent binding between cavsol and (A) PTRF (red), (B) gp41 (green), or (C) PKA (purple) is observed. Cavsol binds with moderate affinity and high specificity. (D) Notably, cavsol does not bind nonspecifically to off-target proteins. All error bars report standard deviation from the mean (n=3).
Fig. 3
Fig. 3
Biophysical characterization of cavsol. (A) CD spectrum of cavsol. The CD spectrum of cavsol reveals a highly helical secondary structure with some disordered regions and β-strands. The spectrum was obtained from averaging six continuous scans (B) Particle size of cavsol at varying concentrations. Analysis by DLS shows a large particle size which increases proportionally with concentration. This suggests that cavsol exists in an oligomeric form. Error bars denote standard deviation (n=3). (C). SEC-MALS analysis of the cavsol protein at three different concentrations. The SEC-MALS/RI/RALS were used to determine the cavsol molecular masses using Malvern OmniSEC software. Chromatograms show the readings from the light scattering at 90° for the three different protein concentrations (120, 361, and 724 μM). The left and right axes represent the light scattering detector reading and molecular mass, respectively. The lines across the peaks indicate molar mass and homogeneity. Calculated molar masses are indicated.
Fig. 4
Fig. 4
Measuring binding between cavsol and either (A) gp41 or (B) PKA by ITC. The upper panel depicts the calorimetric output from the cavsol interaction with either gp41 (45 total injections of gp41) or PKA (38 total injections of PKA). The lower panel depicts integration of the calorimetric output, where the x-axis indicates the molar ratio of gp41 or PKA to cavsol. The least squares fit is shown by the solid line. (C) ITC-derived thermodynamic binding parameters for interactions with cavsol. Error indicates standard error.
Fig. 5
Fig. 5
Localization of WT cav or cavsol fused to mCherry in HEK 293 cells. HEK 293 cells transfected with either (A) WT cav-mCherry, (B) cavsol-mCherry, or (C) non-transfected cells were observed using confocal microscopy. Cells were tested for mCherry fluorescence to identify the mCherry-fused cav, cellmask green fluorescence to identify the cell membrane, DAPI to identify single-stranded DNA (cell nucleus), or with the darkfield setting to identify the cell outline. White arrows represent either WT cav or cavsol fluorescence. The contrast of select samples was adjusted to improve the image quality of select panels. The contrast of the WT Cav and Cavsol-mCherry samples was increased by approximately 40%. The contrast of the WT Cav and Cavsol CellMask Green samples was increased by approximately 20%.
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