Unravelling the molecular structure, self-assembly, and properties of a cephalopod protein variant

Cephalopods, such as the loliginid in Figure 1A, are known for their remarkable ability to rapidly change the color and appearance of their skin. These capabilities are enabled in part by unique structural proteins called reflectins, which play essential roles in optical behavior of cephalopod skin cells. Moreover, reflectins have demonstrated exciting potential as functional materials within the context of biophotonic and bioelectronic systems. Given reflectins’ demonstrated significance from both fundamental biology and applications perspectives, some research effort has been devoted to resolving their three-dimensional (3D) structures. However, the peculiar sequence composition of reflectins has made them extremely sensitive to subtle changes in environmental conditions and prone to aggregation, thus significantly complicating the study of their structure-function relationships and precluding their definitive molecular-level structural characterization. In this work, we have elucidated the structure of a reflectin variant at the molecular level, demonstrated a robust methodology for controlling its assembly and optical properties.

We began our studies by rationally selecting a prototypical reflectin variant (RfA1TV) by using a bioinformatics-guided approach (Figure 1B). Next, we not only produced the variant in high yield and purity but also optimized conditions for maintaining this protein in a monomeric state (Figure 1C). We then probed the protein with small angle X-ray scattering (SAXS) using the Austrian SAXS beamline at the Elettra Synchrotron Laboratory in Trieste, Italy. For this purpose, a well-dispersed solution of RfA1TV was prepared in a low-pH buffer and transferred into a glass capillary, which was positioned in the path of an incident X-ray beam. The X-rays scattered by the solution-borne RfA1TV molecules formed a 2-D pattern on a Pilatus3 1M detector (Figure 1D). Subsequently, radial averaging and image calibration of the two-dimensional data furnished corresponding one-dimensional curves, which were further processed, analyzed, and correlated with other experiments to obtain insight into the protein’s geometry (Figure 1E).

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Image: (A) A camera image of a Doryteuthis pealeii squid. (B) An illustration of the selection of the prototypical truncated reflectin variant (RfA1TV) from full-length Doryteuthis pealeii reflectin A1. (C) A digital camera image of a solution of primarily monomeric RfA1TV (Upper) and a corresponding cartoon of RfA1TV monomers (Lower Inset). (D) An illustration of the SAXS analysis of the reflectin variant, wherein incident X-rays are scattered by the solution-borne proteins to furnish a corresponding scattering pattern. (E)The 3D structure of RfA1TV (random coils – gray, helices – orange, β-strands – purple). 

Credit: This figure has been adapted from M. J. Umerani*, P. Pratakshya* et al.Proc. Natl. Acad. Sci. U.S.A 117, 32891-32901 (2020).