Unraveling 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).

Figure 1. (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). This figure has been adapted from M. J. Umerani*, P. Pratakshya* et al.Proc. Natl. Acad. Sci. U.S.A 117, 32891-32901 (2020).
 

A detailed analysis of the SAXS data obtained for monomeric RfA1TV solutions revealed important information about the protein’s size, global shape and folding state. Figure 2Ashows the background-subtracted experimental scattering intensity I(q) as a function of the scattering vector q, along with the corresponding fit obtained via a unified approach. The unified fit’s low-q (< 0.07 Å⁻¹) Guinier region revealed a radius of gyration (R_g) of 16.6 ± 0.3 Å, which correlated with an experimental R_g/R_h (where R_h is the hydrodynamic radius) ratio of 1.2 that indicated deviation from a globular shape. The fit’s high-q (> 0.07 Å⁻¹) Porod region revealed a Porod exponent of ∼3, which was likewise indicative of a complex shape for the protein. Figure 2B shows the Kratky plot of I(q) × q² as a function of q, which featured a peak at q ≈ 0.12 Å⁻¹followed by a decay and a plateau at intermediate to high q values, suggesting that RfA1TV was at least partially folded. Figure 2C shows the pair-wise distance distribution function P(r), which exhibited a single main peak at a distance of r ≈ 21 Å and tailed to a maximum dimension of D_max≈ 68 Å. The shape of the plot corroborated the notion of a non-spherical, partially folded protein. Further investigation of RfA1TV’s secondary and tertiary structures was carried out using a combination of computational and experimental tools, such as molecular dynamics simulations, dynamic light scattering, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy. Together with the SAXS measurements, these techniques enabled us to resolve the 3D structure of our reflectin variant (Figure 1E). 
This work advances current understanding of reflectins on multiple fronts and addresses several challenges that have hindered reflectins’ technological development to date. Notably, our results provide a better understanding of reflectins' structure-function relationships, thereby underscoring their potential as functional biomaterials. Furthermore, our findings also provide new insights into reflectins’ role in cephalopods’ camouflage abilities. Overall, this work may inform new research directions across the fields of biochemistry, cellular biology, bioengineering, and optics. 
 

Figure 2. (A) A representative plot of the scattering intensity I(q) as a function of the scattering vector q obtained for RfA1TV (black squares), and the corresponding unified fit of the experimental data (orange trace). (B) A Kratky plot of the scattering intensity times the square of the scattering vector I(q) × q2as a function of the scattering vector q for RfA1TV.(C)A representative plot of the pair-wise distance distribution function P(r) as a function of the distance r for RfA1TV. This figure has been adapted from M. J. Umerani*, P. Pratakshya* et al.Proc. Natl. Acad. Sci. U.S.A 117, 32891-32901 (2020).

This research was conducted by the following research team:

Mehran J. Umerani¹, Preeta Pratakshya², Atrouli Chatterjee³, Juana A. Cerna Sanchez⁴, Ho Shin Kim⁵, Gregor Ilc⁶, Matic Kovačič⁶, Christophe Magnan⁷, Benedetta Marmiroli⁸, Barbara Sartori⁸, Albert L. Kwansa⁵, Helen Orins³, Andrew W. Bartlett³, Erica M. Leung³, Zhijing Feng¹, Kyle L. Naughton⁹, Brenna Norton-Baker², Long Phan¹, James Long³, Alex Allevato¹, Jessica E. Leal-Cruz¹, Qiyin Lin¹⁰, Pierre Baldi⁷, Sigrid Bernstorff ¹¹, Janez Plavec⁶, Yaroslava G. Yingling⁵and Alon A. Gorodetsky^1,2,3

¹ Department of Materials Science and Engineering, University of California, Irvine, Irvine, U.S.A.
² Department of Chemistry, University of California, Irvine, Irvine, U.S.A.
³ Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, U.S.A.
⁴ Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, U.S.A.
⁵ Department of Materials Science and Engineering, North Carolina State University, Raleigh, U.S.A.
⁶ Slovenian NMR Centre, National Institute of Chemistry, Ljubljuana, Slovenia
⁷ Department of Computer Science, University of California, Irvine, Irvine, U.S.A.
⁸ Institute of Inorganic Chemistry, Graz University of Technology, Graz, Austria
⁹ Department of Physics and Astronomy, University of California, Irvine, Irvine, U.S.A. 
¹⁰ Irvine Materials Research Institute, University of California, Irvine, U.S.A. 
¹¹ Elettra - Sincrotrone Trieste S.C.p.A., Trieste, Italy

Contact persons:

Alon Gorodetsky, email: