Self-assembly of the cephalopod protein reflectin
Cephalopods (squids, octopus, cuttlefish) have captivated scientists due to their stunning camouflage displays. Recently, these animals have emerged as exciting sources of inspiration for the design of functional materials. Within this context, our group has investigated cephalopod structural proteins known as reflectins. We have studied the self-assembly, structural characteristics, and stimulus response of films from reflectin A1 (RfA1) with grazing-incidence small-angle X-ray scattering (GISAXS). RfA1 self-assembles into prolate nanoparticles in solution, which change shape both during film formation and upon application of an exogenous stimulus. Based on our measurements, we have elucidated the nanostructure of RfA1 films and obtained a rationale for their functionality.
Due to the unique capabilities and characteristics of cephalopods, they have emerged as an exciting source of inspiration for the development of novel materials and technologies. Accordingly, our group has investigated cephalopod structural proteins known as reflectins, which possess an amino acid sequence consisting of characteristic conserved motifs separated by variable linker regions and containing a high percentage of charged and aromatic residues. We have discovered that films from the Doryteuthis pealeii RfA1 isoform function as stimuli-responsive reconfigurable infrared camouflage coatings, feature electrical properties that rival those of well-known artificial proton conductors, and support the proliferation and differentiation of neural stem cells. These indicate that both RfA1 and the extended reflectin protein family hold promise as advanced functional materials. Despite the technological potential of RfA1, a clear relationship between this protein’s high-order structure/ organization in films and in vitro functionality has remained elusive to date. Consequently, there exists an opportunity for further detailed structural characterization of RfA1-based ensembles, with the goal of better understanding their multifaceted functionality and exciting in vitro properties.
Figure 1. a) A schematic of a GISAXS experiment for a RfA1 film. The incident X-rays are scattered by substrate-confined aggregated macromolecules, yielding a 2D pattern (data for RfA1 is shown). b) A 1D plot of the GISAXS intensity I(q) versus the scattering vector qy obtained at different times during the self-assembly/formation of a film from RfA1 nanoparticles. The measurements were obtained at 3 (blue), 7 (green), 11 (purple), and 23 (brown) min after initiation of assembly. The circles represent the experimental data, and the lines represent the simulated scattering intensity profiles. c) A 1D plot of the GISAXS intensity I(q) versus the scattering vector qy obtained for a RfA1 film before and after hydration. The orange circles represent the experimental data obtained before hydration, and the solid orange line represents the simulated scattering intensity profile. The red circles represent the experimental data obtained after hydration, and the solid red line represents the simulated scattering intensity profile.
We studied the formation, nanostructure, and stimulus response of substrate-confined RfA1 using in situ grazing incidence small-angle x-ray scattering (GISAXS), for which the experimental setup is illustrated in Figure 1A. The integrated scattering intensity I(q) versus the horizontal scattering vector qy obtained at various times during the self-assembly of a representative RfA1 film is in turn shown in Figure 1B. To understand the emergence of structure during film formation, we monitored changes in the effective RfA1 nanoparticle geometry by extracting the radius of gyration RG and scattering intensity exponent P- which is related to the particle shape-from the scattering profiles collected at different time points after initiation of film assembly. At t =3 min, corresponding to an experiment on a nearly aqueous system, we calculated an RG of 830 Å, which was approximately twice the radius obtained in solution, which may indicate dimerization of RfA1 nanoparticles, and an exponent P of 1.5, indicative of an elongated spheroidal object. At the final time t =23 min (corresponding to complete loss of solvent), we found a nanoparticle radius RG of 910 Å and an exponent P of 2.1, similar to the value expected for an oblate or plate-like ellipsoid (P =2). The final geometry suggested that the nanoparticles had become flattened upon moving from solution to the interior of the film. Such substantial geometric changes probably resulted from a combination of factors, including the absence of solvation, presence of both lateral and vertical confinement, enhanced interparticle interactions due to crowding, and proximity-induced merger between neighbors. Altogether, our observations and analysis provided mechanistic insight into the formation and nanostructure of RfA1 films, enabling us to postulate a model for nanoparticle self-assembly, which is illustrated in Figure 2.
Figure 2. The hierarchical organization of RfA1 in solution and in films. When dispersed in solution, RfA1 aggregates into interacting nanoparticles with an asymmetric prolate (elongated) geometry. When self-assembled into films, the RfA1 nanoparticles still interact and adopt an oblate (flattened) geometry, which changes slightly upon the application of a stimulus (an increase in relative humidity leading to hydration).
We used in situ GISAXS to evaluate the effect of a water-uptake on the nanostructure of our RfA1 films (water uptake is a prerequisite for protonic conductivity in RfA1 films and has been shown to change the films’ thickness and reflectance). Figure 1C illustrates the experimental and simulated scattering intensity profiles obtained for a typical RfA1 film before and after hydration. Before hydration (relative humidity of < 50%), we extracted a nanoparticle radius RG of 920 Å and an exponent P of 2.0 (plate-like particle) from the simulated scattering profiles. This measurement indicated the RfA1 films were comprised of large oblate ellipsoids, in agreement with our observations during film formation. After hydration (relative humidity of ~90 %), we extracted a nanoparticle radius RG of 890 Å and an exponent P of 1.8 (elongated particle) from the simulated scattering profiles. This measurement indicated that the ellipticity of our nanoparticles had decreased, in agreement with expectations for their transition from a dry to a solvated environment. Altogether, our measurements revealed the nanostructure of RfA1 films in their hydrated state, as illustrated in Figure 2.
This research was conducted by the following research team:
Kyle L. Naughton1,Long Phan2, Erica M. Leung2, Rylan Kautz2, Qiyin Lin3, Yegor Van Dyke2,Benedetta Marmiroli4,Barbara Sartori4,Andy Arvai5, Sheng Li6, Michael E. Pique5, Mahan Naeim2, Justin P. Kerr2, Mercedeez J. Aquino2, Victoria A. Roberts7, Elizabeth D. Getzoff5,8, Chenhui Zhu9, Sigrid Bernstorff10 and Alon A. Gorodetsky2,11
1 Department of Physics and Astronomy, University of California, Irvine, Irvine, USA
2 Department of Chemical Engineering and Materials Science, University of California, Irvine, Irvine, USA
3 Laboratory for Electron and x-ray Instrumentation, University of California, Irvine, Irvine, USA
4 Institute of Inorganic Chemistry, Graz University of Technology, Graz, Austria
5 Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, USA
6 Department of Medicine, University of California, San Diego, La Jolla, USA
7 San Diego Supercomputer Center, University of California, San Diego, La Jolla, USA
8 The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, USA
9 Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, United States
10 Elettra - Sincrotrone Trieste S.C.p.A., Trieste, Italy
11 Department of Chemistry, University of California, Irvine, Irvine, USA
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Reference
Kyle L. Naughton, Long Phan, Erica M. Leung, Rylan Kautz, Qiyin Lin, Yegor Van Dyke, Benedetta Marmiroli, Barbara Sartori, Andy Arvai, Sheng Li, Michael E. Pique, Mahan Naeim, Justin P. Kerr, Mercedeez J. Aquino, Victoria A. Roberts, Elizabeth D. Getzoff, Chenhui Zhu, Sigrid Bernstorff, and Alon A. Gorodetsky, “Self-Assembly of the Cephalopod Protein Reflectin”. Adv. Mater. 28, 8405 (2016), DOI: 10.1002/adma.201601666This paper was selected for "Inside Back Cover" of Advanced Materials. |
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