Cyclable Condensation and Hierarchical Assembly of Metastable Reflectin Proteins, the Drivers of Tunable Biophotonics

Abstract

Reversible changes in the phosphorylation of reflectin proteins have been shown to drive the tunability of color and brightness of light reflected from specialized cells in the skin of squids and related cephalopods. We show here, using dynamic light scattering, electron microscopy, and fluorescence analyses, that reversible titration of the excess positive charges of the reflectins, comparable with that produced by phosphorylation, is sufficient to drive the reversible condensation and hierarchical assembly of these proteins. The results suggest a two-stage process in which charge neutralization first triggers condensation, resulting in the emergence of previously cryptic structures that subsequently mediate reversible, hierarchical assembly. The extent to which cyclability is seen in the in vitro formation and disassembly of complexes estimated to contain several thousand reflectin molecules suggests that intrinsic sequence- and structure-determined specificity governs the reversible condensation and assembly of the reflectins and that these processes are therefore sufficient to produce the reversible changes in refractive index, thickness, and spacing of the reflectin-containing subcellular Bragg lamellae to change the brightness and color of reflected light. This molecular mechanism points to the metastability of reflectins as the centrally important design principle governing biophotonic tunability in this system.

Keywords: biomaterials; biophotonics; intrinsically disordered protein; iridescence; protein aggregation; protein assembly; protein metastability; protein self-assembly; reflectins; tunable.

FIGURE 1.

A, schematic of the four D. opalescens reflectin proteins, showing the number and locations of the two types of canonical reflectin motifs, the regular reflectin motifs (Ref), and the N-terminal reflectin motifs (N-Ref). Previously identified reflectin phosphorylation sites are labeled with stars (7, 8). The GMXX motif in reflectin C is a unique region of increased overall hydrophobicity composed of a four-amino acid repeat, where X represents less conserved locations within the repeat (7). Additionally, the reflectin motif of reflectin C is marked with an asterisk to indicate that it contains substantial deviations in sequence not observed in any other reflectin motifs. Sites of phosphorylation in reflectin C have not yet been identified. B, sequence logos of the N-terminal and regular reflectin motifs. The logograms were formed from alignment of motifs from D. opalescens and the nearly identical D. pealeii reflectins (irregular motif in reflectin C excluded) (31).

FIGURE 2.

A, SDS-PAGE of purified reflectin proteins stained with Coomassie Brilliant Blue. B, turbidity (optical density at λ = 350 nm) of the indicated reflectins as a function of pH. Each measurement was taken 5 min after dilution of water-dissolved reflectin into buffer at the indicated pH. C, insoluble reflectin (samples from A pelleted after centrifugation for 10 min), as determined by absorbance at 280 nm. Insets, results for the BSA control. Shown are typical results from duplicate experiments.

FIGURE 3.

Dynamic light scattering of reflectins as a function of pH. A, intensity distributions of 9 μm reflectin A1 diluted in 5 mm MOPS buffer at the indicated pH values. Representative data from multiple analyses are shown. B, DLS volume distributions of same samples as shown in A. C, time dependence of DLS volumetrically dominant distributions from time of dilution of reflectin A1 into buffer at the indicated pH. Each individual data point is the average of the last 2–3 min of repetitive scattering measurements. Black arrows indicate measurements immediately before acidification with 15 mm acetic acid and subsequent equilibration (black dotted line) prior to the next DLS measurement. D, R_H of individual reflectins or indicated mixtures measured ∼20 min after dilution into buffer at the indicated pH. All measurements were replicated at least three times with samples from at least two separate protein purifications and buffer preparations. Error bars show mean ± S.D. The individual time courses of particle size for all samples resembled those shown in C.

FIGURE 4.

TEMs of reflectin A1 assemblies. A and B, reflectin A1 assembled at pH 6.5, imaged at low and high magnification. DLS of the sample prior to grid application showed R_H = 10 nm (diameter = 20 nm). C and D, reflectin A1 assembled at pH 7.5, imaged at low and high magnification. DLS of the sample prior to grid application showed R_H = 17 nm (diameter = 34 nm).

FIGURE 5.

Reflectin assembly, disassembly, and cyclability. A, dynamic light scattering intensity distribution data of assembly and disassembly of 9 μm reflectin A1 first diluted in 5 mm MOPS buffer at pH 7.0 and then immediately after addition of acid to reach 15 mm acetic acid (pH 4.5). The experiments were repeated multiple times. The results shown are representative of A1 at all assembled pH values. pH values are as indicated. B, volume distribution of the data in A. C, cyclability of assembly and disassembly of reflectin A1 and a mixture of the four reflectins in a ratio corresponding to that of the tunable dorsal iridocytes. Blue arrows indicate points of measurement of reflectin R_H values followed by addition of a molar excess of acetic acid (pH 4.5). Red arrows indicate measurements of reflectin R_H values following partial disassembly. The transition from pH 4.5 to 7.5 was effected by dialysis (see details under “Experimental Procedures”). The R_H shown is for the volumetrically predominant species as determined by DLS. Error bars indicate mean ± S.D. of volumetric data within the experiment shown. The results are representative of closely agreeing duplicate analyses.

FIGURE 6.

Fluorescence spectra of tryptophan residues in reflectin A1 in the unassembled water-solubilized state, incubated at the indicated pH values, and in the acetic acid-reversed state. Excitation λ = 295 nm. A.U., arbitrary units.

FIGURE 7.

A, fluorescence of ANS bound to reflectin A1 (excitation λ = 350 nm). ANS was bound to the reflectin in the unassembled water-solubilized state and bound independently to reflectin at pH 6.5, and that sample was then reacidified to pH 4.5 as described under “Experimental Procedures.” B, fluorescence resonance energy transfer from tryptophan to ANS (excitation at λ = 295 nm). The samples were those described in A. A.U., arbitrary units.

FIGURE 8.

A–D, calculated reflectin net charge versus pH, with assumed histidine pK_as of 5.5 (A and B) and 6.5 (C and D). All titratable amino acid pK_as other than histidine are from Ref. . The data were calculated by spreadsheet with 0.01-pH unit increments.

FIGURE 9.

Sequences of the D. opalescens reflectins with selected, experimentally identified important residues colored. Histidine residues are colored green, and tryptophan residues are colored purple. N-terminal reflectin motifs and regular reflectin motifs are shown highlighted in bordered and unbordered gray, respectively. In reflectin C, the GMXX repeat is highlighted in brown (7). Previously identified physiological phosphorylation sites are shown with a yellow background (7, 8).

FIGURE 10.

A, calculation of net hydrophobic moments within reflectin A1 for two sets of residue angles corresponding to the maximal values observed for α helices (100°) and β sheets (160°) (28). Net moments are shown for 18-residue windows centered at those residues. Calculations were performed using EMBOSS (32). B, helical projections for selected regions of reflectin A1. Yellow, hydrophobic residues; blue, positively charged; red, negatively charged. A vertical line designates the N-terminal residue of the modeled helix. Helical projection images were adapted from the HELIQUEST web server (33).