Neutralization of a Distributed Coulombic Switch Precisely Tunes Reflectin Assembly

Abstract

Reflectin proteins are widely distributed in reflective structures in cephalopods, but only in Loliginid squids are they and the sub-wavelength photonic structures they control dynamically tunable, driving changes in skin color for camouflage and communication. The reflectins are block copolymers with repeated canonical domains interspersed with cationic linkers. Neurotransmitter-activated signal transduction culminates in catalytic phosphorylation of the tunable reflectins' cationic linkers, with the resulting charge-neutralization overcoming Coulombic repulsion to progressively allow condensation and concommitant assembly to form multimeric spheres of tunable size. Structural transitions of reflectins A1 and A2 were analyzed by dynamic light scattering, transmission electron microscopy, solution small angle x-ray scattering, circular dichroism, atomic force microscopy, and fluorimetry. We analyzed the assembly behavior of phospho-mimetic, deletion, and other mutants in conjunction with pH-titration as an in vitro surrogate of phosphorylation to discover a predictive relationship between the extent of neutralization of the protein's net charge density and the size of resulting multimeric protein assemblies of narrow polydispersity. Comparison of mutants shows this sensitivity to neutralization resides in the linkers and is spatially distributed along the protein.These results are consistent with the behavior of a charge-stabilized colloidal system, while imaging of large particles, and analysis of sequence composition, suggest that assembly may proceed through a transient liquid-liquid phase separated intermediate. These results offer insights into the basis of reflectinbased tunable biophotonics and open new paths for the design of new reflectin mutants with tunable properties.

Protein Charge Neutralization is the Proximate Driver Dynamically Tuning a Nanoscale Bragg Reflector

“…These results reveal a quantitatively predictive relationship between net charge density and reflectin assemb size over the range predicted for the physiological phosphorylation. They also confirm the quantitative relationship between charg and size revealed in our analyses of phosphomimetic, deletion, and other mutants [8,11] .…”

“…As shown here by dynamic light scattering (DLS) (Figure 1A), and previously demonstrated [8,11] , pH-titration of reflectin's exce positive charges serves as an in vitro surrogate for the neutralization by phosphorylation in vivo, driving progressive assembly of th purified protein with increasing pH. For the experiments shown here, purified D. opalescens reflectin A1 was first extensive equilibrated by dialysis into 25 mM sodium acetate buffer at pH 4.5; reflectin remains stably monomeric under these acidic condition Assemblies formed by pH neutralization are essentially stable with time.…”

“…Comparison of results from three buffer system demonstrates relatively little buffer-specific effects. Using the known pK a 's of the constituent amino acid residues allows us calculate the net charge density (net positive charge/100 residues) of the protein for each measured value of pH [8,11] , permitti comparison of the observed effects of neutralization by titration with those that might be expected as a result of the in vi phosphorylation with 1, 2, or 3 phosphates per reflectin (as quantitatively measured by 2D electrophoresis and mapped by L MS [2,12] ) (Figure 1B). These results reveal a quantitatively predictive relationship between net charge density and reflectin assemb size over the range predicted for the physiological phosphorylation.…”

“…Multiple mechanisms have been implicated [7][8][9][10][11] , but uncertainty remains about the proximate trigger that first drives assembly. Xie and colleagues showed that small aromatic molecules including imidazole can drive assembly [7] , and suggested a similar process might initiate assembly in the physiological control of photonic behavior [7] .…”

“…In contrast, Izumi et al and DeMartini et al observed that neuronal triggering of the reflectin-mediated photonic changes requires ACh-induced site-specific phosphorylation of the reflectins, and proposed that this phosphorylation overcomes Coulombic repulsion of reflectin's observed excess positive charges to permit condensation and assembly [2,4] . Levenson et al then showed that progressive reduction of reflectin's positive charge -either by pH titration or by genetic engineering -drives proportional assembly of the initially disordered protein [8,11] . However, because titration reduced the charge of the imidazolium ions of histidine residues, and the genetic engineering secondarily affected the net balance of aromatic residues, some questions still remained.…”