Serpin-Driven Green Camouflage and NIR Fluorescence in Frogs

Animals have evolved multiple strategies to generate optical traits and coloration. While most amphibians rely on a three-dimensional arrangement of chromatophores in the skin, hundreds of arboreal frog species achieve leaf-like green color through a different mechanism involving reduced pigmentation, subcutaneous biological mirrors, and high concentrations of the blood-derived pigment biliverdin. Although biliverdin is rapidly excreted in most vertebrates, hylid and centrolenid frogs can retain it through the binding to a serpin-family protein (BBS). Here we show that BBSs bind biliverdin with high affinity (K_d < 10 nM), comparable to hormone-receptor interactions. This interaction alters biliverdin's spectral signature in ways that resemble those of green-leaf pigments. BBSs from different species exhibit distinct biophysical properties, accounting for interspecific differences in color saturation and hue. Unlike most serpins <i>in vivo</i>, BBS of the glassfrog <i>Teratohyla pulverata</i> is naturally cleaved, yielding a highly thermostable, thermodynamically favored protein, without loss of affinity. Using custom-designed hyperspectral photoacoustic tomography (PAT), we demonstrate that BBS is widely distributed throughout the body, contributing to whole-body color and camouflage. Furthermore, we show that BBSs emit near-infrared (NIR) fluorescence (>700 nm) rendering these frogs fluorescent in a spectral region where biological tissues are largely transparent. Together, BBSs shed light on serpin evolution, protein thermostability, amphibian color diversity, and the development of NIR molecular probes.