Title: Electrochemically Driven Structural Dynamics and Spectroscopy of Biomolecular Systems

 

Abstract: The diverse structure and function of biological systems often depend on the dynamic conformations and assemblies of biomolecules. In particular, electrostatic interactions arising from ionizable amino acid residue side chains play a central role in regulating biomolecular structure in vivo through changes in pH or post-translational modifications. Developing methods that can actively trigger and control assembly is therefore essential to understand protein function and to gain insight into the biophysical mechanisms that govern folding and assembly pathways.

 

This work presents an electrochemical approach for precisely modulating electrostatic interactions in biomolecules by neutralizing protonated amino acid residues, thereby enabling the study of protein structural evolution driven by changes in Coulombic interactions and intermolecular assembly. Electrochemistry was combined with in situ spectroscopies (e.g., dynamic light scattering, circular dichroism, ellipsometry, and surface plasmon resonance) to simultaneously trigger, control, and observe protein assembly. This electrochemically coupled spectroscopic approach was first demonstrated on freely diffusing reflectin proteins, the biomolecules responsible for dynamic color change in squid, and revealed that the electrochemical regulation closely mirrors the physiological transitions of reflectin, enabling precise, real-time analysis of conformational and assembly dynamics. These methods were then applied to reflectin thin films, showing that applied voltage modulates both the refractive index and thickness of the films, mimicking osmotic dehydration and shedding light on the color-changing mechanisms in squid skin. Building on this, engineered RM1 (the most conserved motif in reflectin) peptides were investigated using electrochemical correlative spectroscopy, demonstrating that the essential features of reflectin tunability can be recapitulated by a minimal motif-based construct. At the same time, these results highlight the importance of native sequence architecture in promoting efficient and homogeneous assembly. More broadly, this work shows that reflectin behavior is governed by a delicate balance between electrostatic interactions and hierarchical assembly, providing a strong foundation for the future design and control of reflectin-inspired responsive biomaterials.

The approaches developed in this work enable electrical control and real-time observation of biomolecular systems, providing tools to study macromolecular dynamics, uncover biophysical mechanisms, design functional materials, and bridge the biotic–abiotic interface.