No abstract

A photoresponsive surfactant has been used as a means to control protein structure and dynamics with light illumination. This cationic azobenzene surfactant, azoTAB, which undergoes a reversible photoisomerization upon exposure to the appropriate wavelength of light, adopts a relatively hydrophobic, trans structure under visible light illumination and a relatively hydrophilic cis structure under UV light illumination. Small-angle neutron scattering (SANS) and neutron spin echo (NSE) spectroscopy were used to measure the tertiary structure and internal dynamics of lysozyme in the presence of the photosurfactant, respectively. The SANS-based in vitro structures indicate that under visible light the photosurfactant induces partial unfolding that principally occurs away from the active site near the hinge region connecting the α and β domains. Upon UV exposure, however, the protein refolds to a nativelike structure. At the same time, enhanced internal dynamics of lysozyme were detected with the surfactant in the trans form through NSE measurements of the Q-dependent effective diffusion coefficient (D(eff)) of the protein. In contrast, the D(eff) values of lysozyme in the presence of cis azoTAB largely agree with the rigid-body calculation as well as those measured for pure lysozyme, suggesting that the native protein is dormant on the nanosecond time and nanometer length scales. Lysozyme internal motions were modeled by assuming a protein of two (α and β domains) or three (α and β domains and the hinge region) domains connects by either soft linkers or rigid, freely rotating bonds. Protein dynamics were also tracked with Fourier transform infrared spectroscopy through hydrogen-deuterium exchange kinetics, which further demonstrated enhanced protein flexibility induced by the trans form of the surfactant relative to the native protein. Ensemble-averaged intramolecular fluorescent resonance energy transfer measurements similarly demonstrated the enhanced dynamics of lysozyme with the trans form of the photosurfactant. Previous results have shown a significant increase in protein activity in the presence of azoTAB in the trans conformation. Combined, these results provide insight into a unique light-based method of controlling protein structure, dynamics, and function and strongly support the relevance of large domain motions for the activity of proteins.

The effects of a photoresponsive surfactant and light illumination on the complex formed between ribonuclease A (RNase A) and a protein ribonuclease inhibitor (RI) have been investigated to develop a light‐based technique to reactivate an enzyme through surfactant‐induced dissociation of the enzyme‐inhibitor complex. The photoresponsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to reversibly control protein‐inhibitor interactions. In the absence of surfactant, RI binds tightly to RNase A through noncovalent interactions, which inhibits the enzyme activity. Upon addition of the surfactant under visible light, RNase A is reactivated, regaining ~75% of the native activity in the absence of RI. In the presence of the surfactant under UV light, however, the enzyme remains inhibited. Fluorescence spectroscopy, dynamic light scattering, and circular dichroism spectroscopy reveal that RI dramatically unfolds upon addition of the trans form of the surfactant, while RNase A does not undergo noticeable structural changes under the same conditions. This indicates that RNase A reactivation occurs through dissociation of the enzyme‐inhibitor complex arising from surfactant‐induced unfolding of the inhibitor. As a result, photoresponsive surfactant and light illumination can be used as a novel light‐based technique to dissociate enzyme‐inhibitor complexes and, thus, reactivate an inhibited enzyme.