The relevance of amino acid-chromophore interactions in protein conformational changes and their impact on chromophore relaxation dynamics

The lack of control in the protein-chromophore interaction network in designed bio-hybrids is one of the key challenges in the field. This issue makes the photoresponse of the generated bio-hybrids unpredictable, limiting their potential. In this study, we address this challenge by developing a biohybrid system using a small, de novo α3 protein composed of three short α-helices connected by flexible loops. This design creates a simplified protein pocket in which folding is defined by a few amino acids, while the rest can be modified, enabling precise predictions through rational design. Based on this control, we showed a strategy to label the chromophore specifically achieving a well-folded Biohybridand to change the dielectric environment of the chromophore in a selective manner by locating aromatic amino acids at key positions in the pocket based on the structure of the α3. In particular, we incorporated a pyrene chromophore at position 34, centrally located within the pocket in helix 2, and introduced aromatic amino acids—phenylalanine, tyrosine, and tryptophan—at position 18 in helix 1, at 0.3 nm distance from the chromophore. Our findings revealed that these amino acids not only affect the relaxation dynamics of the chromophore but also protein conformation in a correlated manner. Specifically, tryptophan-induced an open protein conformation, while phenylalanine and tyrosine promoted a closed conformation, as evidenced by changes in pyrene emission, which acted as a conformation reporter. Using an iterative approach that combines circular dichroism and protein engineering, we demonstrated that the conformational changes arise from the low folding energy of the α3 protein (~4.3 kcal/mol). This low energy allows the protein's structure to be modulated by a single interaction serving as the sole energy input. Furthermore, transient absorption spectroscopy showed that open conformations exhibited faster triplet-state decay than closed conformations. This study highlights the critical yet often overlooked role of protein-chromophore interactions in modulating Biohybrid photoresponses. It also establishes the α3 protein as an ideal protein model system for studying these interactions with high potential for rational biohybrid design.