Side chain torsion dictates planarity and ionizability of Green fluorescent protein's chromophore leading to spectral perturbations.

Spectral characteristics of Fluorescent Proteins (FP) are well studied and through protein engineering, several FP variants constituting entire visible spectrum have been created. One of the most common mechanisms attributed to spectral shifts in FP are excited state proton transfer (ESPT), hydroxyl moiety protonation and deprotonation, along with chromophore Cis-Trans isomerism. Most widely studied FPs are those derived from avGFP (Aequorea victoria GFP) and Dsred (Discosoma coral). Apart from the above mechanism, certain interacting residues are said to play a vital role in altering the proton transfer pathway leading to numerous spectral variants. Similarly, the hydrogen-bonded networks solely cannot dictate the energy landscape of FPs. Non-bonded interactions also can create secondary harmonic shifts by dipole-dipole inductions. Side chain contacts tend to alter the topological and torsional geometry, thereby disturbing the chromophore's planarity. Side chain torsional variations have almost been unaccounted for their distortions in FP's. We hypothesize the torsional landscape and altered residual interactions are prominent factors for the spectral shifts. Through our 200 ns molecular dynamics investigation we prospect that van der Waals packing in Dsred is more compact than that of avGFP, thus creating a low solvent occupiable environment and reduced solvent interactions having higher red spectral shift. The torsional changes of wild avGFP, S65T avGFP, and Dsred has been studied to comprehend the inter-residual contact distance and the geometrical descriptors.