Final Activity Report Summary - SIMULATING ECFP (Dissecting the fluorescence properties of the Enhanced Cyan Fluorescent Protein by computational analysis of its structure and dynamics) Due to its intrinsic visible fluorescence, the Green Fluorescent Protein (GFP) is used to monitor cellular and biomolecular functions. Its chromophore is formed by cyclisation of three aminoacid residues: Ser65, Tyr66 and Gly67. The substitution of Tyr66 by Trp and few additional mutations lead to the Enhanced Cyan Fluorescent Protein (ECFP), characterised by cyan emission. ECFP is one of the most used donors in FRET imaging which is a technique to monitor molecular interaction, gene expression and other important cellular processes. These important applications are hampered by the incomplete understanding of the complex photophysics of ECFP. In particular, a molecular interpretation of the fluorescence signal is essential for understanding the effect of conformational changes and external factors such as pH, temperature and molecular interactions.In this project, we propose a combination of different methods of theoretical chemistry (electrostatics, molecular dynamics, and quantum chemistry) to understand and explain important physical properties (photophysics, pH and temperature dependence of the dynamics) of ECFP. Understanding the structural basis of the ECFP fluorescence properties will help to design more effective tools for cellular biology and medical diagnostics.We first investigated the protonation of the protein in the two different conformations detected by X-ray crystallography and NMR using electrostatic calculations. Four residues, Glu142, His148, Tyr151 and Glu222, have a significantly different titration behaviour in the two conformations. In particular Glu142 and His148 titrate in the physiological pH range where the conformational change is expected. Thus, these residues are candidates to trigger the conformational change. Additionally, we found that the proportion between the two conformations depend strongly on pH. Moreover this pH dependence correlates very well with the pH dependence of the fluorescence lifetimes of the excited state of the chromophore. This agreement indicates that our electrostatic model is able to explain the pH dependence of these spectroscopic data. There are several residues that titrate in the pH range 4.5 - 10.5 that is of physiological relevance. These residues are potential candidate to explain the pH dependence of the photophysical properties of ECFP. By analysing the Monte Carlo simulations, we found that the following residues are strongly correlated with the conformational change. This result indicates that several residues are involved in the pH-induced conformational change.In parallel, we performed a more fundamental work devoted to clarify the meaning of titration curves of residues in proteins and how they are connected to pK values and protonation free energy. A pure mathematical approach connected to electrostatic calculations was used for this study. We showed that there are at least two different meaningful ways of deriving pK values from titration curves. One relates to thermodynamic data and the other to kinetic data. pK values derived from thermodynamic measurements and kinetic measurements may have different meanings depending on whether the protonation can equilibrate during the reaction or not. We could show under which conditions the two ways are equivalent. This parallel project, besides being of fundamental importance, has also direct implications on the topic of the main project.
