Molecular Dynamics Simulations of Enhanced Green Fluorescent Proteins: Effects of F64L, S65T and T203Y Mutations on the Ground-State Proton Equilibria R. Nifosı ` and V. Tozzini NEST-INFM and Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56126 Pisa, Italy ABSTRACT Molecular dynamics simulations with the Amber force field are carried out to study two mutants of the green fluorescent protein (GFP), namely EGFP (F64L/S65T) and T203Y-EGFP (E 2 GFP). Those variants display an opposite equilib- rium between the structural A and B states, associ- ated with neutral and anionic protonation forms of the chromophore. Configurations of those two states are simulated for each variant and the energetics of their equilibrium in the two mutants is studied by evaluating the change in the relative free energy of A and B states (G AB ) upon T203Y mutation. The resulting G AB agrees with the value inferred from absorption measurements. A comparison of the hy- drogen bond network around the chromophore ra- tionalizes the different population of state A and B in EGFP and E 2 GFP. On the basis of structural and energetic considerations, a mechanism for destabili- zation of the neutral chromophore in S65T mutants is proposed. Simulations of the B state of the S65T variant and of WT GFP are also performed for comparison and to test the force field parameters of the chromophore derived for the present calcula- tions. Possible paths of proton transfer leading to nonfluorescent states of the chromophore are dis- cussed in light of the photodynamical behavior of GFP, as revealed by fluorescence correlation spec- troscopy and single-molecule experiments. Proteins 2003;51:378 –389. © 2003 Wiley-Liss, Inc. Key words: GFP; molecular dynamics simulations; proton equilibria; photodynamics INTRODUCTION In the last decades, the green fluorescent protein (GFP) of the jellyfish Aequorea victoria has found countless applications in cell biology, biochemistry, and biotechnol- ogy. The ability to fluoresce without the need of any external co-factor and the absence of interference with the function of the tagged protein 1 make GFP an ideal marker of gene expression and protein trafficking in living cells. Moreover, GFP shows high thermal stability and good quantum yield thanks to its rigid -barrel fold protecting the chromophore, 2,3 generated by an autocatalytic cycliza- tion and subsequent oxidation of three adjacent residues (Ser65, Tyr66 and Gly67). 1 This mechanism, whose effi- ciency is a key ingredient in GFP functionality, produces a p-hydroxybenzylindene-imidazolidinone, constituted by the phenolic ring of Y66 linked to an imidazolidinone ring by a bridging carbon (see Fig. 1). The absorption spectrum of wild type (WT) GFP consists of a major band at 395 nm (A) and a minor one at 475 nm (B). On the basis of structural analyses 1,4,5 these bands are attributed to different forms of the chromophore, differing in the protonation of the hydroxyl group of the phenolic ring, protonated in the more populated state (correspond- ing to peak A) and deprotonated in the minor one (peak B). To date no experimental evidence for protonation of the imidazolidinone nitrogen has been found, 6 and the bright A and B states are generally assigned to the neutral and anionic chromophore, respectively. States having the imi- dazolidinone nitrogen protonated (namely the cation and the zwitterion) were proposed as possible nonemitting dark states. 7,8 The fluorescence of WT GFP peaks at 505 nm and is almost entirely due to the emission of the anionic chromophore. Indeed, when the A state (with a neutral chromophore) is excited, the phenolic moiety loses the proton, leading to the fluorescence of the anionic species. The destination of the involved proton is some- what debated, the current hypothesis being that it is transferred to the carboxylate group of Glu222 through a network of H-bonds in the chromophore environment. 4 The state resulting from the proton transfer, usually called I, is supposed to be intermediate between the A and B states, namely having the anionic chromophore in an unrelaxed environment, more similar to that of state A. 4 Several GFP mutants have been developed with im- proved photostability and different spectral properties, such as blue or red shifted emission. The double F64L/ S65T mutant, called Enhanced GFP (EGFP), is the most widely used in molecular and cell biology. F64L mutation improves folding efficiency, whereas S65T yields an excita- tion spectrum with a maximum peak at 490 nm, which is more suitable for use in living cells being less energetic than the 395 nm excitation needed for WT GFP. 9 The side chain of residue at position 65, though not directly in- volved in the double ring structure of the chromophore, is located in close contact with the imidazolidinone ring and with Glu222. Because Glu222 is supposed to be anionic in *Correspondence to: Riccardo Nifosı `, NEST-INFM and Scuola Nor- male Superiore, Piazza dei Cavalieri 7, I-56126 Pisa, Italy. E-mail: r.nifosi@nest.sns.it. Received 26 July 2002; Accepted 25 October 2002 Published online 00 Month 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.10335 PROTEINS: Structure, Function, and Genetics 51:378 –389 (2003) © 2003 WILEY-LISS, INC.