How Does the Environment Affect the Absorption Spectrum of the Fluorescent Protein mKeima? Marc Nadal-Ferret, † Ricard Gelabert, † Miquel Moreno,* ,† and Jose ́ M. Lluch †,‡ † Departament de Química and ‡ Institut de Biotecnologia i de Biomedicina, Universitat Autò noma de Barcelona, 08193 Bellaterra, Barcelona, Spain * S Supporting Information ABSTRACT: The absorption spectrum of a fluorescent protein is determined by its chromophore, but the residues that surround it also have a remarkable role, leading to noticeable spectral shifts. We have theoretically analyzed the monomeric protein Keima (mKeima), a red fluorescent protein most remarkable for an outstanding difference between the absorption and emission frequencies, and potentially suited for multicolor imaging applications. In the present work, we have performed excited state electronic calculations on the chromophore with an increasing number of atoms surrounding it, and we have compared these results with the excited states calculations on an ensemble of structures obtained from a molecular dynamics simulation of the complete protein. The importance of the inclusion of the effects of the whole protein in the electronic calculations has been proved, and it is concluded that only with the consideration of the thermal effects can the absorption spectra of the protein be properly characterized. 1. INTRODUCTION Fluorescent proteins (FP) are nowadays a major topic in biochemical and biomedical research since they allow for the study of dynamic processes in live cells. 1-3 In particular, Aequorea victoria Green Fluorescent Protein (GFP, see the Nobel lectures by Shimomura, 4 Chalfie, 5 and Tsien, 6 and references therein) has been the most outstanding for its high quantum yield, its stability, and the autocatalytic formation of its chromophore. 7 Since the discovery of GFP, a huge amount of fluorescent proteins, whose fluorescence covers the full range of the visible electromagnetic spectrum, have been devel- oped. 1-3,8-11 This palette of fluorescent proteins furnishes a powerful tool for multicolor imaging, namely, an approach to the study of the above-mentioned processes in live cells. Therefore, it is crucial to have a deep understanding of the photochemical processes involved in these proteins to be able to track their photophysical and photochemical properties to their structural characteristics. In this sense, a large amount of work has addressed the optical properties of the fluorescent proteins. 12-67 There is solid agreement now that the way in which a chromophore has been matured determines the fluorescent properties of a protein, 68 even if formed by similar amino acids. For instance, the common tripeptide Met-Tyr-Gly is able to form chromophores spanning a 175-nm range for the maxima of emission spectra. 69 Although the nature of the chromophore and its maturation are crucial to account for the fluorescence wavelength, it has been reported that local environmental features such as the position of charged amino acid residues, hydrogen bonds, or hydrophobic interactions can yield shifts of up to 40 nm in absorption and emission maxima. 69 Among the spread of fluorescent proteins, let us focus on the family of Red Fluorescent Proteins (RFP), in particular, the subgroup represented by drFP5833 11 (commercial name DsRed). In this kind of chromophore, the π-system of the GFP chromophore is extended by an additional N-acylimine moiety; however, the mechanism of the chromophore maturation is not fully understood yet. 68 Recently developed proteins like mKeima, 1 LSSmKate1, 2 and LSSmKate2 2 (440- 463 nm absorption, 605-624 nm emission), or proteins like mNeptune, 8 eqFP670 70 , and TagRFP657 71 (592-611 nm absorption, 646-670 nm emission), all of them containing a DsRed-like chromophore, have exhibited novel photochemical properties by means of rationally modifying the chromophore environment. 8 Such a phenomenon suggests that the absorption and fluorescence spectra are affected not only by the chromophore but also by the surrounding residues and the solvent. Considering therefore the fact that the absorption properties of the chromophore are related to the environment, we should bear in mind that a single protein at a given temperature can show different wavelengths, as it displays a collection of configurations in which charges, hydrogen bonding networks, or long-range interactions are constantly changing. In other words, the experimental data available concerning radiation Received: November 15, 2012 Published: February 11, 2013 Article pubs.acs.org/JCTC © 2013 American Chemical Society 1731 dx.doi.org/10.1021/ct301003t | J. Chem. Theory Comput. 2013, 9, 1731-1742