Proton shuttle in green fluorescent protein studied by dynamic simulations

Abstract

As a direct simulation of a multistep proton transfer reaction involving protein residues, the proton relay shuttle between A and I forms of green fluorescent protein (GFP) is simulated in atomic detail by using a special molecular dynamics simulation technique. Electronic excitation of neutral chromophore in wild-type GFP is generally followed by excited-state proton transfer to a nearby glutamic acid residue via a water molecule and a serine residue. Here we show that the second and third transfer steps occur ultrafast on time scales of several tens of femtoseconds. Proton back-shuttle in the ground state is slower and occurs in a different sequence of events. The simulations provide atomic models of various intermediates and yield realistic rate constants for proton transfer events. In particular, we argue that the I form observed spectroscopically under equilibrium conditions may differ from the I form observed as a fast intermediate by an anti to syn rotation of the carboxyl proton of neutral Glu-222.B ecause of its unique photophysical properties-strong green fluorescence without need for an additional cofactorgreen fluorescent protein (GFP) has become a very powerful marker for gene expression, cellular localization, and dynamic intracellular events over the past 5 years (1). Its rich photophysical behavior was characterized quite well by a great variety of spectroscopic techniques (1-4), and its three-dimensional structure was determined by x-ray crystallography (5, 6). Wild-type GFP exhibits two absorption maxima ''A'' and ''B'' at 395 nanometers (nm) and at 475 nm (1) that are present roughly in a 6:1 ratio (1), and that correspond to forms of the protein with either neutral (A) or anionic (B) chromophores. Time-resolved fluorescence spectroscopy identified an additional form ''I*'' that is populated as a fast intermediate after excitation of the A form, A* 3 I* (2, 3). The mechanism of interconversion is likely caused by excited-state proton transfer from the chromophore to a nearby protein residue (2, 3) because the new form emits at a similar wave length as B* and because the transfer rate slows down upon deuteration (2); see Fig. 1 for an overview.Although ''I form'' originally denoted only this fast intermediate, it has become common to use ''I form'' as a term for GFP species that absorb at wavelengths red-shifted with respect to the B form. Surprisingly, recent hole-burning experiments demonstrated that an I form is already present in wild-type GFP samples at room temperature under equilibrium conditions (4). It is currently unclear whether this is the same I form observed in the time-resolved fluorescence experiments. Although the I form has so far not been described in atomic detail, a structural model of I was suggested on the basis of comparisons between the crystal structures of wild-type (A form) and mutant GFP proteins containing the B form (7, 8); see Fig. 2. In this model, Glu-222 is assumed to be protonated in the I form (7), thus requiring a rise of the pK a of Glu-222 by more than 4 ...

Molecular modeling of green fluorescent protein: Structural effects of chromophore deprotonation

“…Helms et al 40 observed that, although in their starting structure Arg168 sticks out to the solution, during the 318-ps simulations it folds back to the protein surface to form a hydrogen bond with Ser147. The anionic chromophore shows additional hydrogen-bonding to His148 and Thr203 but not to Ser205, which instead is hydrogen bonded to W 22 . The increased hydrogenbonding pattern for the anionic chromophore can additionally explain the larger rotational barrier for 2 rotation observed in the ONIOM model(II).…”

“…GFP exhibits strong two-photon absorption (TPA) and therefore has great potential for a wide range of applications, ranging from three-dimensional fluorescence imaging, optical data storage to microfabrication. 18 -21 Previous QM/MM studies have addressed the excited states of the chromophore, [22][23][24][25] but the understanding of the spectra of the protein is far from complete. Although time-dependent density functional theory (TDDFT) calculations with the initial structure derived using a QM/MM (specifically, DFT/ AM1) approach have been recently reported, 23 the nonlinear optical response has not been examined, and accurate calculations of the TPA cross section with an understanding of the structure and energetics in the excited state are still required.…”

Anomalous Acid-Base Equilibria in Biologically Relevant Water Nanopools

“…5) 17 but similar to that of an alcohol (ε = [30][31][32][33][34][35][36][37][38][39][40]. 18,19 On the other hand, biological processes often take place based on proton relay along a hydrogen (H)-bonded chain, [1][2][3][4][20][21][22][23][24] and the dynamics of biological proton relay is determined by the size, the structure, and the motion of a water cluster which is the prime agent in most of biological systems. 3,[13][14][15][24][25][26] Thus, for better understanding of cellular dynamics, it is necessary to investigate the properties of a biologically relevant water nanopool as a biomimetic system of water confined in a cell membrane.…”

“…

Water plays a crucial role in many principal biological phenomena such as enzymatic catalysis and proton pumping through a membrane protein channel.1-4 Moreover, in biological systems, water is usually contained in a small pocket of a membrane, 5-8 and such confined water, which is generally called a water nanopool, [8][9][10] shows peculiar properties differing considerably from the properties of bulk water.

8-16The confinement effect and the enclosing interfacial surfaces of waterpools are the main factors to determine the properties, such as polarity, viscosity, and H-bonding ability, of water nanopools.5-13 For example, the dielectric constant of a water nanopool has been reported to be much lower than that of bulk water (ε = 78.5)17 but similar to that of an alcohol (ε = 30-40).18,19 On the other hand, biological processes often take place based on proton relay along a hydrogen (H)-bonded chain, [1][2][3][4][20][21][22][23][24] and the dynamics of biological proton relay is determined by the size, the structure, and the motion of a water cluster which is the prime agent in most of biological systems. 3,[13][14][15][24][25][26] Thus, for better understanding of cellular dynamics, it is necessary to investigate the properties of a biologically relevant water nanopool as a biomimetic system of water confined in a cell membrane.

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Dynamisches Protonierungsgleichgewicht der in Wasser gelösten Essigsäure

“…Die Transferwahrscheinlichkeiten hängen vom momentanen Donor-AkzeptorAbstand (R DA ) und vom Energieunterschied zwischen den beiden Minima an Donor und Akzeptor (E 12 ) ab. Diese Methode wurde erfolgreich eingesetzt, um den Protonenshuttle im grün fluoreszierenden Protein zu untersuchen [40] und um den Mechanismus zu verstehen, der Aquaporin undurchlässig für Protonen macht. [41] Hier stellen wir die Anwendung von Q-HOP-MD auf die Berechnung des expliziten Protonierungsgleichgewichts der gelösten Essigsäure auf einer Zeitskala von 50 ns bei sinnvollen pH-Bedingungen (pH 1) vor.…”