Maria-Elisabeth Michel-Beyerle

QUANTUM CHEMICAL MODELING OF STRUCTURE AND ABSORPTION SPECTRA OF THE CHROMOPHORE IN GREEN FLUORESCENT PROTEINS

Abstract As a first step towards modeling the green fluorescent protein (GFP), we have carried out absorption spectra calculations on chromophores of both native and mutant proteins using the semiempirical method INDO/S. A number of protonated and deprotonated states of the GFP fluorophore were considered. We find predicted and observed absorbance energies in very good agreement. Based on a comparison of calculated and experimental absorption spectra, we suggest structures for the ground and excited states of the chromophore of GFP. We assign the absorption maximum of GFP at 477 nm to an H-bonded complex of the zwitterion (O Y ,xa0HN,xa0O X ) involving the phenolic oxygen of Tyr66 and its environment; the nitrogen of the heterocyclic ring is protonated in this complex. Another peak at 397 nm is due to excitation of the corresponding protonated form (HO Y ,xa0HN,xa0O X ) + . Calculated transitions in the mutant chromophores (Tyr66→Phe), (Tyr66→Trp), and (Tyr66→His) at 355, 433, and 387 nm, respectively, are close to the corresponding experimental values of 360, 436, and 382 nm, suggesting cationic forms with the nitrogen protonated to be responsible for the absorbance. Proton transfer to or from the phenolic hydroxyl group of the chromophore is shown to be crucial for understanding the absorption and emission spectra of GFP.

STRUCTURE AND ROTATION BARRIERS FOR GROUND AND EXCITED STATES OF THE ISOLATED CHROMOPHORE OF THE GREEN FLUORESCENT PROTEIN

Abstract Semiempirical CISD calculations were carried out on models of differently protonated forms of the isolated chromophore active in the green fluorescent protein (GFP). Electronic excitation (S 0 →S 1 ) is found to considerably alter the equilibrium conformation of the chromophore. Low activation barriers to rotation about the exocyclic bond C γ –C β adjacent to the phenol ring are calculated for the cationic form in both states, S 0 and S 1 , for the neutral chromophore in the S 0 , and for the zwitterion in the S 1 state. It is of interest that only in the protonated state the energy gap between the two potential energy surfaces is small enough to allow internal conversion of the isolated chromophore. We propose that the fast internal conversion observed in GFP mutants is also associated with an avoided crossing of the S 0 and S 1 surfaces of the cation during the rotation around the bond C β –C α .

Protonation effects on the chromophore of green fluorescent protein. Quantum chemical study of the absorption spectrum

Abstract The absorption spectrum of the chromophore of the green fluorescent protein (GFP) was investigated by INDO/S-CI model calculations. Geometrical variations of the hydrogen bonds between the chromophore and its environment were studied in detail. Based on the good agreement between experimental and calculated data, the spectrum of GFP is assigned. The absorptions at 397 and 477 nm are due to the chromophore cation and zwitterion, respectively. The phenolic oxygen of the chromophore is deprotonated in the excited state, but can be protonated or deprotonated in the ground state. The heterocyclic nitrogen remains protonated after excitation.

Absorption spectra of the GFP chromophore in solution: comparison of theoretical and experimental results

Abstract Spectroscopic characteristics of the green fluorescent protein (GFP) and its mutants are controlled through protonation states of the chromophore and the polarity of its environment. Using the semiempirical method NDDO-G, absorption spectra of all possible protonation states of a model chromophore in the gas phase and in polar solution (ethanol) were calculated. Very good agreement between the simulated and experimental spectra has been found. We discuss the following protonation states of the model GFP chromophore: the anion (OY, N, OX)−, the neutral forms (HOY, N, OX) and (OY, N, HOX), the zwitter ion (OY−, HN+, OX), the cations (HOY, HN, OX)+, (OY, HN, HOX)+ and (HOY, N, HOX)+, and the dication (HOY, HN, HOX)2+. Our calculations show that solvatochromic effects on the electronic spectrum of the GFP chromophore can induce red or blue shifts of the absorption, depending on the protonation state. The different spectroscopic characteristics found for the various protonation states may be useful for the interpretation of spectra of intact GFPs.

Energetics of excess electron transfer in DNA

Abstract The energetics of electron transfer in DNA has been estimated via differences of electron affinities (EAs) of nucleobases B in trimers of Watson–Crick pairs 5 ′ -XBY-3 ′ calculated with the semi-empirical method AM1. The EA values of the nucleobases were found to decrease in the order cytosine ( C )≈ thymine ( T )≫ adenine ( A )> guanine ( G ) . The destabilizing effect of the subsequent base Y is more pronounced than that of the preceding base X. We predict that the strongest electron traps are 5 ′ -XCY-3 ′ and 5 ′ -XTY-3 ′ , where X, Y are pyrimidines C and T. These triads exhibit very similar EA values, and therefore, the corresponding anion radical states should be approximately in resonance, favoring efficient transport of excess electrons in DNA.

Similarity of primary radical pair recombination in photosystem II and bacterial reaction centers

We report temperature and magnetic field dependent measurements of the recombination dynamics of the radical pair P680+Pheo− in D1D2cytb559 reaction centers of photosystem II and compare the results to those obtained in bacterial reaction centers. In photosystem II the rate of recombination to the groundstate is found to be slower than in the bacterial reaction centers by a factor of at least 50. This difference arises from the different redox potentials of the pigments of plant and bacterial reaction centers. In contrast, the rate of recombination to the triplet state is similar in all reaction centers, indicating a similar electronic coupling which allows us to conclude upon the structural similarity.

The Recombination Dynamics of the Radical Pair P+H− in External Magnetic and Electric Fields

When electron transfer to the primary quinone is blocked, the radical pair P+H− (P: primary donor, H: bacteriopheophytin at the A-branch) recombines on the 10 ns time scale either to the ground state P or, after hyperfine-induced singlet-triplet-mixing, to the triplet state 3P*. An external magnetic field hinders singlettriplet-mixing, thus reducing the yield of 3P* and slowing the recombination of P+H−. Magnetic field dependent measurements of the recombination dynamics allow the determination of the recombination rates ks and kT and the exchange interaction J. From these parameters free energies, electronic matrix elements and reorganization energies relevant for the fast charge separation and slow charge recombination processes in the reaction center can be determined. In many cases, recombination data constitute the sole experimental access to such parameters, which constitute the basis for the theoretical treatment of electron transfer processes.

Sensitive analysis of the occupancy of the quinone binding site at the active branch of photosynthetic reaction centers

Abstract A sensitive method for determining the degree of the occupancy of the primary quinone site in reaction centers of photosynthetic bacteria is described. By measuring the transient absorbance bleaching of the primary electron donor during the time interval 1 ns to 1 ms after an exciting laser pulse, small amounts of quinone-depleted reaction centers can be detected with an accuracy of about ± 3%. In the case of Rb. sphaeroides , standard preparations typically contain 20% reaction centers in which the quinone binding site at the active branch is not occupied.

Energetics of the Primary Charge Separation in Bacterial Photosynthesis

We consider the free energy relationship for the primary electron transfer (ET) (at T = 295 K) from the electronically excited singler state of the bacteriochlorophyll dimer in the bacterial photosynthetic native reaction center (RC), some of its single-site mutants and chemically engineered RCs containing accessory 132-OH-Ni-bacteriochlorophyll (Ni-B). This analysis resulted in the reasonable value λ1 = 800±250 cm-1 for the medium reorganization energy and ΔG1(N) = -480±180 cm-1 for the energy gap of the native RC. These energetic parameters imply that ET in the native RC conresponds to (nearly) optimal activationless ET. The quantum free energy relation predicts very fast ET time for the Ni-B substituted center ΔG1 ≃ -1500 cm-1 which reflects prosthetic group(s) vibrational excitation induced by ET in the inverted region. The low negative value of ΔG1(N) implies that the dominant room temperature ET mechanism for the native RC involves sequential ET.

Fluorescence enhancement of asCP595 is due to consecutive absorbance of two photons

Colored proteins are widely used as gene markers in biotechnology. Chromophores result from autocatalytic posttranslational reactions involving several amino acids. The protein asCP595 was isolated for the first time from the coral as a weakly fluorescent chromoprotein with a fluorescence maximum at 595 nm. Strong illumination in the blue wing of the low energy absorption band results in a superlinear increase of the fluorescence yield and shifts its fluorescence spectrum by about 10 nm to the red. Time resolved fluorescence measurements using excitation pulses with 10 ps duration revealed a multiexponential decay pattern with time constants in the range from 20 ps to 2.1 ns. The ratio of amplitudes related to the different time constants depends on the intensity of illumination favoring the ns component at high intensities. Transient absorption measurements using ultrashort excitation pulses (150 fs, 1 kHz repetition rate) did not reveal excited states with nanosecond lifetimes as observed in fluorescence upon excitation using 10 ps pulses. This observation leads to the notion that within 10 ps a second photon is absorbed by a state not yet populated within 150 fs. As a consequence we propose two different excited singlet states operative in asCP595, one with low fluorescence quantum yield peaking at 595 nm and one with high fluorescence quantum yield peaking at 605 nm which is populated via the consecutive absorption of two photons at high excitation intensities.