RESUMEN

Biphotochromic proteins simultaneously possess reversible photoswitching (on-to-off) and irreversible photoconversion (green-to-red). High photochemical reactivity of cysteine residues is one of the reasons for the development of "mox"-monomeric and oxidation resistant proteins. Based on site-saturated simultaneous two-point C105 and C117 mutagenesis, we chose C21N/C71G/C105G/C117T/C175A as the moxSAASoti variant. Since its on-to-off photoswitching rate is higher, off-to-on recovery is more complete and photoconversion rates are higher than those of mSAASoti. We analyzed the conformational behavior of the F177 side chain by classical MD simulations. The conformational flexibility of the F177 side chain is mainly responsible for the off-to-on conversion rate changes and can be further utilized as a measure of the conversion rate. Point mutations in mSAASoti mainly affect the pKa values of the red form and off-to-on switching. We demonstrate that the microscopic measure of the observed pKa value is the C-O bond length in the phenyl fragment of the neutral chromophore. According to molecular dynamics simulations with QM/MM potentials, larger C-O bond lengths are found for proteins with larger pKa. This feature can be utilized for prediction of the pKa values of red fluorescent proteins.

Asunto(s)

RESUMEN

Biphotochromic fluorescent protein SAASoti contains five cysteine residues in its sequence and a V127T point mutation transforms it to the monomeric form, mSAASoti. These cysteine residues are located far from the chromophore and might control its properties only allosterically. The influence of individual, double and triple cysteine substitutions of mSAASoti on fluorescent parameters and phototransformation reactions (irreversible green-to-red photoconversion and reversible photoswitching) is studied. A set of mSAASoti mutant forms (C21N, C117S, C71V, C105V, C175A, C21N/C71V, C21N/C175A, C21N/C71G/C175A) is obtained by site-directed mutagenesis. We demonstrate that the C21N variant exists in a monomeric form up to high concentrations, the C71V substitution accelerates photoconversion to the red form and the C105V variant has the maximum photoswitching rate. All C175A-containing variants demonstrate different photoswitching kinetics and decreased photostability during subsequent switching cycles compared with other considered systems. Classical molecular dynamic simulations reveal that the F177 side chain located in the vicinity of the chromophore is considerably more flexible in the mSAASoti compared with its C175A variant. This might be the explanation of the experimentally observed slowdown the thermal relaxation rate, i.e., trans-cis isomerization of the chromophore in mSAASoti upon C175A substitution.

Asunto(s)

RESUMEN

Human carboxylesterases are involved in the protective processes of detoxification during the hydrolytic metabolism of xenobiotics. Knowledge of the molecular mechanisms of substrates hydrolysis in the enzymes active site is necessary for the rational drug design. In this work, the molecular mechanism of the hydrolysis reaction of para-nitrophenyl acetate in the active site of human carboxylesterase was determined using modern methods of molecular modeling. According to the combined method of quantum mechanics/molecular mechanics calculations, the chemical reaction occurs within four elementary steps, including two steps of the acylation stage, and two steps of the deacylation stage. All elementary steps have low energy barriers, with the gradual lowering of the intermediate energies that stimulates reaction in the forward direction. The molecular docking was used to estimate the binding constants of the enzyme-substrate complex and the dissociation constant of enzyme-product complexes. The effective kinetic parameters of the enzymatic hydrolysis in the active site of carboxylesterase are determined by numerical solution of the differential kinetic equations.

Asunto(s)

RESUMEN

The BLUF domain (sensor of blue light using flavin adenine dinucleotide) from a bacterial photoreceptor protein AppA undergoes a cascade of chemical transformations, including hydrogen bond rearrangements around the flavin adenine dinucleotide (FAD) chromophore, in response to light illumination. These transformations are initiated by photoinduced electron and proton transfer from a tyrosine residue to the photoexcited flavin which is assisted by a glutamine residue. According to the recent studies, the proton-coupled electron transfer leads to formation of a radical-pair intermediate Tyr•···FADH• and a tautomeric EE form of glutamine in the ground electronic state. This intermediate is a precursor of the light-induced state of the BLUF photoreceptor implicated in biological signaling. In order to describe evolution of the radical pair, we computed reaction pathways on the ground state potential energy surface employing quantum-chemical calculations in the DFT PBE0/cc-pVDZ approximation for a molecular cluster mimicking the chromophore containing pocket of the AppA BLUF protein. We found a minimum-energy pathway comprised of the following consecutive reaction steps: (1) rotation of the imidic group of the EE glutamine side chain around the Cγ-Cδ bond; (2) flip of the OεH group and formation of the ZE form of the glutamine side chain; and (3) biradical recombination via coupled proton and electron transfer, leading to the ZZ form of the glutamine side chain. The potential-energy barriers for stages 1-3 do not exceed 9 kcal/mol. Energy barrier 3 describing the ZE to ZZ glutamine tautomerization is significantly smaller in the BLUF model than in isolated glutamine, since tautomerization in BLUF is facilitated by electron transfer and radical recombination. Thus, our study shows that tautomerization of the conserved glutamine is coupled to the light-induced electron transfer process in BLUF and, thus, is a viable candidate for the photoactivation mechanism which at present is very much debated.

Asunto(s)

RESUMEN

The quantum mechanical-molecular mechanical (QM/MM) theory was applied to calculate accurate structural parameters, vibrational and optical spectra of bathorhodopsin (BATHO), one of the primary photoproducts of the functional cycle of the visual pigment rhodopsin (RHO), and to characterize reaction routes from RHO to BATHO. The recently resolved crystal structure of BATHO (PDBID: 2G87) served as an initial source of coordinates of heavy atoms. Protein structures in the ground electronic state and vibrational frequencies were determined by using the density functional theory in the PBE0/cc-pVDZ approximation for the QM part and the AMBER force field parameters in the MM part. Calculated and assigned vibrational spectra of both model protein systems, BATHO and RHO, cover three main regions referring to the hydrogen-out-of-plan (HOOP) motion, the C==C ethylenic stretches, and the C--C single-bond stretches. The S(0)-S(1) electronic excitation energies of the QM part, including the chromophore group in the field of the protein matrix, were estimated by using the advanced quantum chemistry methods. The computed structural parameters as well as the spectral bands match perfectly the experimental findings. A structure of the transition state on the S(0) potential energy surface for the ground electronic state rearrangement from RHO to BATHO was located proving a possible route of the thermal protein activation to the primary photoproduct.

Asunto(s)

RESUMEN

The proposed mechanisms of photoinduced reactions in the blue light using flavin chromophore photoreceptor proteins are primarily based on the results of X-ray crystallography and spectroscopy studies. Of particular value are the observed band shifts in optical and vibrational spectra upon formation of the signaling (light-induced) state. However, the same set of experimental data has given rise to contradictory interpretations suggesting different structures of the dark and signaling states. To verify the specific mechanism of light-induced changes involving the rotation/tautomerization transformations with the conserved Gln residue near the flavin chromophore, we performed accurate quantum chemical calculations of the equilibrium structures, vibrational and absorption bands of the model systems mimicking the BLUF domain of flavoprotein AppA. Geometry optimization and calculations of vibrational frequencies were carried out with the QM(B3LYP/cc-pVDZ)/MM(AMBER) approach starting from the representative molecular dynamics (MD) snapshots. The MD simulations were initiated from the available crystal structures of the AppA protein. Calculations of the vertical excitation energies were performed with the scaled opposite spin configuration interaction with single substitutions SOS-CIS(D) method that enables efficient treatment of excited states in large molecular systems. The computed molecular structures as well as the spectral shifts (the red shift by 12÷16 nm in absorption and the downshift by 25 cm(-1) for the C4═O flavin vibrational mode) are in excellent agreement with the experimental results, lending a strong support to the mechanism proposed by Domratcheva et al. (Biophys. J. 2008, 94, 3872).

RESUMEN

Based on computer simulation methods, the molecular dynamics of the rhodopsin chromophore group (11-cis-retinal) has been analyzed. The molecular dynamics has been traced within a 3-ns time interval; thereby 3 x 10(6) discrete conformational states of opsin and rhodopsin were compared and analyzed. It was shown that, within a short time of about 0.3-0.4 ns from the start of simulation, the retinal beta-ionone ring becomes twisted around the C6-C7 bond by approximately 60 degrees compared with that of the initial configuration. The influence of retinal conformation on the positions of the maximum of the absorption band of rhodopsin at the conformational states of t=0 and t=3 ns were estimated using the ab initio methods. The results indicated that the absorption maximum for the final (3-ns) state is shifted by 10 nm toward the long wavelength region compared with the initial state. This suggests that the rhodopsin molecule with its twisted chromophore will possess a considerably lower activation energy than the rhodopsin molecule where the beta-ionone ring is in a planar orientation to the retinal polyene chain.

Asunto(s)