Relaxation Dynamics of Pseudomonas aeruginosa Re^I(C)O_3(α-diimine)(HisX)^+ (X=83, 107, 109, 124, 126)Cu-^(II) Azurins

Photoinduced relaxation processes of five structurally characterized Pseudomonas aeruginosa Re^I(CO)_3(α-diimine)(HisX) (X = 83, 107, 109, 124, 126)Cu^(II) azurins have been investigated by time-resolved (ps−ns) IR spectroscopy and emission spectroscopy. Crystal structures reveal the presence of Re-azurin dimers and trimers that in two cases (X = 107, 124) involve van der Waals interactions between interdigitated diimine aromatic rings. Time-dependent emission anisotropy measurements confirm that the proteins aggregate in mM solutions (D2O, KPi buffer, pD = 7.1). Excited-state DFT calculations show that extensive charge redistribution in the ReI(CO)_3 → diimine ^3MLCT state occurs: excitation of this ^3MLCT state triggers several relaxation processes in Re-azurins whose kinetics strongly depend on the location of the metallolabel on the protein surface. Relaxation is manifested by dynamic blue shifts of excited-state ν(CO) IR bands that occur with triexponential kinetics: intramolecular vibrational redistribution together with vibrational and solvent relaxation give rise to subps, 2, and 8−20 ps components, while the ~10^2 ps kinetics are attributed to displacement (reorientation) of the Re^I(CO)_3(phen)(im) unit relative to the peptide chain, which optimizes Coulombic interactions of the Re^I excited-state electron density with solvated peptide groups. Evidence also suggests that additional segmental movements of Re-bearing β-strands occur without perturbing the reaction field or interactions with the peptide. Our work demonstrates that time-resolved IR spectroscopy and emission anisotropy of Re^I carbonyl−diimine complexes are powerful probes of molecular dynamics at or around the surfaces of proteins and protein−protein interfacial regions

Deciphering the Catalytic Machinery in 30S Ribosome Assembly GTPase YqeH

YqeH, a circularly permuted GTPase (cpGTPase), which is conserved across bacteria and eukaryotes including humans is important for the maturation of small (30S) ribosomal subunit in Bacillus subtilis. Recently, we have shown that it binds 30S in a GTP/GDP dependent fashion. However, the catalytic machinery employed to hydrolyze GTP is not recognized for any of the cpGTPases, including YqeH. This is because they possess a hydrophobic substitution in place of a catalytic glutamine (present in Ras-like GTPases). Such GTPases were categorized as HAS-GTPases and were proposed to follow a catalytic mechanism, different from the Ras-like proteins.MnmE, another HAS-GTPase, but not circularly permuted, utilizes a potassium ion and water mediated interactions to drive GTP hydrolysis. Though the G-domain of MnmE and YqeH share only approximately 25% sequence identity, the conservation of characteristic sequence motifs between them prompted us to probe GTP hydrolysis machinery in YqeH, by employing homology modeling in conjunction with biochemical experiments. Here, we show that YqeH too, uses a potassium ion to drive GTP hydrolysis and stabilize the transition state. However, unlike MnmE, it does not dimerize in the transition state, suggesting alternative ways to stabilize switches I and II. Furthermore, we identify a potential catalytic residue in Asp-57, whose recognition, in the absence of structural information, was non-trivial due to the circular permutation in YqeH. Interestingly, when compared with MnmE, helix alpha2 that presents Asp-57 is relocated towards the N-terminus in YqeH. An analysis of the YqeH homology model, suggests that despite such relocation, Asp-57 may facilitate water mediated catalysis, similarly as the catalytic Glu-282 of MnmE. Indeed, an abolished catalysis by D57I mutant supports this inference.An uncommon means to achieve GTP hydrolysis utilizing a K(+) ion has so far been demonstrated only for MnmE. Here, we show that YqeH also utilizes a similar mechanism. While the catalytic machinery is similar in both, mechanistic differences may arise based on the way they are deployed. It appears that K(+) driven mechanism emerges as an alternative theme to stabilize the transition state and hydrolyze GTP in a subset of GTPases, such as the HAS-GTPases

Tryptophan-Accelerated Electron Flow Through Proteins

Energy flow in biological structures often requires submillisecond charge transport over long molecular distances. Kinetics modeling suggests that charge-transfer rates can be greatly enhanced by multistep electron tunneling in which redox-active amino acid side chains act as intermediate donors or acceptors. We report transient optical and infrared spectroscopic experiments that quantify the extent to which an intervening tryptophan residue can facilitate electron transfer between distant metal redox centers in a mutant Pseudomonas aeruginosa azurin. CuI oxidation by a photoexcited ReI-diimine at position 124 on a histidine(124)-glycine(123)-tryptophan(122)-methionine(121) β strand occurs in a few nanoseconds, fully two orders of magnitude faster than documented for single-step electron tunneling at a 19 angstrom donor-acceptor distance

NOA1 Functions in a Temperature-Dependent Manner to Regulate Chlorophyll Biosynthesis and Rubisco Formation in Rice

NITRIC OXIDE-ASSOCIATED1 (NOA1) encodes a circularly permuted GTPase (cGTPase) known to be essential for ribosome assembly in plants. While the reduced chlorophyll and Rubisco phenotypes were formerly noticed in both NOA1-supressed rice and Arabidopsis, a detailed insight is still necessary. In this study, by using RNAi transgenic rice, we further demonstrate that NOA1 functions in a temperature-dependent manner to regulate chlorophyll and Rubisco levels. When plants were grown at 30°C, the chlorophyll and Rubisco levels in OsNOA1-silenced plants were only slightly lower than those in WT. However, at 22°C, the silenced plants accumulated far less chlorophyll and Rubisco than WT. It was further revealed that the regulation of chlorophyll and Rubisco occurs at the anabolic level. Etiolated WT seedlings restored chlorophyll and Rubisco accumulations readily once returned to light, at either 30°C or 15°C. Etiolated OsNOA1-silenced plants accumulated chlorophyll and Rubisco to normal levels only at 30°C, and lost this ability at low temperature. On the other hand, de-etiolated OsNOA1-silenced seedlings maintained similar levels of chlorophyll and Rubisco as WT, even after being shifted to 15°C for various times. Further expression analyses identified several candidate genes, including OsPorA (NADPH: protochlorophyllide oxidoreductase A), OsrbcL (Rubisco large subunit), OsRALyase (Ribosomal RNA apurinic site specific lyase) and OsPuf4 (RNA-binding protein of the Puf family), which may be involved in OsNOA1-regulated chlorophyll biosynthesis and Rubisco formation. Overall, our results suggest OsNOA1 functions in a temperature-dependent manner to regulate chlorophyll biosynthesis, Rubisco formation and plastid development in rice

Theoretical determination of the standard reduction potential of plastocyanin in vitro

Quantum chemical QM/MM calculations have been performed on the copper-containing blue protein plastocyanin that is involved in the photosynthetic electron transfer. Crystallographic coordinates of the non-hydrogen atoms in the oxidized and reduced forms of poplar plastocyanin were obtained from the Protein Data Bank. The present work required calculations on the oxidized form that has a molecular structure independent of pH, and the reduced form with different structures at pH values 3.8 and 7. At pH 7, both the oxidized and reduced forms of the protein have distorted tetrahedral geometry for the copper-containing active site, the Cu atom being coordinated to two histidine, one cysteine, and one methionine residues. At pH 3.8, the active site of the reduced species is trigonally coordinated to one histidine, one cysteine, and one methionine residues. To optimize the geometry of the system while retaining the constraints of the protein backbone, the ONIOM methodology was adopted so as to treat the active site by the DFT-B3LYP method using the 6-31G basis set, whereas the geometries of the nearby residues as well as six neighboring water molecules were optimized by the MM/UFF method. Then atomic charges for the atoms of the protein (apart from those in the active site) were determined from DFT calculations separately for each amino acid residue using the STO-3G basis set. The atomic charges of the water molecules were computed by the DFT/6-31G(d) method. Finally, the electronic energies were recalculated by the ONIOM technique where the DFT-optimized active site was again treated at the 6-31G level of theory, whereas the rest of the protein, along with the solvent molecules near the active site, were treated by the Amber force field method using the calculated DFT charge on each atom. This treatment effectively allowed us to retain the steric constraints offered by the protein backbone during the optimization process, as well as the effects arising from the interaction of the protein dipoles and the bare charges on the protein with the atomic charges in the complex, thereby accommodating all electronic interactions such as charge-charge, charge-dipole, dipole-dipole, etc. The thermal energies of various oxidized and reduced forms were computed for a slightly simplified model of the active site (the part optimized in ONIOM) by using the DFT-B3LYP methodology. The effective radius of the globular protein plastocyanin was determined from the crystallographic data. The stability of each species arising from its interaction with medium was determined by explicitly calculating the Born charge-dielectric (water) interaction energy and, for the solvated proton, the Debye-Huckel energy of ion-ionic atmosphere interaction. The dielectric constant of water and plastocyanin were taken as 78.5 and 8.0, respectively. The interaction with the medium and the entropy changes are found to play a critical role in determining the reduction potential. The process of reduction of plastocyanin in an aqueous medium involves a very large reorganization of water molecules, and a large entropy change that cannot be computed readily. Hence, the entropy of reduction of plastocyanin was taken from experimental data that are available for pH 7. The free energy change was calculated for the reduction of plastocyanin in water and proton in water. From these values, the standard reduction potential was determined at pH 7. The calculated potential (376 +/- 38 mV) is in excellent agreement with the observed one (379 mV) for the radius of the globular protein 22.37 +/- 0.16 Angstrom. A similar calculation leads us to predict the entropy of reduction of plastocyanin at pH 3.8

Azurin as a Protein Scaffold for a Low-coordinate Nonheme Iron Site with a Small-molecule Binding Pocket

The apoprotein of Pseudomonas aeruginosa azurin binds iron(II) to give a 1:1 complex, which has been characterized by electronic absorption, Mössbauer, and NMR spectroscopies, as well as X-ray crystallography and quantum-chemical computations. Despite potential competition by water and other coordinating residues, iron(II) binds tightly to the low-coordinate site. The iron(II) complex does not react with chemical redox agents to undergo oxidation or reduction. Spectroscopically calibrated quantum-chemical computations show that the complex has high-spin iron(II) in a pseudotetrahedral coordination environment, which features interactions with side chains of two histidines and a cysteine as well as the C═O of Gly45. In the 5A1 ground state, the dz2 orbital is doubly occupied. Mutation of Met121 to Ala leaves the metal site in a similar environment but creates a pocket for reversible binding of small anions to the iron(II) center. Specifically, azide forms a high-spin iron(II) complex and cyanide forms a low-spin iron(II) complex

Structural basis for SHOC2 modulation of RAS signalling.

The RAS-RAF pathway is one of the most commonly dysregulated in human cancers1-3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5-8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11-13, but the structure of SHOC2 and the architecture of the presumptive SHOC2-PP1C-RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2-PP1C-MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway

Phototriggering Electron Flow through Re-I-modified Pseudomonas aeruginosa Azurins

The [Re-I(CO)(3)(4,7-dimethyl-1,10-phenanthroline)(histidine-124)(tryptophan-122)] complex, denoted [Re-I(dmp)(W122)], of Pseudomonas aeruginosa azurin behaves as a single photoactive unit that triggers very fast electron transfer (ET) from a distant (2 nm) Cu-I center in the protein. Analysis of time-resolved (ps-ms) IR spectroscopic and kinetics data collected on [Re-I(dmp)(W122) AzM] (in which M=Zn-II, Cu-II, Cu-I; Az=azurin) and position-122 tyrosine (Y), phenylalanine (F), and lysine (K) mutants, together with excited-state DFT/time-dependent (TD)DFT calculations and Xray structural characterization, reveal the character, energetics, and dynamics of the relevant electronic states of the [Re-I(dmp)(W122)] unit and a cascade of photoinduced ET and relaxation steps in the corresponding Re-azurins. Optical population of [Re-I(imidazole-H124)(CO)(3)]-> dmp (CT)-C-1 states (CT=charge transfer) is followed by around 110 fs intersystem crossing and about 600 ps structural relaxation to a (CT)-C-3 state. The IR spectrum indicates a mixed Re-I(CO)(3), A -> dmp/pi ->pi* (dmp) character for aromatic amino acids A122 (A=W, Y, F) and Re-I(CO)(3)-> dmp metal-ligand charge transfer (MLCT) for [Re-I(dmp)(K122)AzCu(II)]. In a few ns, the (CT)-C-3 state of [Re-I(dmp)(W122)AzM] establishes an equilibrium with the [Re-I(dmp(center dot)-)(W122(center dot+))AzM] charge-separated state, (CS)-C-3, whereas the (CT)-C-3 state of the other Y, F, and K122 proteins decays to the ground state. In addition to this main pathway, (CS)-C-3 is populated by fs-and ps-W(indole)-> Re-II ET from (CT)-C-1 and the initially "hot" (CT)-C-3 states, respectively. The (CS)-C-3 state undergoes a tensof-ns dmpC(center dot-)-> W122(center dot+) ET recombination leading to the ground state or, in the case of the Cu-I azurin, a competitively fast (approximate to 30 ns over 1.12 nm) Cu-I -> W center dot+ ET, to give [Re-I (dmp(center dot-))-(W122)AzCu(II)]. The overall photoinduced Cu-I -> Re(dmp) ET through [Re-I(dmp)(W122)AzCu(I)] occurs over a 2 nm distance in <50 ns after excitation, with the intervening fast (CT)-C-3-(CS)-C-3 equilibrium being the principal accelerating factor. No reaction was observed for the three Y, F, and K122 analogues. Although the presence of [Re(dmp)(W122)AzCu(II)] oligomers in solution was documented by mass spectrometry and phosphorescence anisotropy, the kinetics data do not indicate any significant interference from the intermolecular ET steps. The ground-state dmp-indole pi-pi interaction together with well-matched W/W center dot+ and excited-state [Re-II(CO)(3)(dmp(center dot-))]/[Re-I(CO)(3)(dmp(center dot-))] potentials that result in very rapid electron interchange and (CT)-C-3-(CS)-C-3 energetic proximity, are the main factors responsible for the unique ET behavior of [Re-I(dmp)(W122)]-containing azurins