Proton uptake upon quinone reduction in bacterial reaction centers: IR signature and possible participation of a highly polarizable hydrogen bond network

The light-induced Q _A ^– /Q_A FTIR difference spectra of Rb. sphaeroides and Rp. viridis show very broad positive bands of small amplitude peaking around 2750 cm^–1. Upon ¹H/²H exchange these bands shift to about 2150 cm^–1. Similarly, the Q _B ^– /Q_B spectra exhibit broad continuum bands at 2600 and 2800 cm^–1 shifting to 2100 and 2200 cm^–1 in ²H₂O for Rb. sphaeroides and Rp. viridis, respectively. These continuum bands are tentatively interpreted in terms of highly polarizable hydrogen bonds in a large web of polar bonds involving cofactors, amino acid residues, and structured water molecules. As a working hypothesis, we propose that the protons participating in this web redistribute upon quinone reduction, increasing their concentration around the newly formed charged species, and leading to net proton uptake. Assuming that the precise localization of the mobile protons is dependent on the local electrostatic, this model can explain the apparent discrepancies between some results of FTIR experiments and of electrostatic calculations. Notably, it could help rationalize the observation that mobile protons tend to localize on Glu L212 upon Q_B reduction in Rb. sphaeroides, while for Q_B reduction in Rp. viridis and for Q_A reduction in both Rb. sphaeroides and Rp. viridis, proton uptake by a small number of carboxylic residues is not supported by the FTIR data.     

Proton transfer: it's a stringent process

Proton transfer into and out of proteins is important, both for many enzyme reaction mechanisms and proton pumping across membranes. Recent work on several proteins has revealed stringent requirements for amino-acid side chains and subtle reorganisation of hydrogen-bond networks involving bound water molecules.     

A simple hydrogen bond potential function for conformational studies

Since hydrogen bonding plays an important role in determining energetically favourable conformations of biological molecules, a computationally simple and convenient semiempirical function has been evaluated by constraining a function to conform to the experimentally observed data for isolated simple systems of hydrogen bonded dimers. The function is found to be satisfactory for use in conformational energy calculations.     

An improved hydrogen bond potential: impact on medium resolution protein structures

A new semi-empirical force field has been developed to describe hydrogen-bonding interactions with a directional component. The hydrogen bond potential supports two alternative target angles, motivated by the observation that carbonyl hydrogen bond acceptor angles have a bimodal distribution. It has been implemented as a module for a macromolecular refinement package to be combined with other force field terms in the stereochemically restrained refinement of macromolecules. The parameters for the hydrogen bond potential were optimized to best fit crystallographic data from a number of protein structures. Refinement of medium-resolution structures with this additional restraint leads to improved structure, reducing both the free R-factor and over-fitting. However, the improvement is seen only when stringent hydrogen bond selection criteria are used. These findings highlight common misconceptions about hydrogen bonding in proteins, and provide explanations for why the explicit hydrogen bonding terms of some popular force field sets are often best switched off.     

Proton polarizability and proton transfer in histidine-phosphate hydrogen bonds as a function of cations present: ir investigations

(L -His)_n- dihydrogen phosphate systems are studied by ir spectroscopy in the presence of various cations and as a function of the degree of hydration. Ir continua indicate that (I) OH … N ? O^?…H⁺N (IIR) hydrogen bonds are formed and that these bonds show high proton polarizability, which increases from the Li⁺ to the K⁺ system. In the K⁺?system, His-P_i-P_i chains are formed, showing particularly high proton polarizability due to collective proton motion within both hydrogen bonds. The OH N ? O^?…H^?N equilibria are determined from ir bands. With the Li⁺ system, 55% of the protons are present at the histidine residues; this percentage is smaller with the Na⁺ system (41%), and amounts to only 32% with the K⁺ system. With the increasing degree of hydration the removal of the degeneracy of ν_as?PO^2?₃ vanishes, indicating loosening of the cations from the phosphates. Nevertheless, the hydrogen bond acceptor O atom becomes more negative; a shift of the equilibrium to the right is observed in the OH… N ? O^?…H⁺N bond. This is explained by the strong interaction of the dipole of the hydrogen bonds with the water molecules. All these results show that protons can be shifted easily in these hydrogen bonds due to their high proton polarizability. The transfer equilibria can be controlled easily by local electrical fields. In addition, these results may be of significance when phosphates interact with proteins.     

Proton transfer reactions in native and deionized bacteriorhodopsin upon delipidation and monomerization

We have investigated the role of the native lipids on bacteriorhodopsin (bR) proton transfer and their connection with the cation-binding role. We observe that both the efficiency of M formation and the kinetics of M rise and decay depend on the lipids and lattice but, as the lipids are removed, the cation binding is a much less important factor for the proton pumping function. Upon 75% delipidation using 3-[(cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS), the M formation and decay kinetics are much slower than the native, and the efficiency of M formation is approximately 30%-40% that of the native. Upon monomerization of bR by Trition X-100, the efficiency of M recovers close to that of the native (depending on pH), M formation is approximately 10 times faster, and M decay kinetics are comparable to native at pH 7. The same results on the M intermediate are observed if deionized blue bR (deI bbR) is treated with these detergents (with or without pH buffers present), even though deionized blue bR containing all the lipids has no photocycle. This suggests that the cation(s) has a role in native bR that is different than in delipidated or monomerized bR, even so far as to suggest that the cation(s) becomes unimportant to the function as the lipids are removed.     

Proton transfer and polarizability of hydrogen bonds formed between cysteine and lysine residues

(L -Cys)_n + N-base systems and (L -Cys)_n + (L -Lys)_n systems were studied by ir spectroscopy. It is shown that in the water-free systems, SH ?N ? S^? ?H⁺N hydrogen bonds are formed. With the (L -Cys)_n + N-base systems, both proton-limiting structures in the SH ?N ? S^? ?H⁺N bonds have equal weight when the pK_a of the protonated N-base is 2 pK_a units larger than that of (L -Cys)_n. The same is true with the water-free (L -Cys)_n + (L -Lys)_n system. Thus, with regard to the type of proton potentials present, these hydrogen bonds are proton-transfer hydrogen bonds showing very large proton polarizabilities. This is confirmed by the occurrence of continua in the ir spectra. Small amounts of water open these hydrogen bonds and increase the transfer of the proton to (L -Lys)_n. In the (L -Lys)_n + N-base systems, with increasing proton transfer the backbone of (L -Cys)_n changes from antiparallel β-structure to coil. In (L -Cys)_n + (L -Lys)_n, the conformation is determined by the (L -Lys)_n conformation and changes depending on the chain length of (L -Lys)_n. Finally, the reactivity increase in the active center of fatty acid synthetase, which should be caused by the shift of a proton, is discussed on the basis of the great proton polarizability of the cysteine–lysine hydrogen bonds.     

Two-dimensional gel electrophoresis based technologies for potential biomarkers identification in amniotic fluid: a simple model

To assess the efficiency of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and of two-dimensional electrophoresis and ammoniacal silver staining (2D-E), different amounts of soybean trypsin inhibitor and horse myoglobin were added to amniotic fluid. In this model, a minimum of 5 to 10 ng of "artificial" biomarkers was detected.     

Trans-substitution of the proximal hydrogen bond in myoglobin: I. Structural consequences of hydrogen bond deletion

The structural role of a side-chain to side-chain protein hydrogen bond is examined using trans-substitution of the proximal histidine of myoglobin with methylimidazoles (Barrick, Biochemistry 1994;33:6546-6554). Modification of the chemical structure of exogenous ligands allows this hydrogen bond to be disrupted. Comparison of the crystal structures of H93G myoglobin complexed 4-methylimidazole (4meimd; methylation at carbon 4) and 1-methylimidazole (1meimd; methylation at the adjacent nitrogen, preventing hydrogen bonding between the imidazole ligand and the protein) shows that the polypeptide, heme, and methylimidazole orientations are the same within error. For 4meimd there appear to be major and minor conformations corresponding to different tautomeric states of the ligand. Conformational heterogeneity is also seen in the hyperfine-shifted region of the NMR spectrum of 4meimd complexed with high-spin H93G deoxyMb. The major conformation of the 4meimd ligand and the 1meimd ligand, as seen in the respective crystal structures, are quite similar except that the proximal ligand NH-to-Ser92-OH hydrogen bond is eliminated in the 1meimd complex, and instead the proximal ligand CH is adjacent to the Ser92-OH. Thus, this system provides a means to eliminate the Mb proximal hydrogen bond in a chemically and structurally conservative way.     

The formation of hydrogen bond in the proximal heme pocket of HemAT-Bs upon ligand binding

HemAT-Bs is the heme-based O(2) sensor responsible for aerotaxis control in Bacillus subtilis. In this study, we measured the time-resolved resonance Raman spectra of full-length HemAT-Bs wild-type (WT) and Y133F in the deoxy form and the photoproduct after photolysis of CO-bound form. In WT, the nu(Fe-His) band for the 10 ps photoproduct was observed at higher frequency by about 2 cm(-1) compared with that of the deoxy form. This frequency difference is relaxed in hundreds of picoseconds. This time-dependent frequency shift would reflect the conformational change of the protein matrix. On the other hand, Y133F mutant did not show such a substantial nu(Fe-His) frequency shift after photolysis. Since a hydrogen bond to the proximal His induces an up-shift of the nu(Fe-His) frequency, these results indicate that Tyr133 forms a hydrogen bond to the proximal His residue upon the ligand binding. We discuss a functional role of this hydrogen bond formation for the signal transduction in HemAT-Bs.     

Developmental potential of selectively enucleated immature mouse oocytes upon nuclear transfer

Selective enucleation (SE) was applied to germinal vesicle (GV) oocytes by removing the chromatin attached to nuclear envelope, and leaving the liquid contents of GV in the cytoplast. However, after reconstruction with 1/8 blastomeres or fetal fibroblasts (FFs) neither the maturation efficiency nor the frequency of normal (asymmetric) division was improved as compared with completely enucleated (CE) oocytes. Chromosomal aberrations introduced with somatic nuclei were not rescued in SE oocytes either. On the other hand, timing of maturation division in SE GV oocytes, but not in CE GV oocytes, reconstructed with GV-karyoplasts was like in the control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomeres developed nucleolated donor pronuclei, contrary to CE oocytes. The latter could be rescued with nucleoli-containing nucleus, but not anucleolate nucleus, from a 1/2 blastomere. SE oocytes reconstructed with FFs contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes reconstructed with 1/8 blastomeres or FFs failed to cleave after activation or in vitro fertilization. Control GV oocytes enucleolated before fertilization seized cleavage at the 6-cell stage, as oppose to intact GV oocytes, which in 50.9% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the cleavage divisions. Activation of cumulus-enclosed SE GV oocytes matured in hormone-supplemented medium and fused to 1/2 blastomere-karyoplasts, yielded morulae, and blastocysts in 45.5% and 23.4% of reconstructed oocytes, respectively.     

Coarse-grained free energy functions for studying protein conformational changes: a double-well network model

In this work, a double-well network model (DWNM) is presented for generating a coarse-grained free energy function that can be used to study the transition between reference conformational states of a protein molecule. Compared to earlier work that uses a single, multidimensional double-well potential to connect two conformational states, the DWNM uses a set of interconnected double-well potentials for this purpose. The DWNM free energy function has multiple intermediate states and saddle points, and is hence a “rough” free energy landscape. In this implementation of the DWNM, the free energy function is reduced to an elastic-network model representation near the two reference states. The effects of free energy function roughness on the reaction pathways of protein conformational change is demonstrated by applying the DWNM to the conformational changes of two protein systems: the coil-to-helix transition of the DB-loop in G-actin and the open-to-closed transition of adenylate kinase. In both systems, the rough free energy function of the DWNM leads to the identification of distinct minimum free energy paths connecting two conformational states. These results indicate that while the elastic-network model captures the low-frequency vibrational motions of a protein, the roughness in the free energy function introduced by the DWNM can be used to characterize the transition mechanism between protein conformations.     

The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer.

Electron-transfer flavoprotein (ETF) serves as an intermediate electron carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells. The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen bonded to N(1) of the flavin ring. We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond. The binding constants for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol. The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF containing 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein. These data suggest that the 4'-hydoxyl-N(1) hydrogen bond stabilizes the anionic semiquinone in which negative charge is delocalized over the N(1)-C(2)O region. Transfer of the second electron to 4'-deoxy-FAD reconstituted ETF is extremely slow, and it was very difficult to achieve complete reduction of the flavin semiquinone to the hydroquinone. The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions. This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (相似文献   

Non-empirical valence bond calculation of hydrogen bond energy in polypeptides

An approximate ab-initio valence bond calculation of hydrogen bond energy was carried out and the results are discussed. The total bond energy of a simplified N? H…O structure is calculated for various N? H and N…O distances and the potential energy profiles are obtained. The hydrogen bond energy, ie, the delocalization energy gained by the formation of one hydrogen bond is found to be 8.7–12.0 kcal/mole. The potential energy is characterized by a deep minimum at 1.6–1.8 a.u. from the nitrogen and the second trough is found to be considerably higher than the first.     

The stereochemistry of hydrogen transfer to NADP+ by enzymes acting upon stereoisomeric substrates

The stereochemistry of hydrogen transfer from estradiol-17α and estradiol-17β to NADP⁺ in the presence of chicken liver estradiol-17α and estradiol-17β dehydrogenases was found in both cases to involve the 4-pro-S proton of the pyridine nucleotide. One of these enzymes must therefore use the stereochemically less favorable mode of interaction of steroid with coenzyme.     

Conjugation of polyethylene glycol via a disulfide bond confers water solubility upon a peptide model of a protein transmembrane segment.

The aqueous insolubility of hydrophobic peptides has presented a barrier to the structural characterization of membrane protein transmembrane domains. Since the conjugation of polyethylene glycol is known to modulate the solubility of certain proteins and peptides, we have prepared PEG-a-Cys reagent, a polyethylene glycol derivative which reacts spontaneously with Cys residues to attach polyethylene glycol to polypeptides via a mixed disulfide bond. When desired, the PEG moiety can be readily removed by reduction with tricarboxyethylphosphine. The aqueous solubilizing power of PEG-a-Cys reagent is confirmed with a synthetic hydrophobic peptide model of a generic transmembrane segment-soluble carrier fusion protein.