Molecular mechanisms of pH-driven conformational transitions of proteins: insights from continuum electrostatics calculations of acid unfolding

The acid unfolding of staphylococcal nuclease (SNase) is very cooperative (Whitten and García-Moreno, Biochemistry 2000;39:14292-14304). As many as seven hydrogen ions (H+) are bound preferentially by the acid-unfolded state relative to the native (N) state in the pH range 3.2-3.9. To investigate the mechanism of acid unfolding, structure-based pKa calculations were performed with a variety of continuum electrostatic methods. The calculations reproduced successfully the H+ binding properties of the N state between pH 5 and 9, but they systematically overestimated the number of H+ bound upon acid unfolding. The calculated pKa values of all carboxylic residues in the N state were more depressed than they should be. The discrepancy between the observed and the calculated H+ uptake upon acid unfolding was not improved by using high protein dielectric constants, structures relaxed with molecular dynamics, or other empirical modifications implemented previously by others to maximize agreement between measured and calculated pKa values. This suggests an important role for conformational fluctuations of the backbone as important determinants of pKa values of carboxylic groups. Because no global or subglobal conformational changes have been observed previously for SNase under acidic conditions above the acid-unfolding region, these fluctuations must be local. The acid unfolding of SNase does not seem to involve the disruption of the N state by accruement of intramolecular repulsive interactions, nor the protonation of key ion paired carboxylic residues. It is more consistent with modest contributions from many H+ binding groups, with an important role for local conformational fluctuations in the coupling between H+ binding and the global structural transition.     

Structural properties of proteins specific to the myelin sheath

Summary. The myelin sheath is an insulating membrane layer surrounding myelinated axons in vertebrates, which is formed when the plasma membrane of an oligodendrocyte or a Schwann cell wraps itself around the axon. A large fraction of the total protein in this membrane layer is comprised of only a small number of individual proteins, which have certain intriguing structural properties. The myelin proteins are implicated in a number of neurological diseases, including, for example, autoimmune diseases and peripheral neuropathies. In this review, the structural properties of a number of myelin-specific proteins are described. Author’s address: Dr. Petri Kursula, Department of Biochemistry, University of Oulu, FIN-90014 Oulu, Finland     

Generation of membrane potential during photosynthetic electron flow in chromatophores from Rhodopseudomonas capsulata

1. When cytochrome c₂ is available for oxidation by the photosynthetic reaction centre, the decay of the carotenoid absorption band shift generated by a short flash excitation of Rhodopseudomonas capsulata chromatophores is very slow (half-time approximately 10 s). Otherwise the decay is fast (half-time approximately 1 s in the absence and 0.05 s in the presence of 1,10-ortho-phenanthroline) and coincides with the photosynthetic back reaction.2. In each of these situations the carotenoid shift decay, but not electron transport, may be accelerated by ioniophores. The ionophore concentration dependence suggests that in each case the carotenoid response is due to a delocalised membrane potential which may be dissipated either by the electronic back reaction or by electrophoretic ion flux.3. At high redox potentials, where cytochrome c₂ is unavailable for photo-oxidation, electron transport is believed to proceed only across part of the membrane dielectric. Under such conditions it is shown that the driving force for carbonyl cyanide trifluoromethoxyphenyl hydrazone-mediated H⁺ efflux is nevertheless decreased by valinomycin/K⁺; demonstrating that the [BChl]₂ → Q electron transfer generates a delocalised membrane potential.     

Analysis of oligomeric proteins during unfolding by pH and temperature

During thermal transition and variation of pH, structural properties of 35 proteins and their complexes (bound with substrate and co-factor) were analyzed in detail. During pH alteration, these proteins were shown to have substantial differences in conformations. pH conformers were analyzed in detail. Free energy and other energy parameters were also estimated for these proteins at various pH and temperatures. Detailed structural analysis and binding interfaces of various substrates, inhibitors and cofactor of these proteins were also investigated using docking and molecular dynamic simulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structure-based prediction of DNA-binding sites on proteins using the empirical preference of electrostatic potential and the shape of molecular surfaces

Protein-DNA interactions play an essential role in the genetic activities of life. Many structures of protein-DNA complexes are already known, but the common rules on how and where proteins bind to DNA have not emerged. Many attempts have been made to predict protein-DNA interactions using structural information, but the success rate is still about 80%. We analyzed 63 protein-DNA complexes by focusing our attention on the shape of the molecular surface of the protein and DNA, along with the electrostatic potential on the surface, and constructed a new statistical evaluation function to make predictions of DNA interaction sites on protein molecular surfaces. The shape of the molecular surface was described by a combination of local and global average curvature, which are intended to describe the small convex and concave and the large-scale concave curvatures of the protein surface preferentially appearing at DNA-binding sites. Using these structural features, along with the electrostatic potential obtained by solving the Poisson-Boltzmann equation numerically, we have developed prediction schemes with 86% and 96% accuracy for DNA-binding and non-DNA-binding proteins, respectively.     

Electrostatic potential at the retinal of three archaeal rhodopsins: implications for their different absorption spectra

The color tuning mechanism of the rhodopsin protein family has been in the focus of research for decades. However, the structural basis of the tuning mechanism in general and of the absorption shift between rhodopsins in particular remains under discussion. It is clear that a major determinant for spectral shifts between different rhodopsins are electrostatic interactions between the chromophore retinal and the protein. Based on the Poisson-Boltzmann equation, we computed and compared the electrostatic potential at the retinal of three archaeal rhodopsins: bacteriorhodopsin (BR), halorhodopsin (HR), and sensory rhodopsin II (SRII) for which high-resolution structures are available. These proteins are an excellent test case for understanding the spectral tuning of retinal. The absorption maxima of BR and HR are very similar, whereas the spectrum of SRII is considerably blue shifted--despite the structural similarity between these three proteins. In agreement with their absorption maxima, we find that the electrostatic potential is similar in BR and HR, whereas significant differences are seen for SRII. The decomposition of the electrostatic potential into contributions of individual residues, allowed us to identify seven residues that are responsible for the differences in electrostatic potential between the proteins. Three of these residues are located in the retinal binding pocket and have in fact been shown to account for part of the absorption shift between BR and SRII by mutational studies. One residue is located close to the beta-ionone ring of retinal and the remaining three residues are more than 8 A away from the retinal. These residues have not been discussed before, because they are, partly because of their location, no obvious candidates for the spectral shift among BR, HR, and SRII. However, their contribution to the differences in electrostatic potential is evident. The counterion of the Schiff base, which is frequently discussed to be involved in the spectral tuning, does not contribute to the dissimilarities between the electrostatic potentials.     

Identification of ubiquinone as the secondary electron acceptor in the photosynthetic apparatus of Chromatium vinosum

Extracting Chromatium vinosum chromatophores with light petroleum destroys their ability to perform photochemistry on the second of two closely-spaced actinic flashes, without affecting photochemistry on the first flash. Extraction also increases the likelihood of a back-reaction in which an electron returns from the primary electron acceptor directly to P₈₇₀. These effects probably reflect the removal of a secondary electron acceptor. Extraction does not appear to interfere with the primary photochemical reaction. Reconstituting the extracted chromatophores with the lipid extract or with pure ubiquinone (Q) completely reverses the effects of the extraction. Chromatography of the lipid extract shows that Q is the only active material that it contains in detectable quantity. These observations support the conclusion that Q is the secondary electron acceptor.

Piericidin A, certain alkyl-substituted quinolinequinones, and a substituted 4,7-dioxobenzothiazole inhibit electron transfer between the primary and secondary acceptors. The sensitivity to these inhibitors, and the participation of Q and non-heme iron suggest that the secondary electron-transfer reaction resembles the reactions catalyzed by respiratory dehydrogenases.

The proton uptake that follows flash excitation does not seem to be tightly linked to the reduction of the secondary electron acceptor. It still occurs (though with decreased amplitude) in extracted chromatophores, and even in the presence of inhibitors of the secondary electron-transfer reaction.     

Dependence of the membrane potential of Chara cells on external pH in the presence or absence of internal adenosinetriphosphate

The dependence of the membrane potential (E_m) and the membrane resistance (R_m) of Chara australis R. Brown on the pH of the external medium (pH₀) was studied by controlling the activity of the plasmamembrane H⁺ pump under both light and dark conditions. The activity of the pump was controlled by regulating the internal ATP or Mg²⁺ concentration in tonoplast-free cells prepared by vacuolar perfusion. In these cells, which contained Mg · ATP (mgATP cells), E_m and R_m were very sensitive to pH₀, as in normal cells. E_m was more negative in light than in the dark at all pH₀ values tested. Tonoplast-free cells with very low [ATP]_i (-ATP cells) or [Mg²⁺]_i (-Mg cells) showed very weak dependence of E_m and R_m on pH₀. Thus, the active and not the passive component of E_m was sensitive to pH₀. At the same time, the high permeability of the plasma membrane to H⁺ was questioned. In both-ATP cells and-Mg cells, E_m was scarcely affected and R_m markedly decreased on illumination.Abbreviations CyDTA 1,2-cyclohexanediamine-N,N-tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether)N,N-tetraacetic acid - HK hexokinase     

Analyzing heme proteins using EPR techniques: the heme-pocket structure of ferric mouse neuroglobin

In this work, an electron paramagnetic resonance (EPR) strategy to study the heme-pocket structure of low-spin ferric heme proteins is optimized. Frozen solutions of ferric mouse neuroglobin (mNgb) are analyzed by means of electron spin echo envelope modulation and pulsed electron–nuclear double resonance techniques. The hyperfine and nuclear quadrupole couplings of the directly coordinating heme and histidine nitrogens are derived and are discussed in comparison with known data of other ferric porphyrin compounds. In combination with the hyperfine matrices of the imidazole protons, the ¹⁴N EPR parameters reveal structural information on the heme pocket of mNgb that is in agreement with previous X-ray diffraction data on neuroglobins.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.This paper is dedicated to our coauthor Prof. Arthur Schweiger, who passed away unexpectedly on 4 January 2006.     

Influence of waste water from the paper industry and UV-B radiation on the photosynthetic efficiency of Euglena gracilis

The green flagellate Euglena gracilis has been used as a model organism to elucidate the possible large-scale and short-term effects of waste substances from the pulp and paper industry on photosynthetic efficiency (PE). Different concentrations of waste substances before and after treatment in a cleaning system were studied. The uncleaned sample at concentrations up to 1:10 and the cleaned sample at concentrations up to 1:5 showed stimulating effects on the PE after 7 days of incubation compared to the control. The effects of waste substances on the PE of E. gracilis were also studied in combination with short-term studies (20 and 40 min) of ultraviolet-B radiation (UV-B, 280–320 nm). It was shown that increasing concentrations of the uncleaned sample had continuously stimulating effects on the PE and worked protectively against UV-B radiation. The cleaned sample exhibited no effects, or negative effects, on the PE of E. gracilis together with UV-B radiation compared to the experiments with only UV-B radiation. At the concentration 1:1 of the cleaned sample an increase in the PE was detected due to the high concentration of the coloured substances and a decrease in the UV-B penetration. PE revealed itself to be highly sensitive for detecting toxic effects on E. gracilis and is thus very promising for use in regular toxicity tests of waste water from pulp and paper industry. This revised version was published online in July 2006 with corrections to the Cover Date.     

Redox potentials of the blue copper sites of bilirubin oxidases

The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was > 650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV.     

The role of the photosynthetic apparatus in cold acclimation of <Emphasis Type="Italic">Lolium multiflorum</Emphasis>. Characteristics of novel genotypes low-sensitive to PSII over-reduction

During cold acclimation by higher plants, temperature perception via changes in redox state of Photosystem II (PSII) and subsequent acclimation of the photosynthetic apparatus to cold is very important for achieving freezing tolerance. These properties were studied in two groups (A and B) of the same backcross 3 (BC₃) progeny derived from a triploid hybrid of Festuca pratensis (2×) × Lolium multiflorum (4×) backcrossed three times onto diploid L. multiflorum cultivars. Leaves of Group A plants formed at 20°C at medium-low light were unable to acclimate their photosynthetic apparatus to cold. Compared to Group B, the Group A plants were also more frost sensitive. This acclimation ability correlated with the freezing tolerance of the plants. However, leaves of the same Group A plants developed at 20°C, but under higher-light conditions had increased ability to acclimate their photosynthetic apparatus to cold. It was concluded that Group A plants may have impaired PSII temperature perception, and this then resulted in their poor capability to cold acclimate.     

Generation of semiquinone-[2Fe-2S]+ spin-coupled center at the Qo site of cytochrome bc1 in redox-poised,illuminated photosynthetic membranes from Rhodobacter capsulatus

One of the less understood parts of the catalytic cycle of cytochrome bc₁/b₆f complexes is the mechanism of electronic bifurcation occurring within the hydroquinone oxidation site (Q_o site). Several models describing this mechanism invoke a phenomenon of formation of an unstable semiquinone. Recent studies with isolated cytochrome bc₁ or b₆f revealed that a relatively stable semiquinone spin-coupled to the reduced Rieske cluster (SQ-FeS) is generated at the Q_o site during the oxidation of ubi- or plastohydroquinone analogs under conditions of continuous turnover. Here, we identified the EPR transition of SQ-FeS formed upon oxidation of ubihydroquinone in native photosynthetic membranes from purple bacterium Rhodobacter capsulatus. We observed a significant amount of SQ-FeS generated when the antimycin-inhibited enzyme experiences conditions of non-equilibrium caused by the continuous light activation of the reaction center. We also noted that SQ-FeS cannot be detected under equilibrium redox titrations in dark. The non-equilibrium redox titrations of SQ-FeS indicate that this center has a higher apparent redox midpoint potential when compared to the redox midpoint potential of the quinone pool. This suggests that SQ-FeS is stabilized, which corroborates a recently proposed mechanism in which the SQ-FeS state is metastable and functions to safely hold electrons at the local energy minimum during the oxidation of ubihydroquinone and limits superoxide formation. Our results open new possibilities to study the formation and properties of this state in cytochromes bc under close to physiological conditions in which non-equilibrium is attained by the light activation of bacterial reaction centers or photosystems.     

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~ 165 K) than that of the low spin atom (~ 207 K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17 meV) and higher (17-25 meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~ 6 meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q_A and Q_B) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.     

Isolation of a brain peptide identical to the intestinal PHI (peptide HI)

The isolation of a brain peptide identical to the intestinal peptide PHI (peptide HI) is described. The peptide was isolated from porcine brain extract using a chemical assay method based on its C-terminal isoleucine amide structure. The complete amino acid sequence of the peptide was found to be: His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser-Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence is identical to the intestinal peptide thus demonstrating PHI to be a brain-gut peptide. The role of PHI in the central nervous system as a neurotransmitter or neuromodulator is discussed.