Tyrosine triad at the interface between the Rieske iron–sulfur protein,cytochrome c1 and cytochrome c2 in the bc1 complex of Rhodobacter capsulatus

A triad of tyrosine residues (Y152–154) in the cytochrome c₁ subunit (C1) of the Rhodobacter capsulatus cytochrome bc₁ complex (BC1) is ideally positioned to interact with cytochrome c₂ (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations—A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117 mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation.     

Reductive activation of the heme iron–nitrosyl intermediate in the reaction mechanism of cytochrome c nitrite reductase: a theoretical study

Cytochrome c nitrite reductase catalyzes the six-electron, seven-proton reduction of nitrite to ammonia without release of any detectable reaction intermediate. This implies a unique flexibility of the active site combined with a finely tuned proton and electron delivery system. In the present work, we employed density functional theory to study the recharging of the active site with protons and electrons through the series of reaction intermediates based on nitrogen monoxide [Fe(II)-NO(+), Fe(II)-NO·, Fe(II)-NO(-), and Fe(II)-HNO]. The activation barriers for the various proton and electron transfer steps were estimated in the framework of Marcus theory. Using the barriers obtained, we simulated the kinetics of the reduction process. We found that the complex recharging process can be accomplished in two possible ways: either through two consecutive proton-coupled electron transfers (PCETs) or in the form of three consecutive elementary steps involving reduction, PCET, and protonation. Kinetic simulations revealed the recharging through two PCETs to be a means of overcoming the predicted deep energetic minimum that is calculated to occur at the stage of the Fe(II)-NO· intermediate. The radical transfer role for the active-site Tyr(218), as proposed in the literature, cannot be confirmed on the basis of our calculations. The role of the highly conserved calcium located in the direct proximity of the active site in proton delivery has also been studied. It was found to play an important role in the substrate conversion through the facilitation of the proton transfer steps.     

Light-induced absorption spectra of the D1–D2–cytochrome b 559 complex of Photosystem II: Effect of methyl viologen concentration

The light-induced difference absorption spectra associated to the photo-accumulation of reduced pheophytin a were studied in the isolated D1–D2–Cyt b559 complex in the presence of variable methyl viologen concentrations and different illumination conditions under anaerobiosis. Depending on the methyl viologen/reaction centre ratio, the relative intensities of the spectral bands at 681.5±0.5, 667.0±0.5 and 542.5±0.5 nm were modified. The reduced pheophytin a located at the D1-branch of the complex absorbs at 681.7±0.5 nm, and at least two additional pigment species contribute to the Q_y band of the difference absorption spectra with maxima at 667.0±0.5 and 680.5±0.5 nm. We propose the additional species correspond to a peripheral chlorophyll a and the pheophytin a located at the D2-branch of the complex, respectively. The blue absorbing chlorophyll at 667 nm is susceptible to chemical redox changes with a midpoint reduction potential of +470 mV. The Q_x absorption bands of both pheophytins localised at the D2- and D1-branch of the D1–D2–Cyt b559 complex were at 540.7±0.5 and 542.9±0.5, respectively. The results indicated that the two pheophytin molecules can be photoreduced in the D1–D2–Cyt b559 complex in certain experimental conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

The EPR spectrum of cytochrome b-563 in the cytocrhome bf complex from spinach: (1993) FEBS Letters 164, 71–74

Blood group H antigen with globo-series structure, reacting with the monoclonal antibody MBrl, was isolated and characterized from human blood group O erythrocytes. The structure was identified by methylation analysis, direct probe mass spectrometry, and ¹H-nuclear magnetic resonance spectroscopy as shown below: Fucαl → 2Galβl → 3GalNAcβl → 3Galαl → 4Galβl → 4Glcβl → 1Cer     

Design of novel analogue peptides with potent antibiotic activity based on the antimicrobial peptide,HP (2–20), derived from N-terminus of Helicobacter pylori ribosomal protein L1

HP (2–20) (AKKVFKRLEKLFSKIQNDK) is the antimicrobial sequence derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RPL1). In order to develop novel antibiotic peptides useful as therapeutic agents, potent antibiotic activities against bacteria, fungi and cancer cells without a cytotoxic effect are essential. To this end, several analogues with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, the substitution of Trp for the hydrophobic amino acids, Gln and Asp at positions 17 and 19 of HP (2–20) (Anal 3), caused a dramatic increase in antibiotic activity without a hemolytic effect.In contrast, the decrease of hydrophobicity brought about by substituting Ser for Leu and Phe at positions 12 and 19 of HP (2–20), respectively (Anal 4, Anal 5), did not have a significant effect on the antibiotic activity. The antibiotic effects of these synthetic peptides were further investigated by treating prepared protoplasts of Candida albicans and conducting an artificial liposomal vesicle (PC/PS; 3:1, w/w) disrupting activity test. The results demonstrated that the Anal 3 prevented the regeneration of fungal cell walls and induced an enhanced release of fluorescent dye (carboxyfluorescein) trapped in the artificial membrane vesicles to a greater degree than HP (2–20).The potassium-release test conducted on C. albicans indicated that Anal 3 induced greater amounts of potassium ion to be released than the parent peptide, HP (2–20) did. These results indicated that the hydrophobic region of peptides is prerequisite for its effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.     

3D NMR structure of a complex between the amyloid beta peptide (1–40) and the polyphenol ε-viniferin glucoside: Implications in Alzheimer's disease

Background

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. There is a consensus that Aβ is a pathologic agent and that its toxic effects, which are at present incompletely understood, may occur through several potential mechanisms. Polyphenols are known to have wide-ranging properties with regard to health and for helping to prevent various diseases like neurodegenerative disorders. Thus inhibiting the formation of toxic Aβ assemblies is a reasonable hypothesis to prevent and perhaps treat AD

Methods

Solution NMR and molecular modeling were used to obtain more information about the interaction between the Aβ_1–40 and the polyphenol ε-viniferin glucoside (EVG) and particularly the Aβ residues involved in the complex.

Results

The study demonstrates the formation of a complex between two EVG molecules and Aβ_1–40 in peptide characteristic regions that could be in agreement with the inhibition of aggregation. Indeed, in previous studies, we reported that EVG strongly inhibited in vitro the fibril formation of the full length peptides Aβ_1–40 and Aβ_1–42, and had a strong protective effect against PC12 cell death induced by these peptides.

Conclusion

For the full length peptide Aβ_1–40, the binding sites observed could explain the EVG inhibitory effect on fibrillization and thus prevent amyloidogenic neurotoxicity.

General significance

Even though this interaction might be important at the biological level to explain the protective effect of polyphenols in neurodegenerative diseases, caution is required when extrapolating this in vitro model to human physiology.     

Impact of polyphenols on receptor–ligand interactions by NMR: the case of neurotensin (NT)–neurotensin receptor fragment (NTS1) complex

Abstract

Ligand–receptor interactions can be implicated in many pathological events such as chronic neurodegenerative diseases. Thus, the discovery of molecules disrupting this type of interactions could be an interesting therapeutic approach. Polyphenols are well known for their affinity for proteins and several studies have characterized these direct interactions. But studying the direct influence of multi-therapeutic drugs on a ligand–receptor complex relevant to a neurodegenerative disorder is a challenging issue. Solution NMR, molecular modeling and iterative calculations were used to obtain information about the interaction between a phenolic compound, α-glucogallin (α-2) and a ligand/fragment receptor complex neurotensin (NT) and its receptor NTS1. The α-2 was shown to bind to NT and a peptidic fragment of its NTS1 receptor, independently. Although the formation of the corresponding ligand–receptor complex did not seem to be affected, this experimental modeling protocol will enable the evaluation of other anti-amyloidogenic compounds such as blockers of NT–NTS1 binding. These types of studies help in understanding the specificity and influence in binding and can provide information to develop new molecules with a putative pharmacological interest.

Communicated by Ramaswamy H. Sarma     

Structural peculiarities of the (MHF1–MHF2)4 octamer provide a long DNA binding patch to anchor the MHF–FANCM complex to chromatin: A solution SAXS study

MHF1 and MHF2 are histone-fold-containing FANCM-associated proteins. FANCM is a Fanconi anemia (FA) complementation group protein. We previously obtained high-resolution structures of MHF1–MHF2 (MHF) and MHF in complex with a fragment of FANCM (MHF–FANCM-F). Here, we use small angle X-ray scattering (SAXS) to investigate the solution behaviors of these protein complexes. In combination with crystallographic data, the results of the SAXS study reveal that a long, positively charged patch exposed on the surface of the MHF complex plays a critical role in double-stranded DNA (dsDNA) binding.     

The solution structure of rat Aβ-(1–28) and its interaction with zinc ion: insights into the scarcity of amyloid deposition in aged rat brain

The amyloid -peptide (A) is a major component of insoluble amyloid deposits in Alzheimers disease, and the ability of the -peptide to exist in different conformations is dependent on residues 1–28 [-(1–28)]. However, different from humans, no A amyloid deposition has been found in aged rats brains. Studying the three-dimensional solution structure of rat A-(1–28) and the binding circumstance of Zn²⁺ is beneficial to a clear understanding of the potential role of Zn²⁺ in Alzheimer-associated neuropathogenesis and to suggest why there is no amyloid deposition in aged rats brains. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of rat A-(1–28) and the binding constant of Zn²⁺ to rat A-(1–28). Our results suggest that (1) the three-dimensional solution structure of rat A-(1–28) is more stable than that of human A-(1–28) in DMSO-d₆ and that a helical region from Glu16 to Val24 exists in the rat A-(1–28); (2) the affinity of Zn²⁺ for rat A-(1–28) is lower than that for human A-(1–28) and the NMR data suggest that Arg13, His6, and His14 residues provide the primary binding sites for Zn²⁺; and (3) the proper binding of Zn²⁺ to rat A-(1–28) can induce the peptide to change to a more stable conformation.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0556-xAbbreviations A amyloid -peptide - AD Alzheimers disease - hA-(1–28) human A-(1–28) - rA-(1-28) rat A-(1–28) - REM restrained energy minimization