Cysteine ligand vibrations are responsible for the complex resonance Raman spectrum of azurin

 In the redox center of azurin, the Cu(II) is strongly coordinated to one thiolate S from Cys 112 and two imidazole Ns from His 46 and 117. This site yields a complex resonance Raman (RR) spectrum with >20 vibrational modes between 200 and 1500 cm^–1. We have investigated the effects of ligand-selective isotope replacements on the RR spectrum of Pseudomonas aeruginosa azurin to determine the relative spectral contribution from each of the copper ligands. Growth on ³⁴S-sulfate labels the cysteine ligand and allows the identification of a cluster of bands with Cu–S(Cys) stretching character between 370 and 430 cm^–1 whose frequencies are consistent with the trigonal or distorted tetrahedral coordination in type 1 sites. In type 2 copper-cysteinate sites, the lower ν (Cu–S) frequencies between 260 and 320 cm^–1 are consistent with square-planar coordination. Addition of exogenous ¹⁵N-labeled imidazole or histidine to the His117Gly mutant generates type 1 or type 2 sites, respectively. Because neither the above nor the His46Gly mutant reconstituted with ¹⁵N-imidazole exhibits significant isotope dependence, the histidine ligands can be ruled out as important contributors to the RR spectrum. Instead, a variety of evidence, including extensive isotope shifts upon global substitution with ¹⁵N, suggests that the multiple RR modes of azurin are due principally to vibrations of the cysteine ligand. These are resonance-enhanced through kinematic coupling with the Cu–S stretch in the ground state or through an excited-state A-term mechanism involving a Cu-cysteinate chromophore that extends into the peptide backbone. Received: 29 July 1996 / Accepted: 9 November 1996     

Solution NMR views of dynamical ordering of biomacromolecules

Background

To understand the mechanisms related to the ‘dynamical ordering’ of macromolecules and biological systems, it is crucial to monitor, in detail, molecular interactions and their dynamics across multiple timescales. Solution nuclear magnetic resonance (NMR) spectroscopy is an ideal tool that can investigate biophysical events at the atomic level, in near-physiological buffer solutions, or even inside cells.

Surface Plasmon Resonance kinetic analysis of the interaction between G-quadruplex nucleic acids and an anti-G-quadruplex monoclonal antibody

Background

G-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules. We here aimed at improving the orientation of an anti-G4 antibody on the SPR sensor chip to optimize detection of binding antigens.

Citrinin detection by intensified fluorescence signal of a FRET-based immunosensor using magnetic/silica core–shell

The specific immune-reaction between the anti-citrinin antibody immobilized on the surface of magnetic/silica core–shell (MSCS) and the citrinin–Rho123–BSA conjugate brings the Rho123 fluorophore as an acceptor and the QDs as a donor in close spatial proximity and causes FRET for occurring upon photo-excitation of the QDs. The novelties of this study include: (1) immobilization of the MSCS; (2) large amount of the immobilized QDs, and (3) immobilization of a large amount of Rho123 on the BSA macromolecule. Cd/Te QDs were synthesized by the simultaneous reduction of cadmium chloride and tellurium in the presence of sodium borohydride. Magnetic nanoparticles were synthesized using FeSO₄ and FeCl₃. The prepared magnetic nanoparticles shelled by silica using tetraethoxysilane in the presence of ammonia. Transmission electron microscopy (TEM) analysis was used for investigating shape and monodispersity of the nanoparticles. EDC/NHS was used as a cross linking agent for immobilization of the QDs, conjugation of citrinin to amino groups of BSA, labeling of BSA with Rho123 and also for immobilization of the amino-functionalized MSCS on the immobilized QDs. Immobilization of the anti-citrinin antibody on the surface of the amino-functionalized MSCS was performed by Schiff-base mechanism. By using these three effective strategies, sensitivity of the designed nanobiosensor was incredibly enhanced as a very low limit of detection (up to 0.1 pM). The feasibility of this technique was tested by the detection of citrinin in the spiked human serum. Results showed that there was a linear correlation between the decreased fluorescence intensity of the Rho123 and increased fluorescence intensity of the QDs with increasing concentration of citrinin in the spiked samples in the range of 1–6 pM. According to obtained results, we conclude that this highly sensitive detection scheme is a easy, quick and impressive method that can be used in optical-based nanosensors.     

Resonance Raman analysis of a fluorescently labeled oligonucleotide forming a very stable hairpin

An oligodeoxynucleotide has been synthesized, which mimics an ``antigene' oligonucleotide with a polypyrimidic stretch on its 5′ side and is protected on its 3′ side against nucelases by a naturally forming and very stable hairpin, 5′GCGAAGC3′. The in vitro degradation of the resulting oligonucleotide d(5′TTCTCGCGAAGC3′) has already been studied by fluorescence resonance energy transfer (FRET) (Réfrégiers et al. 1996, J Biomol Struct Dyn 14: 365 – 371). The technique required the grafting of fluorophores at both ends of the oligonucleotide. In the present work we have compared the hairpin formed in the presence and in the absence of such fluorophores. This was achieved by the study of the Raman spectra (excitation at 257 nm) of the oligodeoxynucleotides H, which forms the hairpin (5′TTCTCGCGAAGC3′), and a con-trol C (5′TTCTCCGGAAGC3′) which is unable to form the hairpin. Resonance Raman spectroscopy with 257 nm excitation greatly favors the resonance of purines and therefore the study of the 3′ part of the oligonucleotides. The difference spectrum obtained from resonance Raman spectra of C and H showed marker peaks specific for hairpin formation. The search for these marker peaks in difference spectra involving the Raman spectrum of H labeled by fluorophores and either C or H proved that the fluorophores do not modify the structure of the hairpin but only the vibrations of the two terminal bases on which the fluorophores are grafted. The use of such labeling is then justified in order to allow oligonucleotides protected by a hairpin on their 3′ side to be studied by fluorescence spectroscopy. Received: 13 December 1996 / Accepted: 7 April 1997     

Probing the role of Valine 185 of the D1 protein in the Photosystem II oxygen evolution

In Photosystem II (PSII), the Mn₄CaO₅-cluster of the active site advances through five sequential oxidation states (S₀ to S₄) before water is oxidized and O₂ is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824–6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV–visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking Tyr_Z and the halide. The V185 contributes to the stabilization of S₂ into the low spin (LS), S?=?1/2, configuration. Indeed, in the V185T mutant a high proportion of S₂ exhibits a high spin (HS), S?=?5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S₁Tyr_Z?→?S₂^HSTyr_Z transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S₂^LSTyr_Z?→?S₃Tyr_Z transition was observed and we propose that it occurs in the S₂^LSTyr_Z?→?S₂^HSTyr_Z intermediate step before the S₂^HSTyr_Z?→?S₃Tyr_Z transition occurs. The dramatic slowdown of the S₃Tyr_Z?→?S₀Tyr_Z transition in the V185T mutant does not originate from a structural modification of the Mn₄CaO₅ cluster since the spin S?=?3?S₃ EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.     

Discovery of a novel covalent non-β-lactam inhibitor of the metallo-β-lactamase NDM-1

The inhibition of metallo-β-lactamases (MBL) can prevent the hydrolysis of β-lactam antibiotics and hence is a promising strategy for the treatment of antibiotic resistant infections. In this study, we present a novel reversible covalent inhibitor of the clinically relevant MBL New Delhi metallo-β-lactamase 1 (NDM-1). Electrospray ionization-mass spectrometry (ESI-MS) and single site directed mutagenesis were used to show that the inhibitor forms a covalent bond with Lys224 in the active site of NDM-1. The inhibitor was further characterized using an enzyme inhibition assay, a surface plasmon resonance (SPR) based biosensor assay and covalent docking. The determined inhibition constant (K_I¹) was 580 nM and the inhibition constant for the initial complex (K_I) was 76 μM. To our knowledge, this inhibitor is the first example for a reversible covalent non-β-lactam inhibitor targeting NDM-1 and a promising starting point for the design of potent covalent inhibitors.     

Variable forms of soluble guanylyl cyclase: protein-ligand interactions and the issue of activation by carbon monoxide

1 , the resting Fe(II) state is mainly 6-coordinate and low-spin, and the CO adduct has vibrational frequencies characteristic of a histidine-heme-CO complex in a hydrophobic environment. In contrast, the protein sGC₂ is 5-coordinate, high-spin in the resting state, and the CO adduct has perturbed vibrational frequencies indicative of a negatively polarizing residue in the binding pocket. The differences may result from the need to reconstitute sGC₁ or different isolation procedures for sGC₁ versus sGC₂. However, both sGC₁ and sGC₂ are activated by the same mechanism, namely displacement of the proximal histidine ligand upon NO binding, and neither one is activated by CO. If CO is an activator in vivo, some additional molecular component is required. Received: 11 February 1999 / Accepted: 17 September 1999     

磁共振水成像对脊柱疾病诊断中的应用价值

目的:探讨磁共振水成像技术对脊柱疾病诊断的应用价值。方法:采用快速高级自旋回波(FASE)、重T2WI及脂肪抑制序列对300例病人检查行磁共振椎管水成像(MRmyelography,MRM)。结果:MRM显示正常25例,MRM异常275例,清楚显示原发病变与邻近脊髓腔、脊髓、神经根的相关关系。结论:MRM具有无创伤、无辐射、速度快,不需对比剂,患者易接受的特点。MRM与常规MRI图像结合可获得全面、客观的病变信息,MRM图像可取代X线脊髓造影和CT脊髓造影。     

Nickel(II)-substituted azurin I from Alcaligenes xylosoxidans as characterized by resonance Raman spectroscopy at cryogenic temperature

Metal-substituted blue copper proteins (cupredoxins) have been successfully used to study the effect of metal-ion identity on their active-site properties, specifically the coordination geometry and metal–ligand bond strengths. In this work, low-temperature (77 K) resonance Raman (RR) spectra of the blue copper protein Alcaligenes xylosoxidans azurin I and its Ni(II) derivative are reported. A detailed analysis of all observed bands is presented and responsiveness to metal substitution is discussed in terms of structural and bonding changes. The native cupric site exhibits a RR spectrum characteristic of a primarily trigonal planar (type 1) coordination geometry, identified by the ν(Cu–S)_Cys markers at 373, 399, 409, and 430 cm⁻¹. Replacement of Cu(II) with Ni(II) results in optical and RR spectra that reveal (1) a large hypsochromic shift in the main (Cys)S → M(II) charge-transfer absorption from 622 to 440 nm, (2) greatly reduced metal–thiolate bonding interaction, indicated by substantially lower ν(Ni–S)_Cys stretching frequencies, (3) elevation of the cysteine ν(C _β –S) stretching, amide III, and ρ _s(C _β H₂) scissors vibrational modes, and (4) primarily four-coordinated, trigonally distorted tetrahedral geometry of the Ni(II) site that is marked by characteristic ν(Ni–S)_Cys stretching RR bands at 347, 364, and 391 cm⁻¹. Comparisons of the electronic and vibrational properties between A. xylosoxidans azurin I and its closely structurally related azurin from Pseudomonas aeruginosa are made and discussed. For cupric azurins, the intensity-weighted average M(II)–S(Cys) stretching frequencies are calculated to be ν(Cu–S)_iwa = 406.3 and 407.6 cm⁻¹, respectively. These values decreased to ν(Ni–S)_iwa = 359.3 and 365.5 cm⁻¹, respectively, after Ni(II) → Cu(II) exchange, suggesting that the metal–thiolate interactions are similar in the two native proteins but are much less alike in their Ni(II)-substituted forms.