Mending mutations by in vivo site-directed mutagenesis

In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotidesKren, B.T. et al. (1998)Nat. Med. 4, 285–290Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotideKren, B.T. et al. (1997)Hepatology 25, 1462–1468     

Attraction and the single cell

Selective expression of the eotaxin receptor CCR3 by human T helper 2 cellsSallusto, F. et al. (1997)Science 277, 2005–2007Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localising with eosinophilsGerber, B.O. et al. (1997)Curr. Biol. 7, 836–843Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2sBonecchi, R. et al. (1998)J. Exp. Med. 187, 129–134CCR5 is characteristic of Th1 lymphocytesLoetscher, M. et al. (1998)Nature 391, 344–345     

Winning the war against antibiotic resistance

Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-β-lactamaseToney, J.H. et al. (1998)Chem. Biol. 5, 185–196Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureusBalaban, N. et al. (1998)Science 280, 438–440     

Haemorrhagic shock: endogenous cannabinoids to the rescue

Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shockWagner, J.A. et al. (1997)Nature 390, 518–521     

Isolation of cytochrome P-448 from Saccharomyces cerevisiae H 1022 and characterization by different antibodies

Saccharomyces cerevisiae cells H 1022 grown in a continuous culture under glucose repression and O₂ limitation show a high content of cytochrome P-448. According to the fractionation on octyl-Sepharose and to the reaction with P-448 antibodies of S. cerevisiae and S. uvarum only a single protein band with the molecular weight of 58 kDa could be identified as cytochrome P-448. This is in agreement with the results of Aoyama et al. [(1984) J. Biol. Chem. 259, 1661–1666], but not with the result of King et al. [(1984) Xenobiotica 14, 187–206]. There is dissimilarity of the yeast cytochrome P-448 with that of mammalian P-448 by the antibody reaction despite a suggested similarity in the amino acid sequence [(1984) Xenobiotica 14, 187–206].     

Enhanced salt tolerance of transgenic tobacco plants by co-expression of PcINO1 and McIMT1 is accompanied by increased level of myo-inositol and methylated inositol

Introgression and functional expression of either the PcINO1 (l-myo-inositol 1-phosphate synthase or MIPS coding gene from the wild halophytic rice, Porteresia coarctata) or McIMTI (inositol methyl transferase, IMTI coding gene from common ice plant Mesembryanthemum crystallinum) has earlier been shown to confer salt tolerance to transgenic tobacco plants (Sheveleva et al., Plant Physiol 115:1211–1219, 1997; Majee et al., J Biol Chem 279:28539–28552, 2004). In this communication, we show that transgenic tobacco plants co-expressing PcINO1 and McIMT1 gene either in cytosol or in chloroplasts accumulate higher amount of total inositol (free and methyl inositol) compared to non-transgenic plants. These transgenic plants were more competent in terms of growth potential and photosynthetic activity and were less prone to oxidative stress under salt stress. A positive correlation between the elevated level of total inositol and methylated inositol and the capability of the double transgenic plants to withstand a higher degree of salt stress compared to the plants expressing either PcINO1 or McIMT1 alone is inferred.     

Structural Features of Glycera dibranchiata Monomer Hemoglobins. Primary Sequences of Monomer Hemoglobin Components II and III

Primary sequences for the remaining two members (GMH2, GMH3) of the group of three major monomeric hemoglobins from the marine annelid Glycera dibranchiata have been obtained. Full sequences of each 147-amino acid globin were achieved with a high degree of confidence using standard Edman technology in combination with molecular mass determinations of the intact globins and of the cyanogen bromide cleavage fragments using electrospray ionization mass spectrometry. When minor assumptions concerning Q/E identities are made these new results indicate the likely correspondence of GMG2 with the protein represented by the first Glycera dibranchiata monomer hemoglobin complete sequence [Imamura et al., (1972), J. Biol. Chem. 247, 2785–2797]. When these new sequences are combined with the previously determined primary sequence for the third major monomer hemoglobin, GMH4 [Alam et al., J. Protein Chem. (1994), 13, 151–164], it becomes clear that these three (GMG2–4) are truly distinct proteins, contrary to previous suggestions. Surprisingly, our results show that none of these three primary sequences is identical to the published sequence of the refined monomer hemoglobin crystal structure protein; however, there is a strong correspondence to the GMG2 sequence. The present sequencing results, in combination with the published GMH4 sequence, confirm the presence of a distal Leu in place of the more commonly encountered distal His in all three of the major monomer hemoglobins isolated in this laboratory and indicate that the unusual B10 Phe occurs only in GMH4. Analysis of the sequences presented here, along with comparison of amino acid content for Glycera dibranchiata monomer hemoglobins isolated from three different laboratories, and comparison of NMR results from two laboratories suggest further correspondences which unify disparate published isolations.     

ORIUM: Optimized RDC-based Iterative and Unified Model-free analysis

Residual dipolar couplings (RDCs) are NMR parameters that provide both structural and dynamic information concerning inter-nuclear vectors, such as N–H^N and Cα–Hα bonds within the protein backbone. Two approaches for extracting this information from RDCs are the model free analysis (MFA) (Meiler et al. in J Am Chem Soc 123:6098–6107, 2001; Peti et al. in J Am Chem Soc 124:5822–5833, 2002) and the direct interpretation of dipolar couplings (DIDCs) (Tolman in J Am Chem Soc 124:12020–12030, 2002). Both methods have been incorporated into iterative schemes, namely the self-consistent RDC based MFA (SCRM) (Lakomek et al. in J Biomol NMR 41:139–155, 2008) and iterative DIDC (Yao et al. in J Phys Chem B 112:6045–6056, 2008), with the goal of removing the influence of structural noise in the MFA and DIDC formulations. Here, we report a new iterative procedure entitled Optimized RDC-based Iterative and Unified Model-free analysis (ORIUM). ORIUM unifies theoretical concepts developed in the MFA, SCRM, and DIDC methods to construct a computationally less demanding approach to determine these structural and dynamic parameters. In all schemes, dynamic averaging reduces the actual magnitude of the alignment tensors complicating the determination of the absolute values for the generalized order parameters. To readdress this scaling issue that has been previously investigated (Lakomek et al. in J Biomol NMR 41:139–155, 2008; Salmon et al. in Angew Chem Int Edit 48:4154–4157, 2009), a new method is presented using only RDC data to establish a lower bound on protein motion, bypassing the requirement of Lipari–Szabo order parameters. ORIUM and the new scaling procedure are applied to the proteins ubiquitin and the third immunoglobulin domain of protein G (GB3). Our results indicate good agreement with the SCRM and iterative DIDC approaches and signify the general applicability of ORIUM and the proposed scaling for the extraction of inter-nuclear vector structural and dynamic content.     

Iodination of p-nitrophenyl ethers used as photolabels

A system for radioactive labeling of compounds of biological interest that, due to their low electronic density, cannot be labeled by the standard iodination techniques is described. Using p-nitroanisole as a model, we have prepared 2-[¹²⁵I]iodo-4-nitroanisole by treatment with thallium trifluoroacetate, with later displacement of the thallium by iodide according to A. McKillop et al. (J. Amer. Chem. Soc.93, 4841–4844 (1970)). The labeled iodonitroanisole has been used as a photoactive reagent to label a protein (bovine serum albumin), showing that under the irradiation conditions used, the label is incorporated into the polypeptide mainly through modification of ?-amino groups of the lysine residues.     

Molecular Dynamics Simulations of β-turn Forming Tetra- and Hexapeptides

Abstract

It was previously shown that the structural ensemble of model peptides DDKG and GKDG (H. Ishii et al. Biopolymers 24, 2045–2056, 1985), DEKS (A. Otter et al. J. Biomol. Struct. Dyn. 7, 455–476, 1989) NPGQ (F. R. Carbone et al. Int. J. Pept. Protein. Res. 26, 498–508, 1985), SALN (H. Santa et al. J. Biomol. Struct. Dyn. 16, 1033–1041, 1999), SYPFDV and SYPYDV (J. Yao et al. J. Mol. Biol. 243, 736–753, 1994), VP^DAH and VP^DSH (B. Imperiali et al. J. Am. Chem. Soc. 114, 3182–3188, 1992) in solution contains a significant—or in some cases dominant—proportion of β-turn conformation. In this study, a protein database was searched for the above, unprotected sequences which incorporate only L-amino acid residues. Simulated annealing and 25 ns MD simulations of structures were also performed. The DSSP and STRIDE secondary structure-assigning algorithms and clustering were used to analyze trajectories and i, i+3 hydrogen bonds were also sought. The DSSP analysis showed a fluctuation between β-turn and random meander structure, although bend structures were not detected because of the insufficient length of peptide chains. This alternating trend was confirmed when the STRIDE algorithm was used to analyze trajectories, but STRIDE assigned more turn structures. The population of the strongest clusters was above 40% and the middle structures adopted β-turn structure for most sequences. These results are in good agreement with previous experimental results and support the idea of the ultra-marginal stability of turns in the absence of stabilizing long-range interactions of the neighboring segments of a polypeptide chain. However, interactions between the side-chains in tetrapeptides could also contribute to turn stability and result in unusual stability in some cases. Our observations suggest that such interactions are the consequence rather than the driving force of turn formation.     

Synthesis and biological evaluation of imidazole derivatives as novel NOP/ORL1 receptor antagonists: Exploration and optimization of alternative pyrazole structure

Nonpeptidic small-molecule NOP/ORL1 receptor antagonists with an imidazole scaffold were designed and synthesized to investigate alternatives to the pyrazole analog. Systematic modification of the original pyrazole lead [Kobayashi et al., Bioorg. Med. Chem. Lett. 2009, 19, 3627; Kobayashi et al., Bioorg. Med. Chem. Lett., in press] to change the heterocyclic core, substituted side chain, and pendant functional group demonstrated that examining the structure–activity relationship for novel templates allowed the identification of potent, fully substituted 4-aminomethyl-1H-imidazole and 2-aminomethyl-1H-imidazole. These compounds exhibited excellent potency for ORL1 receptor with minimal P-gp efflux and/or reduced hERG affinity.     

Co2+ binding to α-lactalbumin

α-Lactalbumin possesses multiple Zn²⁺ binding sites, with the strongest site having an affinity constant of 5×10⁵ M^?1 [Permyakovet al. (1991),J. Protein Chem. 100, 577]. The binding of zinc at secondary sites is accompanied by destabilization of the protein structure and progressive protein aggregation. This pronounced destabilization is reflected in a shift of the thermal denaturation transition temperature by more than 40°. The present work examines Co²⁺ binding to bovineα-lactalbumin, where for this analog of Zn²⁺, multiple binding sites were also found from spectrofluorimetric titrations. The strong site Co²⁺ binding constant was 1.3×10⁶ M^?1. However, in contrast to Zn²⁺ binding, Co²⁺ does not cause protein aggregation nor any significant thermal destabilization of the protein. Fluroescence energy transfer measurements between Tb³⁺ in the strong calcium site to Co²⁺ in the strong Zn²⁺ site gave a distance in the range of 14–18 Å, which was in excellent agreement with recent crystallographic data for humanα-lactalbumin [Renet al. (1993), J. Biol. Chem.268, 19292–19298] However, the X-ray structure did not identify the additional zinc sites found from earlier solution studies, presumably due to restrictive crystal packing interactions. The results from the current work confirm that the strong cobalt (zinc) site in solution is the same zinc site elucidated by X-ray crystallography.     

Absorption spectra of chlorophyll a and b in Lhcb protein environment

The spectral forms of the two chlorophyll species in higher plant Photosystem II antenna proteins have been experimentally determined within their protein environment. Recombinant CP29 and LHC II antenna proteins missing individual chromophores were obtained by over-expression in bacteria without any changing of the primary protein sequence and in vitro reconstitution. Difference absorption spectroscopy with respect to the corresponding proteins binding the complete pigment complement yielded the spectral shape and extinction of single chlorophyll a and b. A functional relation of their absorption was given by Gaussian subband decomposition covering the entire Q_x and Q_y optical region together with the absolute value of the molar extinction coefficient. With respect to analogous determinations reported in the literature for organic solvents, this information is valuable for further understanding the in-protein chlorophyll excited states and excited state dynamics: in particular, for the calculation of Förster transfer rates by means of chlorophyll–chlorophyll overlap integral employing the Stepanov relation for emission and single chromophore transition energies according to the results of mutational analysis of chlorophyll binding sites [Bassi et al. (1999) Proc Natl Acad Sci USA 96: 10056–10061; Remelli et al. (1999) J Biol Chem 274: 33510–33521].     

Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant

The green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has become a versatile reporter for monitoring gene expression and protein localization in a variety of cells and organisms. GFP emits bright green light (λ_max = 510 nm) when excited with ultraviolet (UV) or blue light (λ_max = 395 nm, minor peak at 470 nm). The chromophore in GFP is intrinsic to the primary structure of the protein, and fluorescence from GFP does not require additional gene products, substrates or other factors. GFP fluorescence is stable, species-independent and can be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry [Chalfie et al., Science 263 (1994) 802–805; Stearns, Curr. Biol. 5 (1995) 262–264]. The protein appears to undergo an autocatalytic reaction to create the fluorophore [Heim et al., Proc. Natl. Acad. Sci. USA 91 (1994) 12501–12504] in a process involving cyclization of a Tyr⁶⁶ aa residue. Recently [Delagrave et al., Bio/Technology 13 (1995) 151–154], a combinatorial mutagenic strategy was targeted at aa 64 through 69, which spans the chromophore of A. victoria GFP, yielding a number of different mutants with redshifted fluorescence excitation spectra. One of these, RSGFP4, retains the characteristic green emission spectra (λ_max = 505 nm), but has a single excitation peak (λ_max = 490 nm). The fluorescence properties of RSGFP4 are similar to those of another naturally occurring GFP from the sea pansy, Renilla reniformis [Ward and Cormier, Photobiochem. Photobiol. 27 (1978) 389–396]. In the present study, we demonstrate by fluorescence microscopy that selective excitation of A. victoria GFP and RSGFP4 allows for spectral separation of each fluorescent signal, and provides the means to image these signals independently in a mixed population of bacteria or mammalian cells.     

Reductive activation in periplasmic nitrate reductase involves chemical modifications of the Mo-cofactor beyond the first coordination sphere of the metal ion

In Rhodobacter sphaeroides periplasmic nitrate reductase NapAB, the major Mo(V) form (the “high g” species) in air-purified samples is inactive and requires reduction to irreversibly convert into a catalytically competent form (Fourmond et al., J. Phys. Chem., 2008). In the present work, we study the kinetics of the activation process by combining EPR spectroscopy and direct electrochemistry. Upon reduction, the Mo (V) “high g” resting EPR signal slowly decays while the other redox centers of the protein are rapidly reduced, which we interpret as a slow and gated (or coupled) intramolecular electron transfer between the [4Fe–4S] center and the Mo cofactor in the inactive enzyme. Besides, we detect spin–spin interactions between the Mo(V) ion and the [4Fe–4S]^1 + cluster which are modified upon activation of the enzyme, while the EPR signatures associated to the Mo cofactor remain almost unchanged. This shows that the activation process, which modifies the exchange coupling pathway between the Mo and the [4Fe–4S]^1 + centers, occurs further away than in the first coordination sphere of the Mo ion. Relying on structural data and studies on Mo-pyranopterin and models, we propose a molecular mechanism of activation which involves the pyranopterin moiety of the molybdenum cofactor that is proximal to the [4Fe–4S] cluster. The mechanism implies both the cyclization of the pyran ring and the reduction of the oxidized pterin to give the competent tricyclic tetrahydropyranopterin form.     

Cytochrome c and superoxide

Wegerich et al. (J. Biol. Inorg. Chem. 18:429–440, 2013), working with singly modified human cytochromes c, claim to have found a new mechanism for the reduction of iron(III) cytochrome c by superoxide. I show that electron transfer by way of the solvent-accessible haem edge—a mechanism not considered by Wegerich et al.—is still the correct mechanism. Furthermore, several deficiencies in this work preclude any comparisons with other publications on this topic.     

Chemical shift assignments of Ribosomal protein S1 from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

RpsA, also known as ribosomal protein S1, is an essential protein required for translation initiation of mRNAs when their Shine-Dalgarno sequence is degenerated (Sorensen et al. 1998). In addition, RpsA of Mycobacterium tuberculosis (M. tb) is involved in trans-translation, which is an effective system mediated by tmRNA-SmpB to release stalled ribosomes from mRNA in the presence of rare codons (Keiler 2008). Shi et al. found that POA binds to RpsA of Mtb and disrupts the formation of RpsA–tmRNA complex (Shi et al. 2011) and mutations at the C-terminus of RpsA confer PZA resistance. The previous work reported the pyrazinoic acid-binding domain of RpsA (Yang et al. Mol Microbiol 95:791–803, 2015). However, the HSQC spectra of the isolated S1 domain does not overlap with that of MtRpsA280-438, suggesting that substantial interactions occur between the flexible C-terminus and the S1 domain in MtRpsA .To further study the PZA resistance and how substantial interactions influence/affect protein structure, using heteronuclear NMR spectroscopy, we have completed backbone and side-chain ¹H, ¹⁵N, ¹³C chemical shift assignments of MtRpsA280-438 which contains S1 domain and the flexible C-terminus. These NMR resonance assignments provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.