The effect of iodide and chloride on transthyretin structure and stability

Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.     

Dimerization of the plant photoreceptor phototropin is probably mediated by the LOV1 domain

Phototropin is a membrane-bound UV-A/blue light photoreceptor of plants responsible for phototropism, chloroplast migration and stomatal opening. Characteristic are two LOV domains, each binding one flavin mononucleotide, in the N-terminal half and having a serine/threonine kinase domain in the C-terminal half of the molecule. We purified the N-terminal half of oat phototropin 1, containing LOV1 and LOV2 domains, as a soluble fusion protein with the calmodulin binding peptide (CBP) by expression in Escherichia coli. Gel chromatography showed that it was dimeric in solution. While the fusion protein CBP-LOV2 was exclusively monomeric in solution, the fusion protein CBP-LOV1 occurred as monomer and dimer. The proportion of dimer increased on prolonged incubation. We conclude that native phototropin is a dimer and that the LOV1 domain is probably responsible for dimerization.     

X-ray structures of the leucine-binding protein illustrate conformational changes and the basis of ligand specificity

The periplasmic leucine-binding protein is the primary receptor for the leucine transport system in Escherichia coli. We report here the structure of an open ligand-free form solved by molecular replacement and refined at 1.5-A resolution. In addition, two closed ligand-bound structures of the same protein are presented, a phenylalanine-bound form at 1.8 A and a leucine-bound structure at a nominal resolution of 2.4 A. These structures show the basis of this protein's ligand specificity, as well as illustrating the conformational changes that are associated with ligand binding. Comparison with earlier structures provides further information about solution conformations, as well as the different specificity of the closely related leucine/isoleucine/valine-binding protein.     

From gene expression analysis to tissue microarrays: a rational approach to identify therapeutic and diagnostic targets in lymphoid malignancies

Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy for which better treatment strategies are needed. To identify potential diagnostic and therapeutic targets, a signature consisting of MCL-associated genes was selected based on a comprehensive gene expression analysis of malignant and normal B cells. The corresponding protein epitope signature tags were identified and used to raise monospecific, polyclonal antibodies, which were subsequently analyzed on paraffin-embedded sections of malignant and normal tissue. In this study, we demonstrate that the initial selection strategy of MCL-associated genes successfully allows identification of protein antigens either uniquely expressed or overexpressed in MCL compared with normal lymphoid tissues. We propose that genome-based, affinity proteomics, using protein epitope signature tag-induced antibodies, is an efficient way to rapidly identify a number of disease-associated protein candidates of both previously known and unknown identities.     

Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase

Nitric -oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N₂O) (2NO + 2H⁺ + 2e⁻ →N₂O + H₂O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b₃, and a non-heme iron Fe_B. Here, we have studied the reaction between fully reduced NOR and NO using the “flow-flash” technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b₃ occurs with a time constant of ∼1 μs at 1.5 mm NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to Fe_B before ligation to heme b₃. Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s⁻¹ and 3 s⁻¹ at 1.5 mm NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s⁻¹ and 12 s⁻¹ at 75 μm NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.     

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos

The shear stress effect on directional expansion of pro embryogenic masses (PEMs) and suspensor cell development of somatic embryos of Norway spruce (Picea abies) at the proliferation stage was studied by a direct and quantitative image analysis system. The experimental system allowed for detailed observations of the effect of hydrodynamic shear stress in rotating and deforming liquid cultures of proliferating Norway spruce somatic embryos. Briefly, somatic embryos at an early development stage comprised only of clusters of meristematic cells without suspensor cells were fixed on an alginate film. The alginate film was affixed on the bottom of a flow cell and the somatic embryos were subjected to laminar flow through the chamber of the flow cell. Magnified images of the cell clusters were collected every 24 h. The image data was processed based on a normalized cross‐correlation method, capable of measuring morphological and size features of individual cell clusters in both temporal and spatial domains. No suspensor cells developed in the cell clusters under shear stress of 140 s^?1 for the duration of the experiments. Cell clusters in the control cultured in stationary liquid conditions developed suspensor cells after 5–9 days in culture. Furthermore, the radial growth of meristematic cell clusters was inhibited by shear rates of 86 and 140 s^?1, corresponding to shear stress of 0.086 and 0.14 N/m², compared to growth under stationary conditions. The shear rate showed a significant negative correlation to growth rate. Control group showed no preference for direction during growth under static conditions. Biotechnol. Bioeng. 2010; 105: 588–599. © 2009 Wiley Periodicals, Inc.     

Transmembrane topology of FRO2, a ferric chelate reductase from Arabidopsis thaliana

Iron uptake in Arabidopsis thaliana is mediated by ferric chelate reductase FRO2, a transmembrane protein belonging to the flavocytochrome b family. There is no high resolution structural information available for any member of this family. We have determined the transmembrane topology of FRO2 experimentally using the alkaline phosphatase fusion method. The resulting topology is different from that obtained by theoretical predictions and contains 8 transmembrane helices, 4 of which build up the highly conserved core of the protein. This core is present in the entire flavocytochrome b family. The large water soluble domain of FRO2, which contains NADPH, FAD and oxidoreductase sequence motifs, was located on the inside of the membrane.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at This work was supported by grants from the Swedish Research Council (VR) to S. Al-Karadaghi and Hans Hebert.     

Solution structure and novel insights into the determinants of the receptor specificity of human relaxin-3

Relaxin-3 is the most recently discovered member of the relaxin family of peptide hormones. In contrast to relaxin-1 and -2, whose main functions are associated with pregnancy, relaxin-3 is involved in neuropeptide signaling in the brain. Here, we report the solution structure of human relaxin-3, the first structure of a relaxin family member to be solved by NMR methods. Overall, relaxin-3 adopts an insulin-like fold, but the structure differs crucially from the crystal structure of human relaxin-2 near the B-chain terminus. In particular, the B-chain C terminus folds back, allowing Trp(B27) to interact with the hydrophobic core. This interaction partly blocks the conserved RXXXRXXI motif identified as a determinant for the interaction with the relaxin receptor LGR7 and may account for the lower affinity of relaxin-3 relative to relaxin for this receptor. This structural feature is likely important for the activation of its endogenous receptor, GPCR135.     

IGF-1R tyrosine kinase expression and dependency in clones of IGF-1R knockout cells (R-)

Insulin-like growth factor 1 receptor (IGF-1R) plays many crucial roles in cancer, like anti-apoptotic activity and necessity for transformation. IGF-1R knockout cells (R-) represent a useful tool for molecular mapping of biological properties of the receptor. R- cells have been shown to be refractory to transformation by viral and cellular oncogenes, highlighting the necessity of this receptor for transformation. Surprisingly, more recent studies have shown that these cells can undergo spontaneous transformation. This observation raises the question as whether R- cells over the years have acquired some properties mimicking those of IGF-1R. Using an IGF-1R inhibitor (cyclolignan PPP) we have identified clones of R- (R-s) that are sensitive to this compound. Since, PPP is closely related to podophyllotoxin, which is an efficient microtubule inhibitor, we first investigated if such a mechanism could explain the sensitivity to PPP. However, highly purified PPP showed no or very slight tubulin binding. Further analysis of R-s revealed expression of a 90 kDa protein being reactive to IGF-1R beta-subunit antibodies. This protein was weakly but constitutively tyrosine phosphorylated and was downregulated by siRNA targeting IGF-1R. This downregulation was paralleled by decreased R-s survival. Taken together, our study suggests that clones of R- express IGF-1R activity and dependency, which in turn may explain that R- can undergo spontaneous transformation.     

Thermofluor-based high-throughput stability optimization of proteins for structural studies

Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization. The method also allowed optimal stabilizing buffer compositions to be rapidly identified for each protein. Furthermore, the method allowed the identification of potential ligands, physiological or non-physiological, that can be used in subsequent crystallization trials. For this study, the thermally induced melting points were determined in different buffers as well as with additives for a total of 25 Escherichia coli proteins. Crystallization trials were set up together with stabilizing and destabilizing additives identified using thermofluor screening. A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives. This suggests that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.