Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca(2+)-binding protein of the βγ-crystallin superfamily from Methanosarcina acetivorans

βγ-Crystallins are a large superfamily of proteins found in vertebrate eye lens. They are hetero-dimers (linked in tandem by a specific peptide) and are shown to bind calcium. The monomers possess two β-strand rich greek-key motifs. Recently, a structurally closest member to the family of lens βγ-crystallins has been described, for the first time, from the archaea Methanosarcina acetivorans, which is named as M-crystallin. Unlike lens βγ-crystallins, M-crystallin exits as a monomer. Here, we synthesized a dimeric gene of M-crystallin in which two monomers are linked by a 10-amino acid residue coding sequence. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy. The dimer of M-crystallin was overexpressed in Escherichia coli BLR(DE3) strain. The overexpressed protein containing an N-terminal hexa-histidine tag was purified using nickel affinity chromatography and then by size-exclusion chromatography. Further, a method to purify isotopically ((15)N) labeled protein with high yield for NMR studies is reported. The uniformly (15)N-labeled M-crystallin dimer thus produced has been characterized by recording sensitivity enhanced 2D [(15)N-(1)H] HSQC and other optical spectroscopy techniques. Observation of only one set of peaks in the HSQC, along with the structural characterization using optical spectroscopy, suggests that the domains in the dimer possess similar structure as that of the monomer.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N NMR assignments of a bacterial immunoglobulin-like domain (group 2) of a protein of a bacterium <Emphasis Type="Italic">Paenarthrobacter aurescens</Emphasis> TC1

The bacterial immunoglobulin-like (Big) domain is one of the prevalent domain types, which facilitates cell–cell adhesion by assembling into multi-domain architectures. We selected a four Big_2 domain protein (named ‘Arig’) from a Gram positive, Paenarthrobacter aurescens TC1 (known earlier as Arthrobacter aurescens TC1). In an attempt to characterize structural and ligand-binding features of individual Big_2 domains, we have cloned, overexpressed, isolated and purified the second Big_2 domain of Arig along with a few of its adjacent Big_2 domain residues (residue 143 to 269) referred to as ‘Arig2’. The ¹³C/¹⁵N-doubly-labeled His-tagged Arig2 (133 residues long) showed an ordered conformation as revealed by the well dispersed 2D [¹⁵N-¹H]-HSQC spectrum. Subsequently, a suite of heteronuclear 3D NMR experiments has enabled almost complete ¹H, ¹³C and ¹⁵N NMR resonance assignments of Arig2.     

NMR structure of a parallel-stranded DNA duplex at atomic resolution

DNA dodecamers have been designed with two cytosines on each end and intervening A and T stretches, such that the oligomers have fully complementary A:T base pairs when aligned in the parallel orientation. Spectroscopic (UV, CD and IR), NMR and molecular dynamics studies have shown that oligomers having the sequences d(CCATAATTTACC) and d(CCTATTAAATCC) form a parallel-stranded duplex when dissolved at 1:1 stoichiometry in aqueous solution. This is due to the C:C⁺ clamps on either end and extensive mismatches in the antiparallel orientation. The structure is stable at neutral and acidic pH. At higher temperatures, the duplex melts into single strands in a highly cooperative fashion. All adenine, cytosine and thymine nucleotides adopt the anti conformation with respect to the glycosidic bond. The A:T base pairs form reverse Watson–Crick base pairs. The duplex shows base stacking and NOEs between the base protons T(H6)/A(H8) and the sugar protons (H1′/H2′/H2″) of the preceding nucleotide, as has been observed in antiparallel duplexes. However, no NOEs are observed between base protons H2/H6/H8 of sequential nucleotides, though such NOEs are observed between T(CH₃) and A(H8). A three-dimensional structure of the parallel-stranded duplex at atomic resolution has been obtained using molecular dynamics simulations under NMR constraints. The simulated structures have torsional angles very similar to those found in B-DNA duplexes, but the base stacking and helicoid parameters are significantly different.     

Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function

RNA thermometers control the translation of several heat shock and virulence genes by their temperature-sensitive structural transitions. Changes in the structure and dynamics of MiniROSE RNA, which regulates translation in the temperature range of 20–45°C, were studied by site specifically replacing seven adenine residues with the fluorescent analog, 2-aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs were compared in their free versus ribosome-bound states for the first time. Noticeably, position dependence of fluorescence observables, which was prominent at 20°C, was persistent even at 45ºC, suggesting the persistence of structural integrity up to 45ºC. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45°C was ablated in ribosome-bound state, when compared to those at 20°C, underscoring loss of structural integrity at 45°C, in ribosome-bound RNA. Significant increase in the value of mean lifetime for 2-AP corresponding to Shine–Dalgarno sequences, when the temperature was raised from 20 to 45°C, to values seen in the presence of urea at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°C, only when it was ribosome bound. Taken all together, we propose a model where we invoke that ribosome binding of the RNA thermometer critically regulates temperature sensing functions in MiniROSE RNA.     

Initiation of Bacillus subtilis sporulation by the stringent response to partial amino acid deprivation

We have controlled the rates at which three different amino acids were available to auxotrophs of Bacillus subtilis by avoiding active transport of the respective substrate. The active transport of oxomethylvalerate, a precursor of isoleucine, was prevented by a kauA mutation, the uptake of L-aspartate was competed by 20 mM L-glutamate, and D-methionine was used instead of L-methionine. When in this way conditions of partial amino acid deprivation were achieved, a partial "stringent response" occurred which included the increase of ppGpp and pppGpp, and the decrease of GTP; such conditions initiated sporulation. In the corresponding relaxed (relA) mutants, the changes of guanine nucleotides were greatly reduced and no sporulation was observed at any substrate concentration; but addition of decoyinine produced a further decrease of GTP and caused sporulation.     

Cloning and characterization of a highly conserved HMG-like protein (PF16) gene from Plasmodium falciparum.

A novel gene encoding a protein of 147 amino acids (Pf16) has been cloned from Plasmodium falciparum and expressed in E. coli. The protein contains 19 methionines, all of which are localized in the NH2-terminal 35 amino acid residues, and it is also rich in lysine. Pf16 is highly basic, contains a polyacidic domain consisting of aspartic acid and is related to the non-histone high mobility group proteins of higher eukaryotes. The gene is conserved among eight different species of Plasmodium so far examined, suggesting an important function for this gene product in the parasite's life cycle.     

Temperature-dependent oligomerization in M-crystallin: lead or lag toward cataract, an NMR perspective

The oligomerization and/or aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from abnormal disease states like Alzheimer's and Parkinson's disease to the production of inclusion bodies, stability, and delivery of protein drugs. In the case of eye-lens proteins, oligomerization is implicated in cataract formation. In the present study, we have investigated the temperature driven oligomerization of M-crystallin, a close homologue of eye-lens proteins, using NMR spectroscopy and dynamic-light scattering (DLS). The NMR data primarily included R(1), R(2) relaxation rates and nOes of the backbone amide groups recorded at three different temperatures, 25, 20, and 15° C. The major outcome of the study is the two fold increase in the overall tumbling time (τ(c)) of M-crystallin on lowering the temperature from 25 to 15° C. An extrapolation of τ(c) to a further lower temperature (5° C) may lead to a τ(c) of ～19 ns that would correspond to a τ(c) value of a tetrameric M-crystallin. These results also validate the observed changes in the hydrodynamic radius of M-crystallin, determined using DLS data. Further, the temperature-dependent protein dynamics of M-crystallin reveal considerable variation at/near the Ca(2+)-binding sites. A concerted analysis of the temperature dependent relaxation parameters and DLS data reveals that the self-association of the protein is not only a monomer-dimer equilibrium, but also goes to tetramers or other multimeric states. These higher states may co-exist in fast exchange with the monomeric and dimeric M-crystallin at milli-molar to sub-millimolar concentrations and at lower temperature.     

Aspirin in Primary Prevention of Cardiovascular Disease and Cancer: A Systematic Review of the Balance of Evidence from Reviews of Randomized Trials

Background

Aspirin has been recommended for primary prevention of cardiovascular disease (CVD) and cancer, but overall benefits are unclear. We aimed to use novel methods to re-evaluate the balance of benefits and harms of aspirin using evidence from randomised controlled trials, systematic reviews and meta-analyses.

Methods and Findings

Data sources included ten electronic bibliographic databases, contact with experts, and scrutiny of reference lists of included studies. Searches were undertaken in September 2012 and restricted to publications since 2008. Of 2,572 potentially relevant papers 27 met the inclusion criteria. Meta-analysis of control arms to estimate event rates, modelling of all-cause mortality and L''Abbé plots to estimate heterogeneity were undertaken. Absolute benefits and harms were low: 60-84 major CVD events and 34-36 colorectal cancer deaths per 100,000 person-years were averted, whereas 46-49 major bleeds and 68-117 gastrointestinal bleeds were incurred. Reductions in all-cause mortality were minor and uncertain (Hazard Ratio 0.96; 95% CI: 0.90-1.02 at 20 years, Relative Risk [RR] 0.94, 95% CI: 0.88-1.00 at 8 years); there was a non-significant change in total CVD (RR 0.85, 95% CI: 0.69-1.06) and change in total cancer mortality ranged from 0.76 (95% CI: 0.66-0.88) to 0.93 (95% CI: 0.84-1.03) depending on follow-up time and studies included. Risks were increased by 37% for gastrointestinal bleeds (RR 1.37, 95% CI: 1.15-1.62), 54%-66% for major bleeds (Rate Ratio from IPD analysis 1.54, 95% CI: 1.30-1.82, and RR 1.62, 95% CI: 1.31-2.00), and 32%-38% for haemorrhagic stroke (Rate Ratio from IPD analysis 1.32; 95% CI: 1.00-1.74; RR 1.38; 95% CI: 1.01-1.82).

Conclusions

Findings indicate small absolute effects of aspirin relative to the burden of these diseases. When aspirin is used for primary prevention of CVD the absolute harms exceed the benefits. Estimates of cancer benefit rely on selective retrospective re-analysis of RCTs and more information is needed.