Effect of low-energy microwaves on DNA stability in solution

DNA isolated from liver of healthy and tumor-bearing (sarcoma 45) rats was irradiated in water-salt solution with weak microwaves (64.5 GHz, 50 μW/cm²). The heat stability of DNA increased with irradiation time (a raise of 1.5°C in T _m for “tumor” DNA after 90 min, without changes in ΔT), which may be associated with dehydration of the surrounding Na⁺ ions.     

Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles

Sterol structure influences liquid ordered domains in membranes, and the dependence of biological functions on sterol structure can help identify processes dependent on ordered domains. In this study we compared the effect of sterol structure on ordered domain formation in symmetric vesicles composed of mixtures of sphingomyelin, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, and in asymmetric vesicles in which sphingomyelin was introduced into the outer leaflet of vesicles composed of DOPC and cholesterol. In most cases, sterol behavior was similar in symmetric and asymmetric vesicles, with ordered domains most strongly stabilized by 7-dehydrocholesterol (7DHC) and cholesterol, stabilized to a moderate degree by lanosterol, epicholesterol and desmosterol, and very little if at all by 4-cholesten-3-one. However, in asymmetric vesicles desmosterol stabilized ordered domain almost as well as cholesterol, and to a much greater degree than epicholesterol, so that the ability to support ordered domains decreased in the order 7-DHC > cholesterol > desmosterol > lanosterol > epicholesterol > 4-cholesten-3-one. This contrasts with values for intermediate stabilizing sterols in symmetric vesicles in which the ranking was cholesterol > lanosterol ~ desmosterol ~ epicholesterol or prior studies in which the ranking was cholesterol ~ epicholesterol > lanosterol ~ desmosterol. The reasons for these differences are discussed. Based on these results, we re-evaluated our prior studies in cells and conclude that endocytosis levels and bacterial uptake are even more closely correlated with the ability of sterols to form ordered domains than previously thought, and do not necessarily require that a sterol have a 3β-OH group.     

DNA melting in the presence of fluorescent intercalating oxazole yellow dyes measured with a gel-based assay

We measured the effect of the intercalating oxazole yellow DNA dye quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (YO-PRO) and its homodimer (YOYO) on the melting of self-complementary DNA duplexes using a gel-based assay. The assay, which requires a self-complementary DNA sequence, is independent of the optical properties of the molecules in solution. The melting temperature of the DNA is observed to increase in direct proportion to the number of occupied intercalation sites on the DNA, irrespective of whether the dye molecules are in monomer or dimer form. The increase is approximately 2.5 degrees C for each intercalation site occupied in the presence of 38 mM [Na(+)], for dye/duplex ratios in which less than 1/5 of the available intercalation sites are occupied.     

Stable oxygen isotopes in<Emphasis Type="Italic"> Porites</Emphasis> corals monitor weekly temperature variations in the northern Gulf of Aqaba,Red Sea

In order to assess the ability of Porites corals to accurately record environmental variations, high-resolution (weekly/biweekly) coral ¹⁸O records were obtained from four coral colonies from the northern Gulf of Aqaba, which grew at depths of 7, 19, 29, and 42 m along one transect. Adjacent to each colony, hourly temperatures, biweekly salinities, and monthly ¹⁸O of seawater were continuously recorded over a period of 14 months (April 1999 to June 2000). Contrary to water temperature, which shows a regular and strong seasonal variation and change with depth, seawater ¹⁸O exhibits a weak seasonality and little change with depth. Positive correlations between seawater ¹⁸O and salinity were observed. The two parameters were related to each other by the equation ¹⁸O _Seawater (, VSMOW) = 0.281 × Salinity – 9.14. The high-resolution coral ¹⁸O records from this study show a regular pattern of seasonality and are able to capture fine details of the weekly average temperature records. They resolve more than 95% of the weekly average temperature range. On the other hand, attenuation and amplification of coral seasonal amplitudes were recorded in deep, slow-growing corals, which were not related to environmental effects (temperature and/or seawater ¹⁸O) or sampling resolution. We propose that these result from a combined effect of subannual variations in extension rate and variable rates of spine thickening of skeletal structures within the tissue layer. However, no smoothing or distortion of the isotopic signals was observed due to calcification within the tissue layer in shallow-water, fast-growing corals. The calculations from coral ¹⁸O calibrations against the in situ measurements show that temperature (T) is related to coral ¹⁸O ( _c ) and seawater ¹⁸O ( _w ) by the equation T (°C) = –5.38 ( _c – _w ) –1.08. Our results demonstrate that coral ¹⁸O from the northern Gulf of Aqaba is a reliable recorder of temperature variations, and that there is a minor contribution of seawater ¹⁸O to this proxy, which could be ignored.     

van't Hoff enthalpies without baselines

Analysis of thermal melting curves represents one important approach for evaluating protein stability and the consequences of amino acid substitution on protein structure. By use of the van't Hoff relationship, the differential melting curve can be robustly fit to only three parameters, two of which are the underlying physical constants of melting temperature (Tm) and van't Hoff enthalpy (deltaHvH). Calculated Tm and deltaHvH values are insensitive to the choice of pre- and post-transition baselines. Consequently, the method accurately computes Tm and deltaHvH for extremely truncated data sets, in the complete absence of baseline information, and for proteins with low melting temperatures, where the traditional direct approach routinely fails. Moreover, agreement between deltaHvH values obtained using points derived from pre- vs. post-transition data provide an independent method for detecting some classes of non-two-state transitions. Finally, fitting of the differential denaturation curve should prove useful for analysis of abbreviated data sets obtained from high throughput array analysis of protein stability.     

Conformational changes and disease - serpins, prions and Alzheimer''s

Some of the most perplexing disorders in medicine are each now known to arise from the conformational instability of an underlying protein. The consequence is a continuum of pathologies with typically a change in fold leading to ordered aggregation and tissue deposition. The serpins provide a structural prototype for these pathologies and give a perspective on the assessment of current proposals as to the conformational basis of both Alzheimer's disease and the transmissible prion encephalopathies.     

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2′-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2′-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2′-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first β sheet and second β sheet of RNase HI of Escherichia coli might participate in substrate binding.     

Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability

Isocitrate dehydrogenase (IDH) has been studied extensively due to its central role in the Krebs cycle, catalyzing the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate to alpha-ketoglutarate and CO(2). Here, we present the first crystal structure of IDH from a psychrophilic bacterium, Desulfotalea psychrophila (DpIDH). The structural information is combined with a detailed biochemical characterization and a comparative study with IDHs from the mesophilic bacterium Desulfitobacterium hafniense (DhIDH), porcine (PcIDH), human cytosolic (HcIDH) and the hyperthermophilic Thermotoga maritima (TmIDH). DpIDH was found to have a higher melting temperature (T(m)=66.9 degrees C) than its mesophilic homologues and a suboptimal catalytic efficiency at low temperatures. The thermodynamic activation parameters indicated a disordered active site, as seen also for the drastic increase in K(m) for isocitrate at elevated temperatures. A methionine cluster situated at the dimeric interface between the two active sites and a cluster of destabilizing charged amino acids in a region close to the active site might explain the poor isocitrate affinity. On the other hand, DpIDH was optimized for interacting with NADP(+) and the crystal structure revealed unique interactions with the cofactor. The highly acidic surface, destabilizing charged residues, fewer ion pairs and reduced size of ionic networks in DpIDH suggest a flexible global structure. However, strategic placement of ionic interactions stabilizing the N and C termini, and additional ionic interactions in the clasp domain as well as two enlarged aromatic clusters might counteract the destabilizing interactions and promote the increased thermal stability. The structure analysis of DpIDH illustrates how psychrophilic enzymes can adjust their flexibility in dynamic regions during their catalytic cycle without compromising the global stability of the protein.