Differential Isotope Labeling Strategy for Determining the Structure of Myristoylated Recoverin by NMR Spectroscopy

The three-dimensional solution structure of recombinant bovine myristoylated recoverin in the Ca2+-free state has been refined using an array of isotope-assisted multidimensional heteronuclear NMR techniques. In some experiments, the myristoyl group covalently attached to the protein N-terminus was labeled with 13C and the protein was unlabeled or vice versa; in others, both were 13C-labeled. This differential labeling strategy was essential for structural refinement and can be applied to other acylated proteins. Stereospecific assignments of 41 pairs of -methylene protons and 48 methyl groups of valine and leucine were included in the structure refinement. The refined structure was constructed using a total of 3679 experimental NMR restraints, comprising 3242 approximate interproton distance restraints (including 153 between the myristoyl group and the polypeptide), 140 distance restraints for 70 backbone hydrogen bonds, and 297 torsion angle restraints. The atomic rms deviations about the averaged minimized coordinate positions for the secondary structure region of the N-terminal and C-terminal domains are 0.44 ± 0.07 and 0.55 ± 0.18 Å for backbone atoms, and 1.09 ± 0.07 and 1.10 ± 0.15 Å for all heavy atoms, respectively. The refined structure allows for a detailed analysis of the myristoyl binding pocket. The myristoyl group is in a slightly bent conformation: the average distance between C1 and C14 atoms of the myristoyl group is 14.6 Å. Hydrophobic residues Leu28, Trp31, and Tyr32 form a cluster that interacts with the front end of the myristoyl group (C1-C8), whereas residues Phe49, Phe56, Tyr86, Val87, and Leu90 interact with the tail end (C9-C14). The relatively deep hydrophobic pocket that binds the myristoyl group (C14:0) could also accommodate other naturally occurring acyl groups such as C12:0, C14:1, and C14:2 chains.     

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography,Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.     

Crystal violet as an i-motif structure probe for reversible and label-free pH-driven electrochemical switch

A simple pH-induced electrochemical switch based on an i-motif structure is developed by using crystal violet as a selective electrochemical probe for the i-motif structure. Thiol-modified cytosine-rich single-strand oligonucleotide (C-rich ssDNA) can be self-assembled on the gold electrode surface via gold–sulfur interaction. Crystal violet is employed as an electrochemical probe for the i-motif structure because of its capability of binding with the i-motif structure through an end-stacking mode. In acidic aqueous solution, crystal violet may approach the electrode surface owing to the formation of the i-motif structure, resulting in an obvious signal, so-called “ON” state. Whereas in neutral or basic aqueous solution, the i-motif structure unfolds to dissociative single strand, which causes crystal violet to leave from the electrode surface, and a weak signal is obtained, so-called “OFF” state. In addition, in the range of pH 4.6–7.3, the increase in current has a good linear relationship (R = 0.989) with pH value in the testing solutions. This pH-driven electrochemical switch has the advantages of simplicity, sensitivity, high selectivity, and good reversibility. Furthermore, it provides a possible platform for pH measurement.     

A plausible model for bimodal p53 switch in DNA damage response

p53 is a tumor suppressor and the p53 dynamics displays stimulus dependent patterns. Recent evidence suggests a bimodal p53 switch in cell fate decision. However, no theoretical studies have been proposed to investigate bimodal p53 induction. Here we constructed a model and showed that MDM2–p53 mRNA binding might contribute to bimodal p53 switch through an intrinsic positive feedback loop. Lower damage favored pulsing while monotonic increasing was generated with higher damage. Bimodal p53 dynamics was largely influenced by cellular MDM2 and elevated p53/MDM2 ratios with increasing etoposide favor mono-ubiquitination. Our model replicated recent experiments and provided potential insights into dynamic mechanisms of bimodal switch.     

A switch-like dynamic mechanism for the initiation of replicative senescence

Telomeres are specialized structures protecting chromosomes against genome instability. Telomeres shorten with cell division, and replicative senescence is induced when telomeres are badly eroded. Whereas TRF2 (telomeric-repeat binding factor 2), ATM (ataxia telangiectasia mutated) and p53 have been identified involved in senescence induction, how it is triggered remains unclear. Here, we propose an integrated model associating telomere loss with senescence trigger. We characterize the dynamics of telomere shorting and the p53-centered regulatory network. We show that senescence is initiated in a switch-like manner when both the shortest telomere becomes uncapped and the TRF2-ATM-p53-Siah1 positive feedback loop is switched on. This work provides a coherent picture of senescence induction in terms of telomere shortening and p53 activation.     

Lower foraging efficiency of offspring constrains use of optimal habitat in birds with extended parental care

After reproducing successfully, birds with extended parental care form family groups. Despite being the dominant social unit, such family groups have been reported to switch to alternative habitat earlier than adults without offspring, with potential negative carry‐over effects for the next breeding season. Here we test a proposed mechanism for this earlier habitat switch, namely a low foraging efficiency in juveniles. Such a test is best performed under controlled conditions because in the field families may occupy food patches of a different quality than singles or pairs without young. We studied this mechanism in Bewick's Swans Cygnus columbianus bewickii, which trample (or ‘treadle’) for food buried in the sediment. The gross intake rate of juveniles was as low as 60% of that of adults, depending on the burial depth of the food. Trampling effort did not differ between age classes, but differences in intake rate were related to body size, suggesting that larger or heavier birds were trampling more efficiently. Corresponding giving‐up densities in the field were calculated to be c. 60% higher for juveniles than for adults. Our findings are consistent with the hypothesis that the lower foraging efficiency of juveniles may be responsible for the segregation of family groups from adults without offspring.     

"Cold spots" in protein cold adaptation: Insights from normalized atomic displacement parameters (B'-factors)

Many analyses published in the last decade suggest that enzymes isolated from cold-adapted organisms are characterized by a higher flexibility of their molecular structure. Recently, it has been argued that all cold-adapted enzymes with catalytic efficiency greater than that of their mesophilic counterparts display local flexibility or rigidity that are likely to cooperate, each acting on specific areas of the enzyme structure. Here we report an analysis of the normalized thermal B-factor distributions in psychrophilic proteins compared with those of their mesophilic and thermophilic counterparts with the aim to detect statistically significant local variations of relative backbone flexibility possibly linked to cold adaptation. We utilized a strategy based mainly on intra-family comparison of local distribution of normalized B-factors. After careful statistical treatment of data, the picture emerging from our results suggests that the distribution of the flexibility in psychrophilic enzymes is locally more heterogeneous than in their respective mesophilic homologues.     

Synthesis and conformation of an analog of the helix‐loop‐helix domain of the Id1 protein containing the O‐acyl iso‐prolyl‐seryl switch motif

Synthetic peptides reproducing the helix‐loop‐helix (HLH) domains of the Id proteins fold into highly stable helix bundles upon self‐association. Recently, we have shown that the replacement of the dipeptide Val‐Ser at the loop–helix‐2 junction with the corresponding O‐acyl iso‐dipeptide leads to a completely unfolded state that only refolds after intramolecular O → N acyl migration. Herein, we report on an Id HLH analog based on the substitution of the Pro‐Ser motif at the helix‐1–loop junction with the corresponding O‐acyl iso‐dipeptide. This analog has been successfully synthesized by solid‐phase Fmoc chemistry upon suppression of DKP formation. No secondary structure could be detected for the O‐acyl iso‐peptide before its conversion into the native form by O → N acyl shift. These results show that the loop–helix junctions are determinant for the folded/unfolded state of the Id HLH domain. Further, despite the high risk of DKP formation, peptides containing O‐acyl iso‐Pro‐Ser/Thr units are synthetically accessible by Fmoc chemistry. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.