Unexpected plasticity of the quaternary structure of iron-manganese superoxide dismutases

Protein 3D structure can be remarkably robust to the accumulation of mutations during evolution. On the other hand, sometimes a single amino acid substitution can be sufficient to generate dramatic and completely unpredictable structural consequences. In an attempt to rationally alter the preferences for the metal ion at the active site of a member of the Iron/Manganese superoxide dismutase family, two examples of the latter phenomenon were identified. Site directed mutants of SOD from Trichoderma reesei were generated and studied crystallographically together with the wild type enzyme. Despite being chosen for their potential impact on the redox potential of the metal, two of the mutations (D150G and G73A) in fact resulted in significant alterations to the protein quaternary structure. The D150G mutant presented alternative inter-subunit contacts leading to a loss of symmetry of the wild type tetramer, whereas the G73A mutation transformed the tetramer into an octamer despite not participating directly in any of the inter-subunit interfaces. We conclude that there is considerable intrinsic plasticity in the Fe/MnSOD fold that can be unpredictably affected by single amino acid substitutions. In much the same way as phenotypic defects at the organism level can reveal much about normal function, so too can such mutations teach us much about the subtleties of protein structure.     

Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution

Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation, and prefractionation of protein interactions in solution independent of isoelectric point. We demonstrate that this assay is compatible with immunochemical methods and mass spectrometry. The assay was used to investigate interactions with several potential substrates for calreticulin, a chaperone that is involved in different biological aspects through interaction with other proteins. The current analytical assays used to investigate these interactions are mainly spectroscopic aggregation assays or solid phase assays that do not provide a direct visualization of the stable protein complex but rather provide an indirect measure of interactions. Therefore, no interaction studies between calreticulin and substrates in solution have been investigated previously. The results presented here indicate that calreticulin has a preference for substrates with a quaternary structure and primarily β-sheets in their secondary structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis.     

Adaptive plasticity of floral display size in animal-pollinated plants

Plants need not participate passively in their own mating, despite their immobility and reliance on pollen vectors. Instead, plants may respond to their recent pollination experience by adjusting the number of flowers that they display simultaneously. Such responsiveness could arise from the dependence of floral display size on the longevity of individual flowers, which varies with pollination rate in many plant species. By hand-pollinating some inflorescences, but not others, we demonstrate plasticity in display size of the orchid Satyrium longicauda. Pollination induced flower wilting, but did not affect the opening of new flowers, so that within a few days pollinated inflorescences displayed fewer flowers than unpollinated inflorescences. During subsequent exposure to intensive natural pollination, pollen removal and receipt increased proportionally with increasing display size, whereas pollen-removal failure and self-pollination accelerated. Such benefit-cost relations allow plants that adjust display size in response to the prevailing pollination rate to increase their attractiveness when pollinators are rare (large displays), or to limit mating costs when pollinators are abundant (small displays). Seen from this perspective, pollination-induced flower wilting serves the entire plant by allowing it to display the number of flowers that is appropriate for the current pollination environment.     

Perspectives on microbial cell surface display in bioremediation

The display of heterologous proteins on the microbial cell surface by means of recombinant DNA biotechnologies has emerged as a novel approach for bioremediation of contaminated sites. Both bacteria and yeasts have been investigated for this purpose. Cell surface expression of specific proteins allows the engineered microorganisms to transport, bio-accumulate and/or detoxify heavy metals as well as to degrade xenobiotics. These otherwise would not be taken up and transformed by the microbial cell. This review focuses on the application of cell surface displays for the enhanced bio-accumulation of heavy metals by metal binding proteins. It also reviews the biodegradation of xenobiotics by enzymes/proteins expressed on microbial cell surfaces.     

Plastocyanin: possible significance of quaternary structure.

Some properties of the blue copper protein plastocyanin from the green alga Scenedesmus have been investigated and compared with that from spinach, including amino acid composition, isoelectric point and copper content. The protein from Scenedesmus contains two, that from spinach four copper atoms per molecular weight of 40000. A combination of sodium dodecylsulfate/polyacrylamide gel electrophoresis and quantification of sulfhydryl groups indicates a strong preference for a species composed of 4 polypeptide chains of identical amino acid composition representing the enzymically active entity. Due to various treatments the subunits of both plastocyanins are detected as either monomer species alone or as monomer and dimer in a molar ratio of 2 : 1 on sodium dodecylsulfate gels. The four -SH groups per molecule are of different reactivity: two -SH groups can be detected after destruction of the chromophore; two more (forming an S-S bridge in the dimer) become evident after appropriate reduction. A KCN treatment for production of apoprotein is reported and the use of electrodialysis to improve incomplete apoprotein formation. These studies lend support to the proposal of a quaternary structure. Apoproteins were subjected to dodecylsulfate gel analysis, which proved to be an effective means of estimating both the extent of apoprotein formation and its reconstitution to the haloprotein.     

Resonance Raman spectroscopic evidence that carp deoxyhemoglobin remains in a T-like quaternary structure at high pH: implications for cooperativity

Resonance Raman spectroscopy shows the Fe-proximal imidazole stretching band to shift from 215 to 219 cm-1 between human deoxyhemoglobin (deoxy-Hb) and a Hb sample which is 75% oxygenated, demonstrating that the T-R quaternary structure switch can be monitored by resonance Raman spectroscopy in native Hb at equilibrium. For deoxy-Hb from carp, the band is at 215 cm-1 at pH 9 as well as pH 6, contrary to previous reports of an elevated frequency at high pH. The invariance of this frequency over a large affinity difference is in contrast to a recent report of continuously varying vFe-ImH frequencies for human mutant deoxy-Hb's. The band shifts to 219 cm-1 for carp Hb at pH 9 when O2 is bound to only 20% of the hemes. The spectra are consistent with a T-R switch upon binding approximately 0.5 O2 per Hb, on the average, although the number may be higher if the binding affinity is higher for alpha- than for beta-chains. The 0.5 value, in conjunction with the weak cooperativity observed for carp Hb at pH 9, is incompatible with a value of the allosteric constant, L = (T0)/(R0), large enough to prevent the vFe-ImH band from shifting detectably at pH 9 in the absence of O2. The possibility of functionally important intermediate structures is discussed.     

GSDS: 基因结构显示系统

构建了一个用于绘制基因结构示意图的网站系统(http://gsds.cbi.pku.edu.cn/)。用户可提交核酸序列、NCBI核酸序列号或基因外显子位置信息, 得到基因结构示意图; 并可指定在基因结构图上标注某些特定区域。系统允许用户同时输入多个基因, 并指定输出次序和标注区域。结果可用位图和矢量图两种图形格式显示。点击位图格式结果, 可以查看相应序列。系统提供中英文两种用户界面。     

The quaternary structure of streptavidin in urea

We report on the interactions of urea and guanidinium salts with streptavidin. Gel filtration chromatography in 0, 4, 6, and 7 M urea indicates that the streptavidin tetramer remains intact in urea. Biotin alters the electrophoretic mobility of streptavidin whether or not 6 M urea is present. The intrinsic fluorescence of streptavidin is increased and blue-shifted in 6 M urea. The fluorescence changes indicate the absence of unfolding. A conformational response to urea is possible, but much of the fluorescence change is due to urea binding as a weak biotin analog (Ka approximately 1.3 M-1). The resistance to structural perturbation by urea reflects the structural stability of streptavidin's anti-parallel beta-barrel motif. Unfolding is sluggish in 6 M guanidinium hydrochloride (half-time, approximately 50 days). After guanidinium thiocyanate unfolding, streptavidin can be refolded, but the unfolding and refolding transitions are centered at different concentrations of perturbant. Slow unfolding, with a 15th power dependence on guanidinium thiocyanate concentration, may be partially responsible for the noncoincidence of the unfolding and refolding processes. Nonequilibrium behavior is also seen in 6 M urea, as native streptavidin does not unfold and guanidinium thiocyanate unfolded streptavidin does not refold. Refolding does occur at lower concentrations of urea. Guanidinium thiocyanate only slowly unfolds the biotin-streptavidin complex. In the presence of biotin, unfolded streptavidin does not refold in 6 M guanidinium thiocyanate or in 6 M urea.     

Polydendrocytes display large lineage plasticity following focal cerebral ischemia

Polydendrocytes (also known as NG2 glial cells) constitute a fourth major glial cell type in the adult mammalian central nervous system (CNS) that is distinct from other cell types. Although much evidence suggests that these cells are multipotent in vitro, their differentiation potential in vivo under physiological or pathophysiological conditions is still controversial.To follow the fate of polydendrocytes after CNS pathology, permanent middle cerebral artery occlusion (MCAo), a commonly used model of focal cerebral ischemia, was carried out on adult NG2creBAC:ZEG double transgenic mice, in which enhanced green fluorescent protein (EGFP) is expressed in polydendrocytes and their progeny. The phenotype of the EGFP(+) cells was analyzed using immunohistochemistry and the patch-clamp technique 3, 7 and 14 days after MCAo. In sham-operated mice (control), EGFP(+) cells in the cortex expressed protein markers and displayed electrophysiological properties of polydendrocytes and oligodendrocytes. We did not detect any co-labeling of EGFP with neuronal, microglial or astroglial markers in this region, thus proving polydendrocyte unipotent differentiation potential under physiological conditions. Three days after MCAo the number of EGFP(+) cells in the gliotic tissue dramatically increased when compared to control animals, and these cells displayed properties of proliferating cells. However, in later phases after MCAo a large subpopulation of EGFP(+) cells expressed protein markers and electrophysiological properties of astrocytes that contribute to the formation of glial scar. Importantly, some EGFP(+) cells displayed membrane properties typical for neural precursor cells, and moreover these cells expressed doublecortin (DCX)--a marker of newly-derived neuronal cells. Taken together, our data indicate that polydendrocytes in the dorsal cortex display multipotent differentiation potential after focal ischemia.     

M-TASSER: an algorithm for protein quaternary structure prediction

In a cell, it has been estimated that each protein on average interacts with roughly 10 others, resulting in tens of thousands of proteins known or suspected to have interaction partners; of these, only a tiny fraction have solved protein structures. To partially address this problem, we have developed M-TASSER, a hierarchical method to predict protein quaternary structure from sequence that involves template identification by multimeric threading, followed by multimer model assembly and refinement. The final models are selected by structure clustering. M-TASSER has been tested on a benchmark set comprising 241 dimers having templates with weak sequence similarity and 246 without multimeric templates in the dimer library. Of the total of 207 targets predicted to interact as dimers, 165 (80%) were correctly assigned as interacting with a true positive rate of 68% and a false positive rate of 17%. The initial best template structures have an average root mean-square deviation to native of 5.3, 6.7, and 7.4 Å for the monomer, interface, and dimer structures. The final model shows on average a root mean-square deviation improvement of 1.3, 1.3, and 1.5 Å over the initial template structure for the monomer, interface, and dimer structures, with refinement evident for 87% of the cases. Thus, we have developed a promising approach to predict full-length quaternary structure for proteins that have weak sequence similarity to proteins of solved quaternary structure.     

Name that microbe: rapid identification of taxa responsible for individual fragments in fingerprints of microbial community structure

Polymerase chain reaction (PCR)‐based ‘fingerprinting’ methods, such as Terminal restriction fragment length polymorphism, Length Heterogeneity‐Polymerase Chain Reaction (LH‐PCR) and Automated Ribosomal Intergenic Spacer Analysis (ARISA) make possible quantitative studies of microbial community structure and dynamics. Here we outline a strategy for the rapid and cost‐effective isolation of 16S clones corresponding to particular fragment sizes in a fingerprint, based on applying the fingerprinting method to pools of colonies from a clone library. This allows the definitive identification of taxa responsible for the most important bands in the community fingerprint from a full 16S sequence. It offers significant advantages over random selection of clones and removes a significant barrier to the use of these methods.     

Snowshoe hares display limited phenotypic plasticity to mismatch in seasonal camouflage

As duration of snow cover decreases owing to climate change, species undergoing seasonal colour moults can become colour mismatched with their background. The immediate adaptive solution to this mismatch is phenotypic plasticity, either in phenology of seasonal colour moults or in behaviours that reduce mismatch or its consequences. We observed nearly 200 snowshoe hares across a wide range of snow conditions and two study sites in Montana, USA, and found minimal plasticity in response to mismatch between coat colour and background. We found that moult phenology varied between study sites, likely due to differences in photoperiod and climate, but was largely fixed within study sites with only minimal plasticity to snow conditions during the spring white-to-brown moult. We also found no evidence that hares modify their behaviour in response to colour mismatch. Hiding and fleeing behaviours and resting spot preference of hares were more affected by variables related to season, site and concealment by vegetation, than by colour mismatch. We conclude that plasticity in moult phenology and behaviours in snowshoe hares is insufficient for adaptation to camouflage mismatch, suggesting that any future adaptation to climate change will require natural selection on moult phenology or behaviour.     

Evolution of quaternary structure in a homotetrameric enzyme

Dihydrodipicolinate synthase (DHDPS) is an essential enzyme in (S)-lysine biosynthesis and an important antibiotic target. All X-ray crystal structures solved to date reveal a homotetrameric enzyme. In order to explore the role of this quaternary structure, dimeric variants of Escherichia coli DHDPS were engineered and their properties were compared to those of the wild-type tetrameric form. X-ray crystallography reveals that the active site is not disturbed when the quaternary structure is disrupted. However, the activity of the dimeric enzymes in solution is substantially reduced, and a tetrahedral adduct of a substrate analogue is observed to be trapped at the active site in the crystal form. Remarkably, heating the dimeric enzymes increases activity. We propose that the homotetrameric structure of DHDPS reduces dynamic fluctuations present in the dimeric forms and increases specificity for the first substrate, pyruvate. By restricting motion in a key catalytic motif, a competing, non-productive reaction with a substrate analogue is avoided. Small-angle X-ray scattering and mutagenesis data, together with a B-factor analysis of the crystal structures, support this hypothesis and lead to the suggestion that in at least some cases, the evolution of quaternary enzyme structures might serve to optimise the dynamic properties of the protein subunits.     

Benzyloxybenzylammonium chlorides: Simple amine salts that display anticonvulsant activity

Several antiepileptic drugs exert their activities by inhibiting Na⁺ currents. Recent studies demonstrated that compounds containing a biaryl-linked motif (Ar-X-Ar′) modulate Na⁺ currents. We, and others, have reported that compounds with an embedded benzyloxyphenyl unit (ArOCH₂Ar′, OCH₂ = X) exhibit potent anticonvulsant activities. Here, we show that benzyloxybenzylammonium chlorides (⁺H₃NCH₂C₆H₄OCH₂Ar′ Cl^?) displayed notable activities in animal seizure models. Electrophysiological studies of 4-(2′-trifluoromethoxybenzyloxy)benzylammonium chloride (9) using embryonic cortical neurons demonstrated that 9 promoted both fast and slow inactivation of Na⁺ channels. These findings suggest that the potent anticonvulsant activities of the earlier compounds were due, in part, to the benzyloxyphenyl motif and provide support for the use of the biaryl-linked pharmacophore in future drug design efforts.