Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.     

Aerobic methanotrophs from the coastal thermal springs of Lake Baikal

The number, activity, and diversity of aerobic methanotrophic bacteria in the sediments of three coastal thermal springs of Lake Baikal were analyzed. The average number of methanotrophs was 10³–10⁴ cells per 1 cm³ of sediment. The highest number of methanotrophs (10⁸ cells/cm³ of silt) and the highest potential rate of methane uptake [7.7 nmol CH₄/(cm³ day)] were revealed in sediments from the Sukhaya thermal spring. The methods of molecular ecology (DGGE, FISH, analysis of pmoA gene fragments) showed the predominance in most enrichment cultures of methanotrophs of type II, i.e., of the genera Methylocystis and Methylosinus. In only one enrichment culture (from the Sukhaya thermal spring), a type I methanotroph was revealed; its similarity to Methylococcus capsulatus Bath did not exceed 80%. These results demonstrate a widespread occurrence and high activity of the aerobic methanotrophic community in the coastal thermal springs of Lake Baikal.     

Adsorption of trypsin on commercial silica gel

The immobilization of trypsin onto various commercial silica gels was studied. Silica gels were used directly and characterized by mercuric porosimetry. Agitation rates (100–740 rpm) and particles size (35–75 to 250–500 μm) of silica gels did not affect the trypsin immobilization capacity. The pore size (3 to 15 nm) is a limiting factor of the trypsin adsorption onto the mesopores structure of silica gels. The adsorption of trypsin was determined as a function of their initial concentration and multilayer formed at high trypsin concentration.     

Characterization and function analysis of a Halo-alkaline-adaptable Trk K+ uptake system in Alkalimonas amylolytica strain N10

By functional complementation of Escherichia coli mutants defective in potassium (K⁺) uptake, two genes that are required for K+ uptake in halo-alkaliphilic Alkalimonas amylolytica strain N10 were cloned. These two genes, Aa-trkA (1337 bp) and Aa-trkH (1452 bp), were adjacent on the A. amylolytica N10 chromosome and transcribed in opposite directions. Complementation experiments revealed that Aa-TrkA and Aa-TrkH from A. amylolytica strain N10 restored the ability to grow at low K⁺ concentration in E. coli DtrkA and DtrkG DtrkH strains, respectively. In addition, Aa-TrkAH supported the growth of an E. coli DsapD strain, indicating that the ATP-binding protein TrkE was dispensable for the Trk system of A. amylolytica strain N10. The net K⁺ uptake was detected at different pH levels and the critical NaCl concentration indicated that Aa-TrkAH is an alkaline-adaptable and partially halo-adaptable K+ transporter. Kinetics determined by heterogeneous K⁺ transport experiments with an E. coli DtrkA strain revealed that Aa-TrkAH has an alkaline pH optimum close to 8.5 or higher. Site-directed mutagenesis of Aa-TrkH showed that Phe103 and Ser229 play certain key roles in K+ selection and transportation. The molecular chaperones groES-groEL and tig promoted Aa-TrkH and Aa-TrkA overexpression in vitro.     

Cardiopulmonary exercise testing in congenital heart disease: equipment and test protocols

Cardiopulmonary exercise testing (CPET) in paediatric cardiology differs in many aspects from the tests as performed in adult cardiology. Children's cardiovascular responses during exercise testing present different characteristics, particularly oxygen uptake, heart rate and blood pressure response, which are essential in interpreting haemodynamic data. Diseases that are associated with myocardial ischaemia are very rare in children. The main indications for CPET in children are evaluation of exercise capacity and the identification of exercise-induced arrhythmias. In this article we will review exercise equipment and test protocols for CPET in children with congenital heart disease. (Neth Heart J 2009;17:339–44.)     

Competition for nitrogen sources between European beech (Fagus sylvatica) and sycamore maple (Acer pseudoplatanus) seedlings

To investigate the short‐term consequences of direct competition between beech and sycamore maple on root N uptake and N composition, mycorrhizal seedlings of both tree species were incubated for 4 days (i.e. beech only, sycamore maple only or both together) in an artificial nutrient solution with low N availability. On the fourth day, N uptake experiments were conducted to study the effects of competition on inorganic and organic N uptake. For this purpose, multiple N sources were applied with a single label. Furthermore, fine roots were sampled and analysed for total amino acids, soluble protein, total nitrogen, nitrate and ammonium content. Our results clearly show that both tree species were able to use inorganic and organic N sources. Uptake of inorganic and organic N by beech roots was negatively affected in the presence of the competing tree species. In contrast, the presence of beech stimulated inorganic N uptake by sycamore maple roots. Both the negative effect of sycamore maple on N uptake of beech and the positive effect of beech on N uptake of sycamore maple led to an increase in root soluble protein in beech, despite an overall decrease in total N concentration. Thus, beech compensated for the negative effects of the tree competitor on N uptake by incorporating less N into structural N components, but otherwise exhibited the same strategy as the competitor, namely, enhancing soluble protein levels in roots when grown under competition. It is speculated that enhanced enzyme activities of so far unknown nature are required in beech as a defence response to inter‐specific competition.     

Effects of an experimental drought on the functioning of a cacao agroforestry system,Sulawesi, Indonesia

Agroforestry systems may play a critical role in reducing the vulnerability of farmers' livelihood to droughts as tree‐based systems provide several mechanisms that can mitigate the impacts from extreme weather events. Here, we use a replicated throughfall reduction experiment to study the drought response of a cacao/Gliricidia stand over a 13‐month period. Soil water content was successfully reduced down to a soil depth of at least 2.5 m. Contrary to our expectations we measured only relatively small nonsignificant changes in cacao (?11%) and Gliricidia (?12%) sap flux densities, cacao leaf litterfall (+8%), Gliricidia leaf litterfall (?2%), soil carbon dioxide efflux (?14%), and cacao yield (?10%) during roof closure. However, cacao bean yield in roof plots was substantially lower (?45%) compared with control plots during the main harvest following the period when soil water content was lowest. This indicates that cacao bean yield was more sensitive to drought than other ecosystem functions. We found evidence in this agroforest that there is complementary use of soil water resources through vertical partitioning of water uptake between cacao and Gliricidia. This, in combination with acclimation may have helped cacao trees to cope with the induced drought. Cacao agroforests may thus play an important role as a drought‐tolerant land use in those (sub‐) tropical regions where the frequency and severity of droughts is projected to increase.     

Viscous drag as the source of active site perturbation during protease translocation: insights into how inhibitory processes are controlled by serpin metastability

The native form of serine protease inhibitors (serpins) is kinetically trapped in a metastable state, which is thought to play a central role in the inhibitory mechanism. The initial binding complex between a serpin and a target protease undergoes a conformational change that forces the protease to translocate toward the opposite pole. Although structural determination of the final stable complex revealed a detailed mechanism of keeping the bound protease in an inactive conformation, it has remained unknown how the serpin exquisitely translocates a target protease with an acyl-linkage unhydrolyzed. We previously suggested that the acyl-linkage hydrolysis is strongly suppressed by active site perturbation during the protease translocation. Here, we address what induces the transient perturbation and how the serpin metastability contributes to the perturbation. Inhibitory activity of alpha1-antitrypsin (alpha1AT) toward elastase showed negative correlations with medium viscosity and Stokes radius of elastase moiety, indicating that viscous drag directly affects the protease translocation. Stopped-flow measurements revealed that the change in the inhibitory activity is primarily caused by the change in the translocation rate. The native stability of alpha1AT cavity mutants showed a negative correlation with the translocation rate but a positive correlation with the acyl-linkage hydrolysis rate, suggesting that the two kinetic steps are not independent but closely related. The degree of active site perturbation was probed by amino acid nucleophiles, supporting the view that the changes in the acyl-linkage hydrolysis rate are due to different perturbation states. These results suggest that the active site perturbation is caused by local imbalance between a pulling force driving protease translocation and a counteracting viscous drag force. The structural architecture of serpin metastability seems to be designed to ensure the active site perturbation by providing a sufficient pulling force, so the undesirable hydrolytic activity of protease is strongly suppressed during the translocation.     

Key role of coulombic interactions for the folding transition state of the cold shock protein

The cold shock protein CspB shows a five-stranded beta-sheet structure, and it folds rapidly via a native-like transition state. A previous Phi value analysis showed that most of the residues with Phi values close to one reside in strand beta1, and two of them, Lys5 and Lys7 are partially exposed charged residues. To elucidate how coulombic interactions of these two residues contribute to the energetic organisation of the folding transition state we performed comparative folding experiments in the presence of an ionic denaturant (guanidinium chloride) and a non-ionic denaturant (urea) and a double-mutant analysis. Lys5 contributes 6.6 kJ mol(-1) to the stability of the transition state, and half of it originates from screenable coulombic interactions. Lys7 contributes 5.3 kJ mol(-1), and 3.4 kJ mol(-1) of it are screened by salt. In the folded protein Lys7 interacts with Asp25, and the screenable coulombic interaction between these two residues is fully formed in the transition state. This suggests that long-range coulombic interactions such as those originating from Lys5 and Lys7 of CspB can be important for organizing and stabilizing native-like structure early in protein folding.     

Inhibition of intestinal glucose uptake by aporphines and secoaporphines

The effects of aporphines and secoaporphines on glucose uptake by isolated intestinal brush-border membrane vesicles (BBMV) or basolateral membrane vesicles (BLMV) and glucose absorption during in situ intestinal perfusion were studied. Of the tested compounds, N-allylsecoboldine was the most potent glucose uptake inhibitor, with IC50 values of 159 microM and 121 microM, respectively, for uptake by BBMV and BLMV. While thaliporphine competitively inhibited glucose uptake by both membrane preparations, inhibition by N-allylsecoboldine was competitive using BBMV and noncompetitive using BLMV. In addition, N-allylsecoboldine significantly reduced both glucose absorption during in situ intestinal perfusion and blood glucose levels in the oral glucose tolerance test. The results demonstrate that levels of both aporphines and secoaporphines achievable by oral administration have an inhibitory effect on intestinal glucose uptake and suggest that the hypoglycemic effects of these compounds merit attention.