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BBA - Proteins and Proteomics (v.1750, #2)
Characterization of a thermostable alkaline phosphatase from a novel species Thermus yunnanensis sp. nov. and investigation of its cobalt activation at high temperature by Ningping Gong; Chaoyin Chen; Liping Xie; Hongtao Chen; Xianzhi Lin; Rongqing Zhang (pp. 103-111).
A thermostable alkaline phosphatase with high specific activity and thermal resistance was purified from a novel species of Thermus sp. named as Thermus yunnanensis sp. nov. The enzyme contains a single peptide with a molecular mass of about 52 kDa on SDS-PAGE analysis and appears to be a homodimer in solution with the molecular mass of 104 kDa. The optimal pH and temperature for its activities are pH 8.0–10.0 and 70–80 °C, respectively. The catalytic activities of the enzyme are metal ion dependent, and Mg2+, Zn2+ and Co2+ are the main activators. Among these, Co2+ is the most active stimulator and has unique activation effect at high temperature. Metal binding analysis showed the binding of Mg2+ at the metal binding site was easy to loss in the thermoinactivation, and Co2+ was apt to bind at that site and kept the favorable configuration of catalysis, which would result high activation in the incubation with Co2+ at high temperature. According to this study, a model was proposed for the explanation of the activation and the results of actual experiments demonstrated the validity of the model.
Keywords: Thermostable alkaline phosphatase; Thermus yunnanensis; sp. nov.; Thermostability; Cobalt activation
Nucleotide-induced conformational change in the catalytic subunit of the phosphate-specific transporter from M. tuberculosis: Implications for the ATPase structure by Sankalp Gupta; Pradip K. Chakraborti; Dibyendu Sarkar (pp. 112-121).
The nucleotide binding subunit of the phosphate-specific transporter (PstB) from Mycobacterium tuberculosis is a member of the ABC family of permeases, which provides energy for transport through ATP hydrolysis. We utilized the intrinsic fluorescence of the single tryptophan containing protein to study the structural and conformational changes that occur upon nucleotide binding. ATP binding appeared to lead to a conformation in which the tryptophan residue had a higher degree of solvent exposure and fluorescence quenching. Substantial alteration in the proteolysis profile of PstB owing to nucleotide binding was used to decipher conformational change in the protein. In limited proteolysis experiments, we found that ATP or its nonhydrolyzable analog provided significant protection of the native protein, indicating that the effect of nucleotide on PstB conformation is directly associated with nucleotide binding. Taken together, these results indicate that nucleotide binding to PstB is accompanied by a global conformational change of the protein, which involves the helical domain from Arg137 to Trp150. Results reported here provide evidence that the putative movement of the α-helical sub-domain relative to the core sub-domain, until now only inferred from X-ray structures and modeling, is indeed a physiological phenomenon and is nucleotide dependent.
Keywords: ABC ATPase; Conformational change; Nucleotide binding subunit of the mycobacterial phosphate specific-transporter; Nucleotide binding; Phosphate-specific transporter; Tryptophan fluorescence
Ligand-induced thermostability in proteins: Thermodynamic analysis of ANS–albumin interaction by M. Soledad Celej; Sergio A. Dassie; Eleonora Freire; M. Lucia Bianconi; Gerardo D. Fidelio (pp. 122-133).
A comparative thermodynamic study of the interaction of anilinonaphthalene sulfonate (ANS) derivatives with bovine serum albumin (BSA) was performed by using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The chemically related ligands, 1,8-ANS and 2,6-ANS, present a similar affinity for BSA with different binding energetics. The analysis of the binding driving forces suggests that not only hydrophobic effect but also electrostatic interactions are relevant, even though they have been extensively used as probes for non-polar domains in proteins. Ligand association leads to an increase in protein thermostability, indicating that both dyes interact mainly with native BSA. ITC data show that 1,8-ANS and 2,6-ANS have a moderate affinity for BSA, with an association constant of around 1–9×105 M−1 for the high-affinity site. Ligand binding is disfavoured by conformational entropy. The theoretical model used to simulate DSC data satisfactorily reproduces experimental thermograms, validating this approach as one which provides new insights into the interaction between one or more ligands with a protein. By comparison with 1,8-ANS, 2,6-ANS appears as a more “inert� probe to assess processes which involve conformational changes in proteins.
Keywords: Protein stability; Protein unfolding thermodynamics; Protein–ligand interaction; Linked equilibria; ANS derivative
Early stages of salmon calcitonin aggregation: Effect induced by ageing and oxidation processes in water and in the presence of model membranes by Maria Cristina Gaudiano; Marisa Colone; Cecilia Bombelli; Pietro Chistolini; Luisa Valvo; Marco Diociaiuti (pp. 134-145).
The natural ageing- and hydrogen peroxide-induced aggregation of salmon calcitonin were studied in water and in the presence of dipalmitoylphosphatidylcholine (DPPC) liposomes. The early stages of the aggregation process at low protein concentration were investigated by means of Circular Dichroism spectroscopy (CD) and conventional and immunogold labelling Transmission Electron Microscopy (TEM). In buffered water solution, salmon calcitonin showed a two-stage conformational variation related to fibril formation and phase-separation of larger aggregates. A first stage, characterised by small conformational changes but a decrease in dichroic band intensity, was followed by a second stage, 6 days after, leading to higher conformational variations and aggregations. Salmon calcitonin showed a distinct modification in the secondary structure and aggregate morphology in the presence of hydrogen peroxide with respect to natural ageing, indicating that the two aggregation processes (natural and chemical-induced) followed a distinct mechanism. The oxidised forms of the peptide were separated by liquid chromatography. The same study was performed in the presence of DPPC liposomes. The results obtained by conventional and immunogold labelling TEM evidenced that salmon calcitonin in buffered water solution essentially does not enter the liposomes but forms around them a fibril network characterised by the same conformational changes after 6 days. The oxidised sample in the presence of liposomes showed a “fibrils hank�, separated from liposomes. The presence of liposomes did not affect either the aggregation or the conformational modifications yet observed by TEM and CD in water solution.
Keywords: Salmon Calcitonin; Liposomes; Oxidative stress; Aggregation; TEM; CD
Conformational detours during folding of a collapsed state by Daniel E. Otzen (pp. 146-153).
The protein S6 is a useful model to probe the role of partially folded states in the folding process. In the absence of salt, S6 folds from the denatured state D to the native state N without detectable intermediates. High concentrations of sodium sulfate induce the accumulation of a collapsed state C, which is off the direct folding route. However, the mutation VA85 enables S6 to fold from C directly to N through the transition state TSC. According to the denaturant dependence of this reaction, TSC and C are equally compact, but the data are difficult to deconvolute. Therefore, I have measured the heat capacities (Δ Cp) for the D→C and C→TSC transitions. The Δ Cp-values suggest that C needs to increase its surface area in order to fold directly to N. This underlines that it is a misfolded state that can only fold by at least partial unfolding. In contrast to the C-state formed by S6 wildtype, the VA85 C-state is just as compact as the native state, and this may be a prerequisite for direct folding. Individual “gatekeeper� residues may thus play a disproportionately large role in guiding proteins through different folding pathways.
Keywords: Misfolded state; Heat capacity; Detour; Energy landscape; Protein folding; Intermediate
Nitrogenase proteins from Gluconacetobacter diazotrophicus, a sugarcane-colonizing bacterium by Karl Fisher; William E. Newton (pp. 154-165).
Gluconacetobacter diazotrophicus Pal-5 grew well and expressed nitrogenase activity in the absence of NH4+ and at initial O2 concentrations greater than 5% in the culture atmosphere. G. diazotrophicus nitrogenase consisted of two components, Gd1 and Gd2, which were difficult to separate but were purified individually to homogeneity. Their compositions were very similar to those of Azotobacter vinelandii nitrogenase, however, all subunits were slightly smaller in size. The purified Gd1 protein contained a 12:1 Fe/Mo ratio as compared to 14:1 found for Av1 purified in parallel. Both Gd2 and Av2 contained 3.9 Fe atoms per molecule. Dithionite-reduced Gd1 exhibited EPR features at g=3.69, 3.96, and 4.16 compared with 3.64 and 4.27 for Av1. Gd2 gave an S=1/2 EPR signal identical to that of Av2. A Gd1 maximum specific activity of 1600 nmol H2 (min mg of protein)−1 was obtained when complemented with either Gd2 or Av2, however, more Av2 was required. Gd2 had specific activities of 600 and 1100 nmol H2 (min mg protein)−1 when complemented with Av1 and Gd1, respectively. The purified G. diazotrophicus nitrogenase exhibited a narrowed pH range for effective catalysis compared to the A. vinelandii nitrogenase, however, both exhibited maximum specific activity at about pH 7. The Gd-nitrogenase was more sensitive to ionic strength than the Av–nitrogenase.
Keywords: Gluconacetobacter diazotrophicus; Nitrogen-fixation; Nitrogenase; MoFe–protein; Fe–protein
Crystallization of three key glycolytic enzymes of the opportunistic pathogen Cryptosporidium parvum by Olga Senkovich; Haley Speed; Alexei Grigorian; Kelley Bradley; Chodavarapu S. Ramarao; Bessie Lane; Guan Zhu; Debasish Chattopadhyay (pp. 166-172).
Cryptosporidium parvum is one of the major causes of waterborne diseases worldwide. This protozoan parasite depends mainly on the anaerobic oxidation of glucose for energy production. In order to identify the differences in the three-dimensional structure of key glycolytic enzymes of C. parvum and its human host, we have expressed, purified and crystallized recombinant versions of three important glycolytic enzymes of the parasite, namely, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase. Lactate dehydrogenase has been crystallized in the absence and in the presence of its substrates and cofactors, while pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase were crystallized only in the apo-form. X-ray diffraction data have been collected for all crystals.
Keywords: Cryptosporidium parvum; Glycolysis; Lactate dehydrogenase; Glyceraldehyde 3-phosphate dehydrogenase; Pyruvate kinase; Crystallization
Crystallization and preliminary X-ray crystallographic analysis of extracellular giant hemoglobin from pogonophoran Oligobrachia mashikoi by Nobutaka Numoto; Taro Nakagawa; Akiko Kita; Yuichi Sasayama; Yoshihiro Fukumori; Kunio Miki (pp. 173-176).
An extracellular giant hemoglobin of Oligobrachia mashikoi, composed of 24 globins with the molecular mass of ∼400 kDa was crystallized in its intact form. Two crystal forms were obtained by the vapor-diffusion method. Form I crystals obtained using sodium acetate as a precipitant belong to the space group P6122 or P6522, with unit-cell parameters a=112.41, c=621.25 Å, and diffracted X-rays beyond 3.0 Å resolution. Form II crystals obtained using PEG 10000 as a precipitant belong to the space group R32, with unit-cell parameters a=111.50, c=276.84 Å, and diffracted X-rays beyond 2.9 Å resolution. The crystals are suitable for X-ray crystallography to determine the supramacromolecular assembly of this giant hemoglobin.
Keywords: Extracellular hemoglobin; Invertebrate; Pogonophora; Oligobrachia mashikoi; Sulfide binding