幽门螺杆菌Dps蛋白的表达、纯化及活性测定

[目的]表达及纯化重组幽门螺杆菌(Helicobacter pylori) Dps(DNA protection during starvation)蛋白并测定其活性。[方法]依照Dps蛋白的基因序列,设计PCR引物,并以幽门螺杆菌基因组DNA为模板扩增Dps基因。将扩增产物回收后连接到p ET15b然后转化大肠杆菌,涂布在抗性平板,37℃过夜培养,然后使用PCR和测序验证阳性菌落。使用IPTG诱导重组Dps蛋白表达,并通过Ni~(2+)亲和层析纯化。测试Dps蛋白的亚铁氧化酶活性和抗氧化功能。[结果]通过PCR获得全长为438 bp的Dps基因,成功构建重组质粒p ET15b-Dps,它能编码分子量为18. 9 k Da的重组Dps蛋白。使用IPTG诱导目的蛋白表达,然后纯化得到Dps蛋白。Dps蛋白能够快速催化亚铁离子生成铁离子。与对照BSA蛋白相比,Dps蛋白具有较强的抑制氧自由基生成的活性。[结论]构建了重组质粒p ET15b-Dps,纯化获得Dps蛋白并测定了其亚铁氧化酶活性和抗氧化活性。     

Effect of the charge distribution along the “ferritin-like” pores of the proteins from the Dps family on the iron incorporation process

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the “ferritin-like” pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.     

Estimation of Förster's distance between two ends of Dps protein from mycobacteria: distance heterogeneity as a function of oligomerization and DNA binding

Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.     

Iron Translocation into and out of Listeria innocua Dps and Size Distribution of the Protein-enclosed Nanomineral Are Modulated by the Electrostatic Gradient at the 3-fold ��Ferritin-like�� Pores

Elucidating pore function at the 3-fold channels of 12-subunit, microbial Dps proteins is important in understanding their role in the management of iron/hydrogen peroxide. The Dps pores are called “ferritin-like” because of the structural resemblance to the 3-fold channels of 24-subunit ferritins used for iron entry and exit to and from the protein cage. In ferritins, negatively charged residues lining the pores generate a negative electrostatic gradient that guides iron ions toward the ferroxidase centers for catalysis with oxidant and destined for the mineralization cavity. To establish whether the set of three aspartate residues that line the pores in Listeria innocua Dps act in a similar fashion, D121N, D126N, D130N, and D121N/D126N/D130N proteins were produced; kinetics of iron uptake/release and the size distribution of the iron mineral in the protein cavity were compared. The results, discussed in the framework of crystal growth in a confined space, indicate that iron uses the hydrophilic 3-fold pores to traverse the protein shell. For the first time, the strength of the electrostatic potential is observed to modulate kinetic cooperativity in the iron uptake/release processes and accordingly the size distribution of the microcrystalline iron minerals in the Dps protein population.The widely distributed bacterial Dps proteins (1, 2) belong to the ferritin superfamily and are characterized by strong similarities (3) but also distinctive differences with respect to “canonical” ferritins, the ubiquitous iron storage, and detoxification proteins found in biological systems. The structural resemblance is apparent in the overall molecular assemblage because both Dps proteins and ferritins are shell-like oligomers constructed from four-helix bundle monomers. However, Dps proteins are 12-mers of identical subunits that assemble with 23 symmetry, whereas ferritins are built by 24 highly similar or identical subunits related by 432 symmetry. The functional similarities consist in the common capacity to remove Fe(II) from cytoplasm, catalyze its oxidation, and store Fe(III) thus produced in the protein cavity, wherefrom the metal can be mobilized when required by the organism. However, ferritins use molecular oxygen as iron oxidant with the production of hydrogen peroxide, whereas Dps proteins prefer hydrogen peroxide, which is typically about 100-fold more efficient than molecular oxygen (1). This difference is of major importance because it renders Dps proteins capable of removing concomitantly Fe(II) and H₂O₂ whose combination leads to the production of reactive oxygen species via Fenton chemistry (4). This capacity confers H₂O₂ resistance and hence may be a virulence factor in certain pathogens (e.g. Campylobacter jejuni, Streptococcus mutans, and Porphyromonas gingivalis) because the H₂O₂ burst represents one of the first defense lines of the host during infection (5–7).Key to a full understanding of the iron uptake and release processes at a molecular level is the route by which iron enters and exits the protein shell. In both 24-subunit ferritins and 12-subunit Dps proteins, the subunit assemblage creates pores across the protein shell that put the internal cavity in communication with the external medium. In ferritins there are two types of pores: largely hydrophobic ones along the axes with 4-fold symmetry and hydrophilic ones along the axes with 3-fold symmetry. The latter channels are funnel-shaped, with the smaller opening toward the protein cavity, and are lined with conserved glutamic and aspartic residues located in the narrow region of the funnel (8). These 3-fold pores were recognized to provide the route for iron entry into the protein cavity soon after resolution of the horse ferritin x-ray crystal structure (9). Later site-directed mutagenesis studies defined the role of specific residues (Asp¹³¹ and Glu¹³⁴) that line the pore (10, 11), whereas electrostatic calculations related the passage of iron to the existence of a gradient that drives metal ions toward the protein interior cavity (12, 13). More recently, the 3-fold symmetry pores were shown to be involved also in the exit process of iron from the protein cavity. Thus, in H-frog ferritin used as model system, iron exit is affected by local protein unfolding promoted by site-specific mutagenesis of individual amino acid residues (14, 15), by the use of chaotropes (16), and by means of selected peptides designed to bind at these channels (17).In Dps proteins, the protein shell is breached by two types of pores along the 3-fold axes, one type is formed by the N-terminal portion of the monomers and bears a strong similarity to the typical 3-fold channels of 24-subunit ferritins in that it is funnel-shaped, hydrophilic, and lined by conserved, negatively charged residues. It was therefore named “ferritin-like” and assumed to be involved in iron entry into the protein cavity upon resolution of the Listeria innocua x-ray crystal structure (18). The other type of pore is formed by the C-terminal ends of the monomers and was called “Dps type” because it is created at a subunit interface that is unique to Dps proteins. Although somewhat variable in length and in the size of the openings, the Dps type pore is mainly hydrophobic in nature (19).The present paper investigates the role of the ferritin-like pores in the iron uptake and release processes in Dps proteins using the well characterized L. innocua Dps (LiDps) as a model system (18, 20–22). In LiDps, the ferritin-like pores contain a set of three aspartate residues, Asp¹²¹, Asp¹²⁶, and Asp¹³⁰ that would be encountered in succession by a metal ion that is attracted by the electrostatic gradient they create and moves down the funnel-shaped pore toward the protein cavity (Fig. 1). Asp¹³⁰, which is located in the narrowest part of the funnel, is conserved significantly among Dps proteins (∼ 80%), whereas Asp¹²¹ and Asp¹²⁶ are less conserved (Fig. 1). Such considerations were used in the design of site-specific variants D121N, D126N, D130N, and D121N/D126N/D130N to elucidate the function of the ferritin-like pores in Dps proteins.Open in a separate windowFIGURE 1.Dps proteins sequences and conservation of the aspartate residues that line the 3-fold ferritin-like pores in L. innocua Dps. A, alignment of multiple Dps sequences from different bacteria: LiDps, non-heme iron-binding ferritin (L. innocua Clip11262]; EcDps, DNA-binding protein Dps (E. coli); HpDps, neutrophil-activating protein (Helicobacter pylori); YpDps, ferritin family protein (Yersinia pestis Angola); GtDps, DNA-protecting protein (Geobacillus thermodenitrificans NG80–2); RmDps, ferritin, and Dps (Ralstonia metallidurans CH34); AtDps, DNA protection during starvation conditions (Agrobacterium tumefaciens str. C58); TeDps Dps family DNA-binding stress response protein (Thermosynechococcus elongatus BP-1); PaDps, putative DNA-binding protein, Dps (Psychrobacter arcticus 273-4); TfDps, hypothetical protein Tfu_0799 (Thermobifida fusca YX); PhDps, DNA-binding DPS protein (Pseudoalteromonas haloplanktis TAC125); SoDps, Dps family protein (Shewanella oneidensis MR-1); BaDps1, general stress protein 20U (Bacillus anthracis str. Ames); BaDps2 general stress protein (B. anthracis str. Ames); LlDps, non-heme iron-binding ferritin (Lactococcus lactis subsp. lactis Il1403); VcDps, DPS family protein (Vibrio cholerae MZO-3); and StDps, DNA-binding ferritin-like protein (oxidative damage protectant) (Streptococcus thermophilus LMD-9). Residues forming the Dps catalytic center are highlighted in pale blue (His³¹, His⁴³, Asp⁵⁸, and Glu⁶² in LiDps); 3-fold pores aspartate residues are highlighted in yellow and marked in bold type. Alignment has been created with ClustalW2 (34). B, view of the junction of three monomers forming the 3-fold ferritin-like pore. C, Asp¹²¹, Asp¹²⁶, and Asp¹³⁰ aspartate residues comprised the pore area. D, three-dimensional view of the pore colored by charge. Red, negatively charged residues; blue, positively charged residues; white, uncharged residues. E, schematic representation of the vertical section of the pore. The images were created with PyMol (35).The results demonstrate that iron uses the LiDps ferritin-like pores to enter and leave the protein shell and hence that these pores have the same role as the structurally similar 3-fold channels in 24-subunit ferritins. LiDps residue Asp¹³⁰ is the most important determinant of the negative electrostatic gradient because of its location in the narrow part of the pores. Importantly, the data show for the first time that the electrostatic gradient at the pores modulates cooperativity in the iron uptake process and influences the size distribution of the iron core (23). The effect of the electrostatic gradient can be explained in terms of the electrostatic interaction effects between the fixed negative charges of the aspartate residues at the pores and the mobile positive charges of iron ions.