Structural Basis for the Aminoacid Composition of Proteins from Halophilic Archea

Proteins from halophilic organisms, which live in extreme saline conditions, have evolved to remain folded at very high ionic strengths. The surfaces of halophilic proteins show a biased amino acid composition with a high prevalence of aspartic and glutamic acids, a low frequency of lysine, and a high occurrence of amino acids with a low hydrophobic character. Using extensive mutational studies on the protein surfaces, we show that it is possible to decrease the salt dependence of a typical halophilic protein to the level of a mesophilic form and engineer a protein from a mesophilic organism into an obligate halophilic form. NMR studies demonstrate complete preservation of the three-dimensional structure of extreme mutants and confirm that salt dependency is conferred exclusively by surface residues. In spite of the statistically established fact that most halophilic proteins are strongly acidic, analysis of a very large number of mutants showed that the effect of salt on protein stability is largely independent of the total protein charge. Conversely, we quantitatively demonstrate that halophilicity is directly related to a decrease in the accessible surface area.     

Protein Hypersaline Adaptation: Insight from Amino Acids with Machine Learning Algorithms

Traditional bioinformatics methods performed systematic comparison between the halophilic proteins and their non-halophilic homologues, to investigate the features related to hypersaline adaptation. Therefore, proposing some quantitative models to explain the sequence-characteristic relationship of halophilic proteins might shed new light on haloadaptation and help to design new biocatalysts adapt to high salt concentration. Five machine learning algorithm, including three linear and two non-linear methods were used to discriminate halophilic and their non-halophilic counterparts and the prediction accuracy was encouraging. The best prediction reliability for halophilic proteins was achieved by artificial neural network and support vector machine and reached 80 %, for non-halophilic proteins, it was achieved by linear regression and reached 100 %. Besides, the linear models have captured some clues for protein halo-stability. Among them, lower frequency of Ser in halophilic protein has not been report before.     

Cloning,expression, and efficient purification in Escherichia coli of a halophilic nucleoside diphosphate kinase from the moderate halophile Halomonas sp. #593

Most typical halophilic enzymes from extremely halophilic archaea require high concentrations of salt for their activity and stability. These enzymes are inactive in Escherichia coli unless refolded in the presence of salts in vitro. In this report, we describe cloning of the ndk gene of nucleoside diphosphate kinase from a moderately halophilic eubacterium and overexpression of the protein in E. coli as an N-terminal hexa-His fusion to facilitate its purification on Ni-NTA affinity resin. We demonstrate evidence that the protein is properly folded and exhibits the same specific activity and stability as the native protein from Halomonas cells.     

Polypeptide elongation factor Tu from Halobacterium marismortui

A GDP-binding protein of 60 kDa from Halobacterium marismortui has been purified to homogeneity. The purification has been carried out in high-salt buffers or in 50% glycerol buffers to protect the halophilic protein from denaturation. Evidence that this protein is the halophilic elongation factor Tu (hEF-Tu) is provided by the high homology of its N terminus with the corresponding sequences of other EF-Tus, and by immunological studies. Like some other EF-Tus the native protein can be cleaved with trypsin without concomitant loss of GDP-binding ability. The molecular mass of this hEF-Tu is higher than that for the corresponding factors from other sources including the halobacterium Halobacterium cutirubrum. The protein possesses typical halophilic characteristics, in that it is stable and active in 3 M KCl or 2 M (NH4)2SO4. Some other properties, like autofragmentation under sample treatment before SDS-PAGE, are described.     

Characterization of a novel complex from halophilic archaebacteria, which displays chaperone-like activities in vitro

We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro. P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits. The protein shows no significant homology to any known protein. P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner. Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability. In vivo experiments showed that P45 accumulates when cells are exposed to a low salt environment. We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.     

Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. β-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several “difficult-to-express” proteins as a BLA fusion protein and verified biological activities of human interleukin 1α and human neutrophil α-defensin, HNP-1.     

Functional implications related to the gene structure of the elongation factor EF-Tu from Halobacterium marismortui.

The primary structure of the gene for the elongation factor EF-Tu from the halophilic archaebacterium Halobacterium marismortui (hEF-Tu) is described. It is the first gene of a halophilic elongation factor EF-Tu to be sequenced. When the sequence of hEF-Tu is compared to that of homologous proteins from other organisms, the highest identity (61%) is found with EF-Tu from Methanococcus vannielii, a non-halophilic archaebacterium. In the search for halophilic characteristics therefore the most appropriate comparison is with the M. vannielii sequence. The excess of acidic amino acid residues in the hEF-Tu sequence (already observed in the composition of other halophilic proteins) results to a large extent from changes of Lys, Asn or Gln to Asp or Glu. A structural analysis algorithm applied to the halophilic sequence places these acidic residues on the surface of the protein. The corresponding residues in the crystal structure of the first domain of EF-Tu from E. coli (the only EF-Tu structure available) are grouped in patches on the protein surface, in each of which several residues that may be far apart in the sequence come quite close to each other in the tertiary structure.     

An efficient proteomics based strategy for the functional characterization of a novel halophilic enzyme from Halobacterium salinarum

The extremely halophilic archaeon, Halobacterium salinarum grows in environments containing over 25% NaCl. The enzymes of this organism have thus been adapted to be active and stable in hypersaline conditions, which makes them strong candidates as robust industrial enzymes. In this study, the proteomics approach was applied to screen novel halophilic enzymes. We focused initially on proteins that are differentially expressed under different salt concentrations in culture media. After two-dimensional gel electrophoresis over a pH 3.5-4.5 range, 29 differentially expressed protein spots were identified by tandem mass spectrometry and six of these had no similarity to preexisting genes of known function. To predict the function of them, we used various bioinformatic methods. Among other proteins, we selected Vng0487h, which showed a high similarity to acetyltransferases. As a step toward assaying the enzymatic activity of this protein, we cloned the Vng0487h gene of H. salinarum and expressed and purified the recombinant protein with a glutathione-S-transferase (GST) tag in Escherichia coli. Using a GST-pulldown assay, a protein fragment derived from E. coli could interact with recombinant Vng0487h, and was identified to be the ribosomal protein L3. This protein showed high sequence homology with ribosomal protein L7/12 from E. coli and ribosomal protein L13p from H. salinarum. This suggests that Vng0487h acetylates a subunit of ribosomal protein, possibly L13p, in H. salinarum. During the present study, an efficient procedure was established to screen novel halophilic enzymes, and to predict and assess their functions.     

嗜盐古生菌AJ4中BR蛋白基因部分片段和16S rRNA基因序列研究

为了研究分析新疆阿尔金山国家自然保护区阿牙克库木湖嗜盐古生菌物种与细菌视紫红质(bacteriorhodopsin ,BR)蛋白资源 ,对分离纯化到的极端嗜盐古生菌AJ4 ,采用PCR方法扩增出其 16SrRNA基因 (16SrDNA)和编码螺旋C至螺旋G的BR蛋白基因片断 ,并测定了基因的核苷酸序列 .通过BR蛋白部分片段序列分析表明 ,BR蛋白中对于完成质子泵功能以及与视黄醛结合的关键性氨基酸残基均为保守序列 ,位于膜内侧的序列比位于膜外侧的序列更保守 ;基于BR蛋白基因和16SrDNA序列的同源性比较以及 16SrDNA序列的系统发育学研究表明 ,AJ4是Haloarcula属中新成员 .由此建立了一种快速筛选具有新BR蛋白的新嗜盐古生菌的方法 .     

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions.

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25 degrees C. Replacement of K+ with Na+ effectively prevented the lysis by SCN-.K+ salts of NO3-, Br- and I-, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN- than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN- is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na+ observed here was considered to be manifested by specific interactions of Na+ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.     

Characterisation of a highly stable α-amylase from the halophilic archaeon Haloarcula hispanica

Intracellular and extracellular proteins from halophilic archaea face very saline conditions and must be able to maintain stability and functionality at nearly saturated salt concentrations. Haloarchaeal proteins contain specific adaptations to prevent aggregation and loss of activity in such conditions, but these adaptations usually result in a lack of stability in the absence of salt. Here, we present the characterisation of a secreted -amylase (AmyH) from the halophilic archaeon Haloarcula hispanica. AmyH was shown to be very halophilic but, unusually for a halophilic protein, it retained activity in the absence of salt. Intrinsic fluorescence measurements and activity assays showed that AmyH was very stable in high-salt buffer and even maintained stability upon the addition of urea. Urea-induced denaturation was only achieved in the absence of NaCl, demonstrating clearly that the stability of the protein was salt-dependent. Sequencing of the amyH gene showed an amino acid composition typical of halophilic proteins and, moreover, the presence of a signal peptide containing diagnostic features characteristic of export via the Twin-arginine translocase (Tat). Analysis of the export of AmyH showed that it was translocated post-translationally, most likely in a folded and active conformation, confirming that AmyH is a substrate of the Tat pathway.     

Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein

Malate dehydrogenase from the extreme halophilic bacterium, Salinibacter ruber (Sr MalDH) was purified and characterised as a tetramer by sedimentation velocity measurements, showing the enzyme belongs to the LDH-like group of MalDHs. In contrast to most other halophilic enzymes, which unfold when incubated at low salt concentration, Sr MalDH is completely stable in absence of salt. Its amino acid composition does not display the strong acidic character specific of halophilic proteins. The enzyme displays a strong KCl-concentration dependent variation in K(m) for oxaloacetate, but not for the NADH co-factor. Its activity is reduced by high salt concentration, but remains sufficient for the enzyme to sustain catalysis at approximately 30% of its maximal rates in 3 M KCl. The properties of the protein were compared with those from other LDH-like MalDHs of bacterial and archaeal origins, showing that Sr MalDH in fact behaves like a non-halophilic enzyme.     

Molecular bases of protein halotolerance

Halophilic proteins are stable and function at high salt concentration. Understanding how these molecules maintain their fold stable and avoid aggregation under harsh conditions is of great interest for biotechnological applications. This mini-review describes what is known about the molecular determinants of protein halotolerance. Comparisons between the sequences of halophilic/non-halophilic homologous protein pairs indicated that Asp and Glu are significantly more frequent, while Lys, Ile and Leu are less frequent in halophilic proteins. Homologous halophilic and non-halophilic proteins have similar overall structure, secondary structure content, and number of residues involved in the formation of H-bonds. On the other hand, on the halophilic protein surface, a decrease of nonpolar residues and an increase of charged residues are observed. Particularly, halophilic adaptation correlates with an increase of Asp and Glu, compensated by a decrease of basic residues, mainly Lys, on protein surface. A thermodynamic model, that provides a reliable explanation of the salt effect on the conformational stability of globular proteins, is presented.     

Trizol-based method for sample preparation and isoelectric focusing of halophilic proteins

A persisting complication in the development of well-resolved two-dimensional PAGE maps of halophilic proteins is their natural incompatibility with isoelectric focusing (IEF). The complete desalting of samples, which is necessary for IEF, tends to aggregate halophilic proteins, often requires relatively large amounts of starting material due to significant loss of sample, and is relatively time-consuming. Here, we describe a method of preparing protein samples from the haloarchaeon Haloferax volcanii that not only desalts the samples thoroughly but also drastically reduces the amount of protein loss associated with previous sample preparation methods and prevents protein aggregation during the removal of salt. This method of sample preparation, which incorporates Trizol (phenol/guanidine isothiocyanate), can easily be extended to analyze halophilic proteins from other organisms.     

Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase

Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting l-methionine, l-norleucine and l-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.     

On the composition and nature of the bulk protein of extremely halophilic bacteria

Summary Comparative studies have been carried out on the amino acid composition of the bulk protein of the two main types of extremely halophilic bacteria and their non-halophilic, bacteriological counterparts. The protein of all the extreme halophiles tested was relatively high in aspartic and glutamic acids and low in lysine and alanine. The results have furnished a basis for a discussion of the molecular mechanisms which might be involved in the evolution of an extremely halophilic organism from a non-halophilic organism.Research Fellow of The Norwegian Research Council for Science and the Humanities.     

Proteomic insight into phenolic adaptation of a moderately halophilic Halomonas sp. strain AAD12

A gram-negative, moderately halophilic bacterium was isolated from ?amalt? Saltern area, located in the Aegean Region of Turkey. Analysis of its 16S rRNA gene sequence and physiological characteristics showed that this strain belonged to the genus Halomonas ; hence, it was designated as Halomonas sp. strain AAD12. The isolate tolerated up to 800 mg?L(-1) phenol; however, at elevated concentrations, phenol severely retarded cell growth. The increase in lag phase with increasing phenol concentrations indicated that the microorganism was undergoing serious adaptative changes. To understand the physiological responses of Halomonas sp. strain AAD12 to phenol, a 2-dimensional electrophoresis approach combined with mass spectrometric analysis was used. This approach showed that the expression of 14 protein spots were altered as phenol concentration increased from 200 to 800 mg?L(-1). Among the identified proteins were those involved in protein biosynthesis, energy, transport, and stress metabolism. So far, this is the first study on phenolic adaptation of a gram-negative, moderately halophilic bacteria using proteomic tools. The results provided new insights for understanding the general mechanism used by moderately halophilic bacteria to tolerate phenol and suggested the potential for using these microorganisms in bioremediation.     

新疆艾比湖嗜盐菌的细菌视紫红质和16S rRNA基因序列的研究

为了研究分析嗜盐古生菌物种与细菌视紫红质(BR)蛋白基因资源,从40份土壤、湖水及淤泥样品中分离出148株嗜盐菌,对其中6株菌采用聚合酶链式反应(PCR)方法对其编码螺旋C至螺旋G的蛋白基因片段和16SrRNA基因进行了扩增,并测定了基因的核苷酸序列。与已报道的相应片段进行对比,ABDH10,ABDH1I和ABDH40中的螺旋C至螺旋G的蛋白与其他菌株差异显著。基于16SrRNA序列的同源性比较以及系统发育学研究表明,ABDH10和ABDH40是Natronorubrum属下的新成员和Natrinema属下的新成员,ABDH40的16SrRNA序列已登录到GenBank,其序列号为AY989910。ABDH11中的螺旋C至螺旋G的蛋白与其他菌株差异显著。