Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa

Poor protein solubility is a common problem in high-resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all 20 amino acids to protein solubility has not been done. Here, 20 variants at the completely solvent-exposed position 76 of ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II beta-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine.     

Anaerobic degradation of organic compounds at high salt concentrations

A number of obligately anaerobic fermentative bacteria are known to degrade a variety of organic substrates such as sugars, amino acids, and others, in the presence of high salt concentrations (up to 3–4 M) to products such as hydrogen, CO₂, acetate and higher fatty acids, and ethanol. Our understanding of the fate of these products in hypersaline environments is still extremely limited. The occurrence of bacterial sulfate reduction is well established at salt concentrations of up to 24%; however, the bacteria involved have not yet been isolated in pure culture, and the range of electron donors used is unknown. Halophilic or halotolerant methanogenic bacteria using hydrogen/CO₂ or acetate as energy source are notably absent; methanogenesis under hypersaline conditions is probably limited to such substrates as methanol and methylamines, which cannot be expected to be major products of anaerobic degradation of most organic compounds.     

Resurrection of Ancestral Malate Dehydrogenases Reveals the Evolutionary History of Halobacterial Proteins: Deciphering Gene Trajectories and Changes in Biochemical Properties

Extreme halophilic Archaea thrive in high salt, where, through proteomic adaptation, they cope with the strong osmolarity and extreme ionic conditions of their environment. In spite of wide fundamental interest, however, studies providing insights into this adaptation are scarce, because of practical difficulties inherent to the purification and characterization of halophilic enzymes. In this work, we describe the evolutionary history of malate dehydrogenases (MalDH) within Halobacteria (a class of the Euryarchaeota phylum). We resurrected nine ancestors along the inferred halobacterial MalDH phylogeny, including the Last Common Ancestral MalDH of Halobacteria (LCAHa) and compared their biochemical properties with those of five modern halobacterial MalDHs. We monitored the stability of these various MalDHs, their oligomeric states and enzymatic properties, as a function of concentration for different salts in the solvent. We found that a variety of evolutionary processes, such as amino acid replacement, gene duplication, loss of MalDH gene and replacement owing to horizontal transfer resulted in significant differences in solubility, stability and catalytic properties between these enzymes in the three Halobacteriales, Haloferacales, and Natrialbales orders since the LCAHa MalDH. We also showed how a stability trade-off might favor the emergence of new properties during adaptation to diverse environmental conditions. Altogether, our results suggest a new view of halophilic protein adaptation in Archaea.     

Microbial degradation of pollutants at high salt concentrations

Though our knowledge on microbial degradation of organic pollutants at high salt concentrations is still limited, the list of toxic compounds shown to be degraded or transformed in media of high salinity is growing. Compounds transformed aerobically include saturated and aromatic hydrocarbons (by certain archaeobacteria), certain aromatic compounds, organophosphorus compounds, and formaldehyde (by halotolerant eubacteria). Anaerobic microbial transformations of toxic compounds occurring at high salt concentrations include reduction of nitroaromatic compounds, and possibly transformation of chlorinated aromatic compounds.     

Thermodynamic stability of a cold-adapted protein, type III antifreeze protein, and energetic contribution of salt bridges

A thermodynamic analysis of a cold-adapted protein, type III anti-freeze protein (AFP), was carried out. The results indicate that the folding equilibrium of type III AFP is a reversible, unimolecular, two-state process with no populated intermediates. Compared to most mesophilic proteins whose folding is two-state, the psychrophilic type III AFP has a much lower thermodynamic stability at 25 degrees C, approximately 3 kcal/mol, and presents a remarkably downshifted stability-temperature curve, reaching a maximum of 5 kcal/mol around 0 degrees C. Type III AFPs contain few and non-optimally distributed surface charges relative to their mesophilic homologs, the C-terminal domains of sialic acid synthases. We used thermodynamic double mutant cycles to evaluate the energetic role of every surface salt bridge in type III AFP. Two isolated salt bridges provided no contribution to stability, while the Asp36-Arg39 salt bridge, involved in a salt bridge network with the C-terminal carboxylate, had a substantial contribution (approximately 1 kcal/mol). However, this contribution was more than counteracted by the destabilizing effect of the Asp36 carboxylate itself, whose removal led to a net 30% increase in stability at 25 degrees C. This study suggests that type III AFPs may have evolved for a minimally acceptable stability at the restricted, low temperature range (around 0 degrees C) at which AFPs must function. In addition, it indicates that salt bridge networks are used in nature also for the stability of psychrophilic proteins, and has led to a type III AFP variant of increased stability that could be used for biotechnological purposes.     

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.     

Effect of high light intensities on oxygen evolution and the light activation of NADP-malate dehydrogenase in intact spinach chloroplasts

The factors limiting the photosynthetic carbon metabolism of intact spinach (Spinacia oleracea L.) chloroplasts after a high-light pretreatment have been studied. Photosynthetic CO₂ fixation was decreased and became more sensitive to the inhibitory effect of the cyclic-electron-flow inhibitor, antimycin A. Depending on the extent of photoinhibition, changing the balance of linear to cyclic electron flow by adding oxaloacetate and antimycin A either did not relieve, or partially relieved the photoinhibitory effect. The decrease in CO₂ fixation appeared to be the consequence of either a limitation by photosystem-II activity (in the case of moderate inhibition) or, at least partially an unfavourable balance between the linear and cyclic electron flows (in the case of strong inhibition). The light activation of NADP-malate dehydrogenase (EC 1.1.1.82) was decreased only in the presence of CO₂, i.e. when there was strong competition for reducing power; otherwise, it was unaffected by photoinhibitory treatments, in accordance with its low energy requirement.Abbreviations Chl chlorophyll - NADP-MDH NADP-dependent malate dehydrogenase - PFD photon flux density - PSI, II photosystem I, II     

Mutational reversions during adaptive protein evolution

Adaptation is often regarded as the sequential fixation of individually, intrinsically beneficial mutations. Contrary to this expectation, we find a surprisingly large number of evolutionary trajectories on which natural selection first favors a mutation, then favors its removal, and later still favors its ultimate restoration during the course of antibiotic resistance evolution. The existence of reversion trajectories implies that natural selection may not follow the most parsimonious path separating two alleles, even during adaptation. Altogether, this discovery highlights the unusual and potentially circuitous routes natural selection can follow during adaptation.     

Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study

Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG-GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG-GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG-GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG-GCSF and 20kPEG-GCSF. It was observed that even 5kPEG-GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG-GCSF. Biological activity measurements demonstrated that both 5kPEG-GCSF and 20kPEG-GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.     

Computational protein design with explicit consideration of surface hydrophobic patches

De novo protein design requires the identification of amino-acid sequences that favor the target-folded conformation and are soluble in water. One strategy for promoting solubility is to disallow hydrophobic residues on the protein surface during design. However, naturally occurring proteins often have hydrophobic amino acids on their surface that contribute to protein stability via the partial burial of hydrophobic surface area or play a key role in the formation of protein-protein interactions. A less restrictive approach for surface design that is used by the modeling program Rosetta is to parameterize the energy function so that the number of hydrophobic amino acids designed on the protein surface is similar to what is observed in naturally occurring monomeric proteins. Previous studies with Rosetta have shown that this limits surface hydrophobics to the naturally occurring frequency (～28%), but that it does not prevent the formation of hydrophobic patches that are considerably larger than those observed in naturally occurring proteins. Here, we describe a new score term that explicitly detects and penalizes the formation of hydrophobic patches during computational protein design. With the new term, we are able to design protein surfaces that include hydrophobic amino acids at naturally occurring frequencies, but do not have large hydrophobic patches. By adjusting the strength of the new score term, the emphasis of surface redesigns can be switched between maintaining solubility and maximizing folding free energy.     

Contribution of surface salt bridges to protein stability: guidelines for protein engineering

The small globular protein, ubiquitin, contains a pair of oppositely charged residues, K11 and E34, that according to the three-dimensional structure are located on the surface of this protein with a spatial orientation characteristic of a salt bridge. We investigated the strength of this salt bridge and its contribution to the global stability of the ubiquitin molecule. Using the "double mutant cycle" analysis, the strength of the pairwise interactions between K11 and E34 was estimated to be favorable by 3.6kJ/mol. Further, the salt bridge of the reverse orientation, i.e. E11/K34, can be formed and is found to have a strength (3.8kJ/mol) similar to that of the K11/E34 pair. However, the global stability of the K11/E34 variant of ubiquitin is 2.2kJ/mol higher than that of the E11/K34 variant. The difference in the contribution of the opposing salt bridge orientations to the overall stability of the ubiquitin molecule is attributed to the difference in the charge-charge interactions between residues forming the salt bridge and the rest of the ionizable groups in this protein. On the basis of these results, we concluded that surface salt bridges are stabilizing, but their contribution to the overall protein stability is strongly context-dependent, with charge-charge interactions being the largest determinant. Analysis of 16 salt bridges from six different proteins, for which detailed experimental data on energetics have been reported, support the conclusions made from the analysis of the salt bridge in ubiquitin. Implications of these findings for engineering proteins with enhanced thermostability are discussed.     

Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine‐rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His‐tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low‐pH harsh conditions, however, His‐HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid‐hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full‐length His‐HP when incubated with 10–20% 2,2,2‐trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage.     

微生物高盐渗透适应策略及其耐盐强化研究进展

高盐废水因具有硬度高、可生化性差、水质成分复杂等特点,是较难处理的工业废水之一。现有物化处理技术存在运行成本高、处理效率低、二次污染重等诸多瓶颈。耐盐/嗜盐微生物可在高盐环境下进行正常生理代谢,因此,开发经济、高效、可靠的高盐废水生物处理技术有望成为高盐废水处理的主流方向之一。本文系统综述了耐盐/嗜盐微生物盐溶、胞内小分子相容溶质积累、蛋白质稳定和细胞表面稳定等高渗透压适应策略。由于嗜盐微生物存在生长条件苛刻、功能微生物种类稀缺等问题,因此,耐盐微生物在高盐废水处理的未来应用空间更大。最新研究发现强化调控技术(电、光、磁)可提升微生物的高渗透压适应能力,其中电调控技术或是未来高盐废水生物处理的重点研究方向。     

Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic organisms usually are dimers. To investigate the potential contribution of the extra dimer-dimer interface in CaMDH with respect to thermal stability, we have engineered an intersubunit disulfide bridge designed to strengthen dimer-dimer interactions. The resulting mutant (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a 200-fold increase in half-life at 75 degrees C and an increase of 15 deg. C in apparent melting temperature compared to the wild-type. The crystal structure of the mutant (solved at 1.75 A resolution) was essentially identical with that of the wild-type, with the exception of the added inter-dimer disulfide bridge and the loss of an aromatic intra-dimer contact. Remarkably, the mutant and the wild-type had similar temperature optima and activities at their temperature optima, thus providing a clear case of uncoupling of thermal stability and thermoactivity. The results show that tetramerization may contribute to MDH stability to an extent that depends strongly on the number of stabilizing interactions in the dimer-dimer interface.     

粉尘螨线粒体样苹果酸脱氢酶基因cDNA克隆和分子特征分析

摘要 目的：获取粉尘螨线粒体样苹果酸脱氢酶蛋白（mitochondrial-like malate dehydrogenase，mMDH）的编码基因并了解其分子特征。方法：以粉尘螨Total RNA为模板，RT-PCR扩增获得mMDH编码基因后、构建原核表达质粒，转化E.coil BL21(DE3)感受态细胞中，IPTG诱导表达，SDS-PAGE和Western Blot鉴定目的蛋白表达情况。采用NCBI、EXPASY在线生物信息学软件分析该基因编码蛋白质的生物学特征。结果：获得粉尘螨线粒体样苹果酸脱氢酶蛋白的编码基因，全长1032bp。构建的原核表达质粒pET28a(+)-mMDH，经转化和诱导表达后，SDS-PAGE和Western Blot可见目的蛋白条带。该基因编码的蛋白质由343个氨基酸组成，相对分子质量（Mr）36014.54Da，亚细胞定位主要在细胞质和细胞核，含有34个磷酸化位点（19个丝氨酸，12个苏氨酸和3个酪氨酸）。糖基化预测结果显示其含有2个N-糖基化位点（123位存在糖基化位点NASI和151位存在糖基化位点NSTV）和1个0-糖基化位点。二级结构主要为?琢-螺旋和无规则卷曲。三维建模可观察到该蛋白为二聚体结构，CD-Search保守区域分析后显示其属于NADB-Rossmann家族，具有MDH-glyoxysomal-mitochondrial结构域。PyMol可视化后可在三维结构中观察到保守区域位点。将该基因推导出的氨基酸序列进行Blast获得同源基因，粉尘螨与屋尘螨、梅氏嗜霉螨进化关系较近，独成一簇。结论：获得粉尘螨线粒体样苹果酸脱氢酶蛋白的编码基因全长及其原核表达质粒，并对其生物学特征进行分析，为进一步探讨该基因的生理功能、开发尘螨控制措施奠定基础。     

The role of protein stability, solubility, and net charge in amyloid fibril formation

Ribonuclease Sa and two charge-reversal variants can be converted into amyloid in vitro by the addition of 2,2,2-triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of well-characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation.     

Effects of salt bridges on protein structure and design.

Theoretical calculations (Hendsch ZS & Tidor B, 1994, Protein Sci 3:211-226) and experiments (Waldburger CD et al., 1995, Nat Struct Biol 2:122-128; Wimley WC et al., 1996, Proc Natl Acad Sci USA 93:2985-2990) suggest that hydrophobic interactions are more stabilizing than salt bridges in protein folding. The lack of apparent stability benefit for many salt bridges requires an alternative explanation for their occurrence within proteins. To examine the effect of salt bridges on protein structure and stability in more detail, we have developed an energy function for simple cubic lattice polymers based on continuum electrostatic calculations of a representative selection of salt bridges found in known protein crystal structures. There are only three types of residues in the model, with charges of -1, 0, or + 1. We have exhaustively enumerated conformational space and significant regions of sequence space for three-dimensional cubic lattice polymers of length 16. The results demonstrate that, while the more highly charged sequences are less stable, the loss of stability is accompanied by a substantial reduction in the degeneracy of the lowest-energy state. Moreover, the reduction in degeneracy is greater due to charges that pair than for lone charges that remain relatively exposed to solvent. We have also explored and illustrated the use of ion-pairing strategies for rational structural design using model lattice studies.     

Interactions of macromolecules with salt ions: an electrostatic theory for the Hofmeister effect

Salting-out of proteins was discovered in the nineteenth century and is widely used for protein separation and crystallization. It is generally believed that salting-out occurs because at high concentrations salts and the protein compete for solvation water. Debye and Kirkwood suggested ideas for explaining salting-out (Debeye and MacAulay, Physik Z; 1925;131:22-29; Kirkwood, In: Proteins, amino acids and peptides as ions and dipolar ions. New York: Reinhold; 1943. p 586-622). However, a quantitative theory has not been developed, and such a theory is presented here. It is built on Kirkwood's idea that a salt ion has a repulsive interaction with an image charge inside a low dielectric cavity. Explicit treatment is given for the effect of other salt ions on the interaction between a salt ion and its image charge. When combined with the Debye-Hückel effect of salts on the solvation energy of protein charges (i.e., salting-in), the characteristic curve of protein solubility versus salt concentration is obtained. The theory yields a direct link between the salting-out effect and surface tension and is able to provide rationalizations for the effects of salt on the folding stability of several proteins.     

Proteomic analysis of age-dependent changes in protein solubility identifies genes that modulate lifespan

While it is generally recognized that misfolding of specific proteins can cause late‐onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry‐based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)‐insoluble conformations during aging in Caenorhabditis elegans. SDS‐insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS‐insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS‐insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were measured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging.     

Activity, stability and structural studies of lactate dehydrogenases adapted to extreme thermal environments

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate with concomitant oxidation of NADH during the last step in anaerobic glycolysis. In the present study, we present a comparative biochemical and structural analysis of various LDHs adapted to function over a large temperature range. The enzymes were from Champsocephalus gunnari (an Antarctic fish), Deinococcus radiodurans (a mesophilic bacterium) and Thermus thermophilus (a hyperthermophilic bacterium). The thermodynamic activation parameters of these LDHs indicated that temperature adaptation from hot to cold conditions was due to a decrease in the activation enthalpy and an increase in activation entropy. The crystal structures of these LDHs have been solved. Pairwise comparisons at the structural level, between hyperthermophilic versus mesophilic LDHs and mesophilic versus psychrophilic LDHs, have revealed that temperature adaptation is due to a few amino acid substitutions that are localized in critical regions of the enzyme. These substitutions, each having accumulating effects, play a role in either the conformational stability or the local flexibility or in both. Going from hot- to cold-adapted LDHs, the various substitutions have decreased the number of ion pairs, reduced the size of ionic networks, created unfavorable interactions involving charged residues and induced strong local disorder. The analysis of the LDHs adapted to extreme temperatures shed light on how evolutionary processes shift the subtle balance between overall stability and flexibility of an enzyme.