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1.
Elucidation of factors responsible for enhanced thermal stability of proteins: a structural genomics based study 总被引:11,自引:0,他引:11
Understanding the molecular basis for the enhanced stability of proteins from thermophiles has been hindered by a lack of structural data for homologous pairs of proteins from thermophiles and mesophiles. To overcome this difficulty, complete genome sequences from 9 thermophilic and 21 mesophilic bacterial genomes were aligned with protein sequences with known structures from the protein data bank. Sequences with high homology to proteins with known structures were chosen for further analysis. High quality models of these chosen sequences were obtained using homology modeling. The current study is based on a data set of models of 900 mesophilic and 300 thermophilic protein single chains and also includes 178 templates of known structure. Structural comparisons of models of homologous proteins allowed several factors responsible for enhanced thermostability to be identified. Several statistically significant, specific amino acid substitutions that occur going from mesophiles to thermophiles are identified. Most of these are at solvent-exposed sites. Salt bridges occur significantly more often in thermophiles. The additional salt bridges in thermophiles are almost exclusively in solvent-exposed regions, and 35% are in the same element of secondary structure. Helices in thermophiles are stabilized by intrahelical salt bridges and by an increase in negative charge at the N-terminus. There is an approximate decrease of 1% in the overall loop content and a corresponding increase in helical content in thermophiles. Previously overlooked cation-pi interactions, estimated to be twice as strong as ion-pairs, are significantly enriched in thermophiles. At buried sites, statistically significant hydrophobic amino acid substitutions are typically consistent with decreased side chain conformational entropy. 相似文献
2.
Priyakumar UD 《Journal of biomolecular structure & dynamics》2012,29(5):1-11
Abstract The role of salt bridges in chromatin protein Sso7d, from S. solfataricus has previously been shown to be crucial for its unusual high thermal stability. Experimental studies have shown that single site mutation of Sso7d (F31A) leads to a substantial decrease in the thermal stability of this protein due to distortion of the hydrophobic core. In the present study, we have performed a total of 0.2 μs long molecular dynamics (MD) simulations on F31A at room temperature, and at 360 K, close to the melting temperature of the wild type (WT) protein to investigate the role of hydrophobic core on protein stability. Sso7d-WT was shown to be stable at both 300 and 360 K; however, F31A undergoes denaturation at 360 K, consistent with experimental results. The structural and energetic properties obtained using the analysis of MD trajectories indicate that the single mutation results in high flexibility of the protein, and loosening of intramolecular interactions. Correlation between the dynamics of the salt bridges with the structural transitions and the unfolding pathway indicate the importance of both salt bridges and hydrophobic in effecting thermal stability of proteins in general. 相似文献
3.
A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones 总被引:19,自引:5,他引:14
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《The Journal of cell biology》1995,131(1):33-44
Interaction of chromatin with the nuclear envelope and lamina is thought to help determine higher order chromosome organization in the interphase nucleus. Previous studies have shown that nuclear lamins bind chromatin directly. Here we have localized a chromatin binding site to the carboxyl-terminal tail domains of both A- and B-type mammalian lamins, and have characterized the biochemical properties of this binding in detail. Recombinant glutathione-S-transferase fusion proteins containing the tail domains of mammalian lamins C, B1, and B2 were analyzed for their ability to associate with rat liver chromatin fragments immobilized on microtiter plate wells. We found that all three lamin tails specifically bind to chromatin with apparent KdS of 120-300 nM. By examining a series of deletion mutants, we have mapped the chromatin binding region of the lamin C tail to amino acids 396- 430, a segment immediately adjacent to the rod domain. Furthermore, by analysis of chromatin subfractions, we found that core histones constitute the principal chromatin binding component for the lamin C tail. Through cooperativity, this lamin-histone interaction could be involved in specifying the high avidity attachment of chromatin to the nuclear envelope in vivo. 相似文献
4.
Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a Ka of 3.02 × 104 M−1 and 1.52 × 104 M−1, respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding. 相似文献
5.
Shingo Hata Katsura Izui Hiroshi Kouchi 《The Plant journal : for cell and molecular biology》1998,13(2):267-273
Three different cDNAs for phosphoenolpyruvate carboxylase (PEPC) were isolated from soybean root nodules. The full-length cDNA of the most abundant isoform (GmPEPC7) was very similar to another one (GmPEPC15), the nucleotide sequence of which is identical to that of a reported clone (gmppc1) (Vazquez-Tello, A., Whittier, R.F., Kawasaki, T., Sugimoto, T., Kawamura, Y., Shibata, D. (1993) Plant Physiol. 103, 1025–1026). In the coding region, the newly isolated GmPEPC7 and the previously reported were gmppc1 99% and 98% identical at the amino acid and nucleotide levels, respectively. In contrast, they exhibited only 39% identity in the 3′ non-coding region, indicating that they are encoded by distinct genes. Northern blot analysis with 3′ non-coding regions as isoform-specific probes showed that GmPEPC7 is nodule-enhanced whereas GmPEPC15 (gmppc1) is expressed in most soybean tissues. The third clone (GmPEPC4) was much less homologous to the above two clones and thus was not further characterized. It was also shown by in situ hybridization that the nodule-enhanced isoform is expressed in all cell types in nodules, including in Bradyrhizobium-infected and uninfected cells and cortical cells. A relatively strong hybridization signal was detected in the vascular bundle pericycle. Southern blot analysis indicated that there are only two PEPC genes exhibiting a high degree of similarity in the soybean genome, one for the nodule-enhanced GmPEPC7 and the other for the constitutively expressed gmppc1. A phylogenetic tree based on the amino acid sequences of soybean PEPCs and nodule-enhanced PEPCs of alfalfa and pea suggested that the soybean nodule-enhanced isoform evolved from the housekeeping PEPC gene after the ureid-translocating and amide-translocating legumes diverged from each other. 相似文献
6.
Rotsaert Frederik A. Pikus Jeremie D. Fox Brian G. Markley John L. Sanders-Loehr Joann 《Journal of biological inorganic chemistry》2003,8(3):318-326
The diiron ferredoxins have a common diamond-core structure with two bridging sulfides, but differ in the nature of their terminal ligands: either four cysteine thiolates in the Fe(2)S(2) ferredoxins or two cysteine thiolates and two histidine imidazoles in the Rieske ferredoxins. Contributions of the bridging (b) and terminal (t) ligands to the resonance Raman spectra of the Fe(2)S(2) ferredoxins have been distinguished previously by isotopic substitution of the bridging sulfides. We now find that uniform (15)N-labeling of Anabaena Fe(2)S(2) ferredoxin results in shifts of -1 cm(-1) in the Fe-S(t) stretching modes at 282, 340, and 357 cm(-1). The (15)N dependence is ascribed to kinematic coupling of the Fe-S(Cys) stretch with deformations of the cysteine backbone, including the amide nitrogen. No (15)N dependence occurs for the nu(Fe-S(b)) modes at 395 and 426 cm(-1). Similar effects are observed for the Rieske center in T4MOC ferredoxin from the toluene-4-monooxygenase system of Pseudomonas mendocina. Upon selective (15)N-labeling of the alpha-amino group of cysteine, the vibrational modes at 321, 332, 350, and 362 cm(-1) all undergo shifts of -1 to -2 cm(-1), thereby identifying them as combinations of nu(Fe-S(t)) and delta(Cys). These same four modes undergo similar isotope shifts when T4MOC ferredoxin is selectively labeled with (15)N-histidine ((15)N in either the alpha1,delta1 or delta1,epsilon2 positions). Thus, the Fe-S(Cys) stretch must also be undergoing kinematic coupling with vibrations of the Fe-His moiety. The extensive kinematic coupling of iron ligand vibrations observed in both the Fe(2)S(2) and Rieske ferredoxins presumably arises from the rigidity of the protein framework and is reminiscent of the behavior of cupredoxins. In both cases, the structural rigidity is likely to play a role in minimizing the reorganization energy for electron transfer. 相似文献
7.
Murphy GS Mills JL Miley MJ Machius M Szyperski T Kuhlman B 《Structure (London, England : 1993)》2012,20(6):1086-1096
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8.
Priyakumar UD 《Journal of biomolecular structure & dynamics》2012,29(5):961-971
The role of salt bridges in chromatin protein Sso7d, from S. solfataricus has previously been shown to be crucial for its unusual high thermal stability. Experimental studies have shown that single site mutation of Sso7d (F31A) leads to a substantial decrease in the thermal stability of this protein due to distortion of the hydrophobic core. In the present study, we have performed a total of 0.2 s long molecular dynamics (MD) simulations on F31A at room temperature, and at 360 K, close to the melting temperature of the wild type (WT) protein to investigate the role of hydrophobic core on protein stability. Sso7d-WT was shown to be stable at both 300 and 360 K; however, F31A undergoes denaturation at 360 K, consistent with experimental results. The structural and energetic properties obtained using the analysis of MD trajectories indicate that the single mutation results in high flexibility of the protein, and loosening of intramolecular interactions. Correlation between the dynamics of the salt bridges with the structural transitions and the unfolding pathway indicate the importance of both salt bridges and hydrophobic in effecting thermal stability of proteins in general. 相似文献
9.
10.
We have analyzed sequence covariation in an alignment of 266 non-redundant SH3 domain sequences using chi-squared statistical methods. Artifactual covariations arising from close evolutionary relationships among certain sequence subgroups were eliminated using empirically derived sequence diversity thresholds. This covariation detection method was able to predict residue-residue contacts (side-chain centres of mass within 8 A) in the structure of the SH3 domain with an accuracy of 85 %, which is greater than that achieved in many previous covariation studies. In examining the positions involved most frequently in covariations, we discovered a dramatic over-representation of a subset of five hydrophobic core positions. This covariation information was used to design second and third site substitutions that could compensate for highly destabilizing hydrophobic core substitutions in the Fyn SH3 domain, thus providing experimental data to validate the covariation analysis. The testing of our covariation detection method on 15 other alignments showed that the accuracy of contact prediction is highly variable depending on which sequence alignment is used, and useful levels of prediction accuracy were obtained with only approximately one-third of alignments. The results presented here provide insight into the difficulties inherent in covariation analysis, and suggest that it may have limited usefulness in tertiary structure prediction. On the other hand, our ability to use covariation analysis to design stabilizing combinations of hydrophobic core substitutions attests to its potential utility for gaining deeper insight into the stability determinants and functional mechanisms of proteins with known three-dimensional structures. 相似文献
11.
12.
Inhibition of alphabeta epithelial sodium channels by external protons indicates that the second hydrophobic domain contains structural elements for closing the pore
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We have examined the effect of extracellular protons on the activity of epithelial sodium channels (ENaCs). We found that alphabeta channels, but not alphabetagamma or alphagamma channels, are inhibited by low extracellular pH. External protons induced short and long closed states that markedly decreased the open probability of alphabeta channels. External protons did not change the single-channel conductance or amiloride binding. Analysis of the proton-induced changes on the kinetics of single channels indicates that at least two protons sequentially bind to the extracellular domain at sites that are not in the ion pathway. Conformational changes induced by protonation of those sites are transmitted to the second hydrophobic domain (M2) of the subunits to induce closure of the pore. The results suggest that elements located in the carboxy-terminal half of M2 participate in the gating mechanism of ENaCs. 相似文献
13.
We study the dependence of folding time, nucleation site, and stability of a model beta-hairpin on the location of a cross-strand hydrophobic pair, using a coarse-grained off-lattice model with the aid of Monte Carlo simulations. Our simulations have produced 6500 independent folding trajectories dynamically, forming the basis for extensive statistical analysis. Four folding pathways, zipping-out, middle-out, zipping-in, and reptation, have been closely monitored and discussed in all seven sequences studied. A hydrophobic pair placed near the beta-turn or in the middle section effectively speed up folding; a hydrophobic pair placed close to the terminal ends or next to the beta-turn encourages stability of the entire chain. 相似文献
14.
15.
Kuhfahl S Hauburger A Thieme T Groppe J Ihling C Tomic S Schutkowski M Sinz A Schwarz E 《Biochemical and biophysical research communications》2011,408(2):300-305
Proregions of bone morphogenetic proteins (BMPs) fulfill important biological functions as intramolecular chaperones and for extracellular targeting of the mature signal ligand. Knowledge of the structures of the proregions would contribute to a more comprehensive picture of the biological activities of the pro-forms of BMP growth factors. In this study, a protease resistant core domain of the proregions of three BMPs was identified. For a more detailed analysis, the core domain of the BMP-2 proregion was recombinantly produced. Unfolding/refolding experiments and spectroscopic analyses proved that the core domain can be obtained as a folded entity. Binding of the core domain to the mature growth factor was demonstrated by SPR. Via peptide microarray analysis, residues within the core fragment could be identified that engage in binding to the dimeric, mature domain. Our study reveals that diverse members of the BMP family share a common, independently folding core domain within the large proregions peculiar to TGF-β superfamily members that may serve as a scaffold for folding and assembly of the dimeric proprotein complexes. 相似文献
16.
Annexin 2 binds and aggregates biological membranes in a Ca(2+)-dependent manner. This protein exists as a monomer (p36) or as a heterotetramer (p90) in which two p36 chains are associated with a dimer of p11, a member of the S100 protein family. Protein kinase C phosphorylates the protein at the level of the N-terminal tail on serines 11 and 25, thereby modifying its oligomeric structure and its properties of membrane aggregation. To analyze these effects, the properties of a series of mutants in which serines 11 and 25 were replaced by alanine and/or glutamic acid were investigated. The affinity for p11 light chain was decreased in the S11E mutants. Glutamic acid residues in positions 11 or 25 did not change membrane binding, either in the tetrameric or in the monomeric form. On the other hand, these mutations affected the aggregation properties of the two forms. For the tetramer, the aggregation efficiency was decreased but not the Ca(2+) sensitivity, whereas the latter was affected in the case of the monomer. The effects were stronger in the S11E mutants, and they were cumulative in the double mutant. They suggest a different conformation of the N-terminal domain in the mutants (and in the phosphorylated protein), a hypothesis which is supported by proteolysis experiments. This conformational change would affect aggregation by the monomer through a dimerization step. 相似文献
17.
Crystal structure of the oligomerization domain of NSP4 from rotavirus reveals a core metal-binding site 总被引:6,自引:0,他引:6
Bowman GD Nodelman IM Levy O Lin SL Tian P Zamb TJ Udem SA Venkataraghavan B Schutt CE 《Journal of molecular biology》2000,304(5):861-871
During the maturation of rotaviral particles, non-structural protein 4 (NSP4) plays a critical role in the translocation of the immature capsid into the lumen of the endoplasmic reticulum. Full-length NSP4 and a 22 amino acid peptide (NSP4(114-135)) derived from this protein have been shown to induce diarrhea in young mice in an age-dependent manner, and may therefore be the agent responsible for rotavirally-induced symptoms. We have determined the crystal structure of the oligomerization domain of NSP4 which spans residues 95 to 137 (NSP4(95-137)). NSP4(95-137) self-associates into a parallel, tetrameric coiled-coil, with the hydrophobic core interrupted by three polar layers occupying a and d-heptad positions. Side-chains from two consecutive polar layers, consisting of four Gln123 and two of the four Glu120 residues, coordinate a divalent cation. Two independent structures built from MAD-phased data indicated the presence of a strontium and calcium ion bound at this site, respectively. This metal-binding site appears to play an important role in stabilizing the homo-tetramer, which has implications for the engagement of NSP4 as an enterotoxin. 相似文献
18.
Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus 总被引:9,自引:0,他引:9
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Rouillé Y Helle F Delgrange D Roingeard P Voisset C Blanchard E Belouzard S McKeating J Patel AH Maertens G Wakita T Wychowski C Dubuisson J 《Journal of virology》2006,80(6):2832-2841
Due to the recent development of a cell culture model, hepatitis C virus (HCV) can be efficiently propagated in cell culture. This allowed us to reinvestigate the subcellular localization of HCV structural proteins in the context of an infectious cycle. In agreement with previous reports, confocal immunofluorescence analysis of the subcellular localization of HCV structural proteins indicated that, in infected cells, the glycoprotein heterodimer is retained in the endoplasmic reticulum. However, in contrast to other studies, the glycoprotein heterodimer did not accumulate in other intracellular compartments or at the plasma membrane. As previously reported, an association between the capsid protein and lipid droplets was also observed. In addition, a fraction of labeling was consistent with the capsid protein being localized in a membranous compartment that is associated with the lipid droplets. However, in contrast to previous reports, the capsid protein was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Surprisingly, no colocalization was observed between the glycoprotein heterodimer and the capsid protein in infected cells. Electron microscopy analyses allowed us to identify a membrane alteration similar to the previously reported "membranous web." However, no virus-like particles were found in this type of structure. In addition, dense elements compatible with the size and shape of a viral particle were seldom observed in infected cells. In conclusion, the cell culture system for HCV allowed us for the first time to characterize the subcellular localization of HCV structural proteins in the context an infectious cycle. 相似文献
19.
Lactoperoxidase (LPO) belongs to the mammalian peroxidase family and catalyzes the oxidation of halides, pseudo-halides and a number of aromatic substrates at the expense of hydrogen peroxide. Despite the complex physiological role of LPO and its potential involvement in carcinogenic mechanisms, cystic fibrosis and inflammatory processes, little is known on the folding and structural stability of this protein. We have undertaken an investigation of the conformational dynamics and catalytic properties of LPO during thermal unfolding, using complementary biophysical techniques (differential scanning calorimetry, electron spin resonance, optical absorption, fluorescence and circular dichroism spectroscopies) together with biological activity assays. LPO is a particularly stable protein, capable of maintaining catalysis and structural integrity up to a high temperature, undergoing irreversible unfolding at 70 degrees C. We have observed that the first stages of the thermal denaturation involve a minor conformational change occurring at 40 degrees C, possibly at the level of the protein beta-sheets, which nevertheless does not result in an unfolding transition. Only at higher temperature, the protein hydrophobic core, which is rich in alpha-helices, unfolds with concomitant disruption of the catalytic heme pocket and activity loss. Evidences concerning the stabilizing role of the disulfide bridges and the covalently bound heme cofactor are shown and discussed in the context of understanding the structural stability determinants in a relatively large protein. 相似文献
20.
Iwasaki T Kounosu A Tao Y Li Z Shokes JE Cosper NJ Imai T Urushiyama A Scott RA 《The Journal of biological chemistry》2005,280(10):9129-9134
Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Rieske-type ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe3+/2+ center at the engineered local metal-binding site (Zn2+ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds. 相似文献