首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 9 毫秒
1.
The crystal structure of a family I.3 lipase from Pseudomonas sp. MIS38 in a closed conformation was determined at 1.5A resolution. This structure highly resembles that of Serratia marcescens LipA in an open conformation, except for the structures of two lids. Lid1 is anchored by a Ca2+ ion (Ca1) in an open conformation, but lacks this Ca1 site and greatly changes its structure and position in a closed conformation. Lid2 forms a helical hairpin in an open conformation, but does not form it and covers the active site in a closed conformation. Based on these results, we discuss on the lid-opening mechanism.  相似文献   

2.
3.
The interfacial activation mechanism of family I.3 lipase from Pseudomonas sp. MIS38 (PML), which has two α-helical lids (lid1 and lid2), was investigated using a combination of X-ray crystallography and molecular dynamics (MD) simulation. The crystal structure of PML in an open conformation was determined at 2.1 Å resolution in the presence of Ca2+ and Triton X-100. Comparison of this structure with that in the closed conformation indicates that both lids greatly change their positions and lid1 is anchored by the calcium ion (Ca1) in the open conformation. This structure was not seriously changed even when the protein was dialyzed extensively against the Ca2+-free buffer containing Triton X-100 before crystallization, indicating that the open conformation is fairly stable unless a micellar substance is removed. The crystal structure of the PML derivative, in which the active site serine residue (Ser207) is diethylphosphorylated by soaking the crystal of PML in the open conformation in a solution containing diethyl p-nitrophenyl phosphate, was also determined. This structure greatly resembles that in the open conformation, indicating that PML structure in the open conformation represents that in the active form. MD simulation of PML in the open conformation in the absence of micelles showed that lid2 closes first, while lid1 maintains its open conformation. Likewise, MD simulation of PML in the closed conformation in the absence of Ca2+ and in the presence of octane or trilaurin micelles showed that lid1 opens, while lid2 remains closed. These results suggest that Ca1 functions as a hook for stabilization of a fully opened conformation of lid1 and for initiation of subsequent opening of lid2.  相似文献   

4.
Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (kcat/KM,IPM) for the substrate IPM has an unusual temperature dependence, showing a local minimum at ∼35°C. In search of an explanation, we measured the individual constants kcat and KM,IPM as a function of temperature, and found that the van 't Hoff plot of KM,IPM shows sigmoid-like transition in the 20-40°C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30°C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.  相似文献   

5.
6.
Hevein, a well-studied lectin from the rubber tree Hevea brasiliensis, is the title representative of a broad family of chitin-binding polypeptides. WAMP-1a, a peptide isolated from the wheat Triticum kiharae, shares considerable similarity with hevein. The peptide possesses antifungal, antibacterial activity and is thought to play an important role in the defense system of wheat. Importantly, it features a substitution of the conserved serine residue to glycine reducing its carbohydrate-binding capacity. We used NMR spectroscopy to derive the spatial structure of WAMP-1a in aqueous solution. Notably, the mutation was found to strengthen amphiphilicity of the molecule, associated with its mode of action, an indication of the hevein domain multi-functionality. Both primary and tertiary structure of WAMP-1a suggest its evolutionary origin from the hevein domain of plant chitinases.  相似文献   

7.
Choi WC  Kim MH  Ro HS  Ryu SR  Oh TK  Lee JK 《FEBS letters》2005,579(16):3461-3466
Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn2+-binding coordination. To elucidate the role of the Zn2+, we disrupted the Zn2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50 degrees C. The mutations also abolished the Zn2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn2+ at 60 degrees C, whereas the mutant enzymes exhibited no response to Zn2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn2+ on lipase L1 at elevated temperatures.  相似文献   

8.
The inhibitory switch (IS) domain of p21-activated kinase 1 (PAK1) stabilizes full-length PAK1 in an inactive conformation by binding to the PAK1 kinase domain. Competitive binding of small guanosine triphosphatases to the IS domain disrupts the autoinhibitory interactions and exposes the IS domain binding site on the surface of the kinase domain. To build an affinity reagent that selectively binds the activated state of PAK1, we used molecular modeling to reengineer the isolated IS domain so that it was soluble and stable, did not bind to guanosine triphosphatases and bound more tightly to the PAK1 kinase domain. Three design strategies were tested: in the first and second cases, extension and redesign of the N-terminus were used to expand the hydrophobic core of the domain, and in the third case, the termini were redesigned to be adjacent in space so that the domain could be stabilized by insertion into a loop in a host cyan fluorescent protein (CFP). The best-performing design, called CFP-PAcKer, was based on the third strategy and bound the kinase domain of PAK1 with an affinity of 400 nM. CFP-PAcKer binds more tightly to a full-length variant of PAK1 that is stabilized in the “open” state (Kd = 3.3 μM) than to full-length PAK1 in the “closed” state (undetectable affinity), and binding can be monitored with fluorescence by placing an environmentally sensitive fluorescence dye on CFP-PAcKer adjacent to the binding site.  相似文献   

9.
Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HII were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol− 1 as compared with the wild-type protein. The removal of each -CH2- group burial decreased the stability by 5.1 kJ mol− 1 on average in the mutant proteins of Tk-RNase HII examined. This is comparable with the value of 5.3 kJ mol− 1 obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HII examined exhibited faster unfolding compared with the wild-type protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HII. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.  相似文献   

10.
Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization.  相似文献   

11.
RecA protein recognises two complementary DNA strands for homologous recombination. To gain insight into the molecular mechanism, the thermodynamic parameters of the DNA binding have been characterised by isothermal calorimetry. Specifically, conformational changes of protein and DNA were searched for by measuring variations in enthalpy change (DeltaH) with temperature (heat capacity change, DeltaC(p)). In the presence of the ATP analogue ATPgammaS, the DeltaH for the binding of the first DNA strand depends upon temperature (large DeltaC(p)) and the type of buffer, in a way that is consistent with the organisation of disordered parts and the protonation of RecA upon complex formation. In contrast, the binding of the second DNA strand occurs without any pronounced DeltaC(p), indicating the absence of further reorganisation of the RecA-DNA filament. In agreement with these findings, a significant change in the CD spectrum of RecA was observed only upon the binding of the first DNA strand. In the absence of nucleotide cofactor, the DeltaH of DNA binding is almost independent of temperature, indicating a requirement for ATP in the reorganisation of RecA. When the second DNA strand is complementary to the first, the DeltaH is larger than that for non-complementary DNA strand, but less than the DeltaH of the annealing of the complementary DNA without RecA. This small DeltaH could reflect a weak binding that may facilitate the dissociation of only partly complementary DNA and thus speed the search for complementary DNA. The DeltaH of binding DNA sequences displaying strong base-base stacking is small for both the first and second binding DNA strand, suggesting that the second is also stretched upon interaction with RecA. These results support the proposal that the RecA protein restructures DNA, preparing it for the recognition of a complementary second DNA strand, and that the recognition is due mainly to direct base-base contacts between DNA strands.  相似文献   

12.
There is a limited understanding of the folding of multidomain membrane proteins. Lactose permease (LacY) of Escherichia coli is an archetypal member of the major facilitator superfamily of membrane transport proteins, which contain two domains of six transmembrane helices each. We exploit chemical denaturation to determine the unfolding free energy of LacY and employ Trp residues as site-specific thermodynamic probes. Single Trp LacY mutants are created with the individual Trps situated at mirror image positions on the two LacY domains. The changes in Trp fluorescence induced by urea denaturation are used to construct denaturation curves from which unfolding free energies can be determined. The majority of the single Trp tracers report the same stability and an unfolding free energy of approximately + 2 kcal mol− 1. There is one exception; the fluorescence of W33 at the cytoplasmic end of helix I on the N domain is unaffected by urea. In contrast, the equivalent position on the first helix, VII, of the C-terminal domain exhibits wild-type stability, with the single Trp tracer at position 243 on helix VII reporting an unfolding free energy of + 2 kcal mol− 1. This indicates that the region of the N domain of LacY at position 33 on helix I has enhanced stability to urea, when compared the corresponding location at the start of the C domain. We also find evidence for a potential network of stabilising interactions across the domain interface, which reduces accessibility to the hydrophilic substrate binding pocket between the two domains.  相似文献   

13.
The stabilities toward thermal and chemical denaturation of three recombinant isoforms of human apolipoprotein E (r-apoE2, r-apoE3 and r-apoE4), human plasma apoE3, the recombinant amino-terminal (NT) and the carboxyl-terminal (CT) domains of plasma apoE3 at pH 7 were studied using near and far ultraviolet circular dichroism (UV CD), fluorescence and size-exclusion chromatography. By far UV CD, thermal unfolding was irreversible for the intact apoE isoforms and consisted of a single transition. The r-apoE3 was found to be less stable as compared to the plasma protein and the stability of recombinant isoforms was r-apoE4相似文献   

14.
The study aimed to define the key factors involved in the modulation of actinomycete mannanases. We focused on the roles of carbohydrate-binding modules (CBMs) and bivalent ions. To investigate the effects of these factors, two actinomycete mannanase genes were cloned from Streptomyces thermoluteus (StManII) and Streptomyces lividans (SlMan). CBMs fused to mannanase catalytic domains do not affect the thermal stability of the proteins. CBM2 of StManII increased the catalytic efficiency toward soluble-mannan and insoluble-mannan by 25%–36%, and CBM10 of SlMan increased the catalytic efficiency toward soluble-mannan by 40%–50%. Thermal stability of wild-type and mutant enzymes was enhanced by calcium and manganese. Thermal stability of SlMandC was also slightly enhanced by magnesium. These results indicated that bivalent ion-binding site responsible for thermal stability was in the catalytic domains. Thermal stability of mannanase differed in the kinds of bivalent ions. Isothermal titration calorimetry revealed that the catalytic domain of StManII bound bivalent ions with a Ka of 5.39 ± 0.45 × 103–7.56 ± 1.47 × 103 M− 1, and the catalytic domain of SlMan bound bivalent ions with a Ka of 1.06 ± 0.34 × 103–3.86 ± 0.94 × 103 M− 1. The stoichiometry of these bindings was consistent with one bivalent ion-binding site per molecule of enzyme. Circular dichroism spectrum revealed that the presence of bivalent ions induced changes in the secondary structures of the enzymes. The binding of certain bivalent ion responsible for thermal stability was accompanied by a different conformational change by each bivalent ion. Actinomycete mannanases belong to GHF5 which contained various hemicellulases; therefore, the information obtained from mannanases applies to the other enzymes.  相似文献   

15.

Background

Interaction of putative anticancer agent sanguinarine with two quadruplex forming sequences, human telomeric DNA (H24) and NHE III1 upstream of the P1 promoter of c-myc (Pu27), has been studied to understand the structural basis of the recognition.

Methods

Absorption, fluorescence and circular dichroism spectroscopy have been employed to characterize the association. Energetics of the interaction was studied by isothermal titration and differential scanning calorimetry. TRAP assay was done to assess the inhibitory potential of sanguinarine.

Results

Absorption and fluorescence studies show that sanguinarine has high binding affinity of ~ 105 M− 1 for both sequences. Binding stoichiometry is 2:1 for H24 and 3:1 for Pu27. Results suggest stacking interaction between planar sanguinarine moiety and G-quartets. Circular dichroism spectra show that sanguinarine does not cause structural perturbation in the all-parallel Pu27 but causes a structural transition from mixed hybrid to basket form at higher sanguinarine concentration in case of H24. The interaction is characterized by total enthalpy–entropy compensation and high heat capacity values. Differential scanning calorimetry studies suggest that sanguinarine binding increases the melting temperature and also the total enthalpy of transition of both quadruplexes. TRAP results show that sanguinarine effectively blocks telomerase activity in a concentration dependent manner in cell extracts from MDAMB-231 breast cancer cell lines.

Conclusion

These results suggest that there is a difference in the structural modes of association of sanguinarine to the quadruplexes.

General significance

It helps to understand the role of quadruplex structures as a target of small molecule inhibitors of telomerase.  相似文献   

16.
The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate-binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg(2+)) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20 degrees C, 5 mM pyruvate (with 2 mM Mg(2+)) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg(2+) is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 10(8) to < 5 x 10(5) or 3 x 10(7) M(-1), respectively. Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg(2+) also increases domain coupling and the dimerization constant approximately 42-fold. With 2 mM Mg(2+) at 15-25 degrees C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K(A)' approximately 10(6) M(-1) (deltaG' = -8.05 +/- 0.05 kcal/mole and deltaH = +3.9 kcal/mole at 20 degrees C); deltaC(p) = -0.33 kcal K(-1) mole(-1). The binding of PEP to EI(H189A) is synergistic with that of Mg(2+). Thus, physiological concentrations of PEP and Mg(2+) increase, whereas pyruvate and Mg(2+) decrease the amount of dimeric, active, dephospho-enzyme I.  相似文献   

17.
To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and alphaTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.  相似文献   

18.
Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2 Å and in complex with phenylmethylsulfonyl fluoride at 1.8 Å resolution. In both structures, bMGL adopts an α/β hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism.  相似文献   

19.
The hydrophobic cavity of lipocalin-type prostaglandin D synthase (L-PGDS) has been suggested to accommodate various lipophilic ligands through hydrophobic effects, but its energetic origin remains unknown. We characterized 18 buffer-independent binding systems between human L-PGDS and lipophilic ligands using isothermal titration calorimetry. Although the classical hydrophobic effect was mostly detected, all complex formations were driven by favorable enthalpic gains. Gibbs energy changes strongly correlated with the number of hydrogen bond acceptors of ligand. Thus, the broad binding capability of L-PGDS for ligands should be viewed as hydrophilic interactions delicately tuned by enthalpy–entropy compensation using combined effects of hydrophilic and hydrophobic interactions.  相似文献   

20.
Computational protein design procedures were applied to the redesign of the entire sequence of a 51 amino acid residue protein, Drosophila melanogaster engrailed homeodomain. Various sequence optimization algorithms were compared and two resulting designed sequences were experimentally evaluated. The two sequences differ by 11 mutations and share 22% and 24% sequence identity with the wild-type protein. Both computationally designed proteins were considerably more stable than the naturally occurring protein, with midpoints of thermal denaturation greater than 99 degrees C. The solution structure was determined for one of the two sequences using multidimensional heteronuclear NMR spectroscopy, and the structure was found to closely match the original design template scaffold.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号