We find that a conserved mutation residue Glu to residue Asp (E303D), which both have the same polar and charged properties, makes Kit2.1 protein lose its function. To understand the mechanism, we identify three int...We find that a conserved mutation residue Glu to residue Asp (E303D), which both have the same polar and charged properties, makes Kit2.1 protein lose its function. To understand the mechanism, we identify three interactions which control the conformation change and maintain the function of the Kit2.1 protein by combining homology modeling and molecular dynamics with targeted molecular dynamics. We find that the E303D mutation weakens these interactions and results in the loss of the related function. Our data indicate that not only the amino residues but also the interactions determine the function of proteins.展开更多
Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy(FLIM), which...Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy(FLIM), which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin(FPN), a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on Cterminal of FPN(FPN-eGFP) and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn^(2+) treatment,suggesting a preference conformation of FPN in Mn^(2+) exposure. It is also observed that the lifetime(rather than intensity) measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn^(2+) treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.展开更多
Proteins are crucial to most biological processes, such as enzymes, and in various catalytic processes a dynamic motion is required. The dynamics of protein are embodied as a conformational change, which is closely re...Proteins are crucial to most biological processes, such as enzymes, and in various catalytic processes a dynamic motion is required. The dynamics of protein are embodied as a conformational change, which is closely related to the flexibility of protein. Recently, nanopore sensors have become accepted as a low cost and high throughput method to study the features of proteins. In this article, we used a SiN nanopore device to study the flexibility of T7 RNA polymerase(RNAP) and its complex with DNA promoter. By calculating full-width at half-maximum(FWHM) of Gaussian fits to the blockade histograms, we found that T7 RNAP becomes more flexible after binding DNA promoter. Moreover, the distribution of fractional current blockade suggests that flexibility alters due to a breath-like change of the volume.展开更多
Introduction Integrins are a large family of adhesion molecules broadly expressed on the surface of a wide variety of cells as heterodimers. Binding of integrins to ligands provides not only mechanical anchorage for t...Introduction Integrins are a large family of adhesion molecules broadly expressed on the surface of a wide variety of cells as heterodimers. Binding of integrins to ligands provides not only mechanical anchorage for the cell to another cell or展开更多
The thermostability of some proteins in weak cation-exchange chromatography was investigated at 20-80 ℃. The results show that there is a fixed thermal denaturation transition temperature for each protein. The appear...The thermostability of some proteins in weak cation-exchange chromatography was investigated at 20-80 ℃. The results show that there is a fixed thermal denaturation transition temperature for each protein. The appearance of the thermal transition temperature indicates that the conformations of the proteins are destroyed seriously. The thermal behavior of the proteins in weak cation-exchange and hydrophobic interaction chromatographies were compared in a wide temperature range. It was found that the proteins have a higher thermostability in a weak cation-exchange chromatography system. The thermodynamic parameters(Δ H 0, Δ S 0) of those proteins were determined by means of Vant Hoff relationship(ln k -1/ T ). According to standard entropy change(Δ S 0), the conformational change of the proteins was judged in the chromatographic process. The linear relationships between Δ H 0 and Δ S 0 can be used to evaluate 'compensation temperature'( β ) at the protein denaturation and identify the identity of the protein retention mechanism in weak cation-exchange chromatography.展开更多
It is known that urea and guanidine hydrochloride(GuHC1) induce conformational change of proteins in a certain range of molar ratios. In our research, a-naphthylamine(NA) above 10^-4 mol/L at pH 7.0 was discovered...It is known that urea and guanidine hydrochloride(GuHC1) induce conformational change of proteins in a certain range of molar ratios. In our research, a-naphthylamine(NA) above 10^-4 mol/L at pH 7.0 was discovered to perturb the conformation of CopC, a copper resistant protein with a Greek fl-barrel motif; this was reflected by the greater fluorescence quenching and red-shifted emission peak of CopC. The conformation change of CopC was also verified in acrylamide collision experiment by comparing quenching levels of CopC in the presence or the abscence of NA. Comparison of emission change of CopC-Cu^2+ with that of apo-CopC showed a consistent result with their denaturation in GuHCl. And decreasing fluorescence polarization of CopC with increasing NA concentration is consistent with a more extend conformation of CopC. Interaction mechanism was also explored.展开更多
Small molecule aptamers discovered by traditional selection methods usually lack conformational changes upon target binding.This limits the use of aptamers as molecular probes for small molecule detection and regulato...Small molecule aptamers discovered by traditional selection methods usually lack conformational changes upon target binding.This limits the use of aptamers as molecular probes for small molecule detection and regulatory elements of genetic circuits.Here,we report a new method called capture and in vitro transcription-systematic evolution of ligands by exponential enrichment(CIVT-SELEX)to select DNA aptamers that can not only bind to small molecule ligands but also undergo significant conformational changes.Through this method,we select a structure-switching aptamer of uridine-5′-diphosphate(UDP).Taking advantage of its conformational changes,we first construct a UDP-responsive transcriptional switch by inserting the aptamer in a genetic circuit and demonstrate that it can respond to the addition of UDP and regulate the transcription of downstream genes.We also build a UDP aptamer-based biosensor that can be used for active glycosyltransferase screening.We believe this method can provide a universal platform for selecting small molecule aptamers with conformational changes and expand the use of aptamers in small molecule detection and genetic regulation.展开更多
Fluorescence lifetime and anisotropy has become a prevalent tool to detect the structure change and motility property of proteins. YgaP is the only membrane-integrated rhodanese in E. coli. The sulfur transfer process...Fluorescence lifetime and anisotropy has become a prevalent tool to detect the structure change and motility property of proteins. YgaP is the only membrane-integrated rhodanese in E. coli. The sulfur transfer process has been characterized by various studies. However, the mechanism of the outward transportation of SCN-remains unclear. In this work, we examined the fluorescence lifetime and anisotropy of site-specific incorporated unnatural amino acid 7-HC to study the conformational change of YgaP upon SCN-binding. We also compared the fluorescence changes between detergent-wrapped environment in DPC and intact native membrane environment in SMA. Our results suggested the presence of at least two different conformations in YgaP protein. Both the residues in the middle of TMH2 and the residues near extracellular side play important roles in the binding and/or output of SCN-. SMA is a good material to reflect the in situ conformation changes of protein than micelles.展开更多
Genistein can induce not only differentiation but also apoptosis in HL60 human leukemia cells. During the differentiation and apoptosis, the HL60 cells undergo some conformational changes. Fourier transform infrared (...Genistein can induce not only differentiation but also apoptosis in HL60 human leukemia cells. During the differentiation and apoptosis, the HL60 cells undergo some conformational changes. Fourier transform infrared (FT IR) was employed to detect those changes. The results showed that the contents of DNA/protein and glycoprotein/protein increased. The α helix of the membrane protein also increased. In addition, the CO (H) stretching mode of serine, threonine and tyrosine residues of the cell proteins also changed. Those conformational changes suggest some mechanisms for how genistein affects the HL60 cells.展开更多
The refractive index increment, dynamic and static laser light scattering, intrinsic viscosity [η] and Huggins constant (KH) of nylon 12 have been measured in m-cresol and sulphuric acid/water system at 10-60℃. Th...The refractive index increment, dynamic and static laser light scattering, intrinsic viscosity [η] and Huggins constant (KH) of nylon 12 have been measured in m-cresol and sulphuric acid/water system at 10-60℃. The intrinsic viscosity, Rn, Rg, A2, and (〈 S 〉2)^1/2 (calculated from viscosity data) and "a" values of nylon 12 are found to be higher in m-cresol than in sulphuric acid. All these parameters decrease with the increase in water contents in sulphuric acid. The refractive index increment, KH and activation energy show an opposite trend to that of [η]. The intrinsic viscosity, RH, Rg, A2, and (〈 S 〉2)^1/2 have maximum values around 30-40℃ in sulphuric acid/water system, whereas in m-cresol they fall at about 20℃. It has been concluded that the variation in size, interaction parameter (second virial coefficient), [η] and KH of the polymer solutions with the alteration in solvent composition and temperature are the out come of change in thermodynamic quality of solvents, selective adsorption, hydrogen bonding and conformational transitions. It has also been concluded that the increase in temperature first enhances the quality of the solvent, encourages hydrogen bonding and specific adsorption, and then deteriorates, bringing conformational transitions in the polymer molecules. However, the addition of water to sulphuric acid continuously deteriorates the solvent quality. This characteristic of the solvent system brings conformational changes in the polymer especially at low temperatures.展开更多
Negative thermal expansion(NTE) behavior has roused wide interest for the control of thermomechanical properties of functional materials.Although NTE behaviors have been found in kinds of compounds,it remains challeng...Negative thermal expansion(NTE) behavior has roused wide interest for the control of thermomechanical properties of functional materials.Although NTE behaviors have been found in kinds of compounds,it remains challenging for polymers to achieve intrinsic NTE property.In this work,we systematically studied the conformational change of dibenzocyclooctadiene(DBCOD) derivatives between chair(C) and twist-boat(TB) forms based on density-functional theo ry(DFT) calculations,and found clear evidence of the relationship between the structure of DBCOD units and the thermal contraction behavior of the related polymers.In order to obtain the polymer with NTE property,two conditions should be met for the thermal contracting DBCOD related units as follows:(i) the TB conformation can turn into C conformation as the temperature increases,and(ii) the volume of C conformation is smaller than that of TB conformation.This rule should offer a guidance to exploration of the new intrinsic NTE polymers in the future.展开更多
Phospholipid hydroperoxide glutathione peroxidase is an antioxidant enzyme that has the highest capability of reducing membrane-bound hydroperoxy lipids as compared to free organic and inorganic hydroperoxides amongst...Phospholipid hydroperoxide glutathione peroxidase is an antioxidant enzyme that has the highest capability of reducing membrane-bound hydroperoxy lipids as compared to free organic and inorganic hydroperoxides amongst the glutathione peroxidases.In this study,urea-induced effects on the inactivation and unfolding of a recombinant phospholipid hydroperoxide glutathione peroxidase(PHGPx)from Oryza sativa were investigated by means of circular dichroism and fluorescence spectroscopy.With the increase of urea concentration,the residual activity of OsPHGPx decreases correspondingly.When the urea concentration is above 5.0 mol/L,there was no residual activity.In addition,the observed changes in intrinsic tryptophan fluorescence,the binding of the hydrophobic fluorescence probe ANS,and the far UV CD describe a common dependence on the concentration of urea suggesting that the conformational features of the native OsPHGPx are lost in a highly cooperative single transition.The unfolding process comprises of three zones:the native base-line zone between 0 and 2.5 mol/L urea,the transition zone between 2.5 and 5.5 mol/L urea,and the denatured base-line zone above 5.5 mol/L urea.The transition zone has a midpoint at about 4.0 mol/L urea.展开更多
It has been reported that inactivation occurs before noticeable conformational changes can be detected during denaturation of creatine kinase and other enzymes. Therefore, Tsou suggested that enzyme active sites may d...It has been reported that inactivation occurs before noticeable conformational changes can be detected during denaturation of creatine kinase and other enzymes. Therefore, Tsou suggested that enzyme active sites may display more conformational flexibility than the enzyme molecules as a whole. In the present investigation, the conformational changes of Penaeus penicillatus acid phosphatase during denaturation in urea solutions were studied by following changes in the intrinsic fluorescence, ultraviolet difference absorption, and circular dichroism spectra. Inactivation of the enzyme in urea solutions was compared with unfolding of the enzyme molecule. The results show that the extent of unfolding in guanidine solutions measured by several different methods closely coincides with each other and that slightly lower concentrations of guanidine are required to bring about inactivation than are required to produce significant conformational changes of the enzyme molecule. At the same concentrations, the inactivation rate constants are markedly faster than the rate constants for unfolding of the enzyme. The above results suggest that the active sites of this enzyme display more conformational flexibility than the enzyme molecule as a whole.展开更多
Encapsulation of biomolecules inside a carbon nanotube (CNT) has attracted great interest because it could enable the delivery of nanoscale pharmaceutical drugs with CNT-based devices. Using a molecular dynamics sim...Encapsulation of biomolecules inside a carbon nanotube (CNT) has attracted great interest because it could enable the delivery of nanoscale pharmaceutical drugs with CNT-based devices. Using a molecular dynamics simulation, we investigate the dynamic process by which human immunodeficiency virus (HIV) replication inhibitor peptides (HRIPs) are encapsulated in a water solution contained inside a CNT. The van der Waals attraction between the HRIPs and the CNT and the root-mean-square deviation are used to analyse the evolution of the encapsulation. It is found that the interaction between the HRIPs and the CNT is the main driving force for the encapsulation process, which does not cause an obvious conformational change to the HRIPs.展开更多
The gene of enzyme(Ape1547) was cloned from hyperthermophilic archaeon Aeropyrum pernix K1 and expressed in Escherichia coil.The effect of calcium cation on the properties of Ape1547 was studied.Ape1547 exhibits bot...The gene of enzyme(Ape1547) was cloned from hyperthermophilic archaeon Aeropyrum pernix K1 and expressed in Escherichia coil.The effect of calcium cation on the properties of Ape1547 was studied.Ape1547 exhibits both peptidase activity and esterase activity.The fluorescence spectrum shows that calcium cation quenches the fluorescence of the enzyme through static quenching mechanism,indicating that calcium cation was bound to the enzyme.Based on the study of calcium cation on CD ellipticity of Ape1547 by circular dichroism,we concluded that the change of enzyme structure induced by calcium cation may be responsible for the change of enzyme activity.Calcium cation has dual effects on Ape1547:it could activate the enzyme activity when its concentration was 0.1 mol/L,and the enzyme had the highest activity;however,when its concentration was higher than 0.2 mol/L,the enzyme activity was inhibited.The results indicate that the activity center of peptidase activity might involve more amino acid residues than that of esterase activity.展开更多
The effects of oligodeoxynucleotide (ODN) on the conformation of basic fibroblast growth factor (bFGF) were studied by spectral method. The results showed that ODN destabilized the protein.
OBJECTIVE The chemokine-like receptor 1(CMKLR1,Chem R23) is a functional receptor for chemerin,the chemerin-derived nonapeptide(C9),and the amyloid β peptide 1-42(Aβ_(42)).Because these peptides share little sequenc...OBJECTIVE The chemokine-like receptor 1(CMKLR1,Chem R23) is a functional receptor for chemerin,the chemerin-derived nonapeptide(C9),and the amyloid β peptide 1-42(Aβ_(42)).Because these peptides share little sequence homology,studies were conducted to investigate their pharmacological properties and regulation at CMKLR1.METHODS Cells expressing CMKLR1 were incubated with Aβ_(42) before stimulation with a strong agonist,the C9 peptide.Calcium mobilization,c AMP inhibition and MAP kinase activation were measured.Intramolecular FRET were determined using CMKLR1 constructs with an ECFP attached to the C-terminus and a Fl As H binding motif embedded in the first intracellular loop(IL1).RESULTS Binding of both Aβ_(42) and the C9 peptide induced CMKLR1 internalization,but only the Aβ_(42)-induced receptor internalization involved clathrin-coated pits.Likewise,Aβ_(42) but not C9 stimulated β-arrestin 2 translocation to plasma membranes.A robust Ca^(2+)flux was observed following C9 stimulation,whereas Aβ_(42) was ineffective even at micromolar concentrations.Despite its low potency in calcium mobilization assay,Aβ_(42) was able to alter C9-induced Ca^(2+) flux in dose-dependent manner:a potentiation effect at 100 pmol·L^(-1) of Aβ_(42) was followed by a suppression at 10 nmol·L^(-1) and further potentiation at 1 μmol·L^(-1).This unusual and biphasic modulatory effect was also seen in the C9-induced ERK phosphorylation but the dose curve was opposite to that of Ca^(2+) flux and c AMP inhibition,suggesting a reciprocal regulatory mechanism.Intramolecular FRET assay confirmed that Aβ_(42) modulates CMKLR1 rather than its downstream signaling pathways.CONCLUSION These findings suggest Aβ_(42) as an allosteric modulator that can both positively and negatively regulate the activation state of CMKLR1 in a manner that differs from existing allosteric modulatory mechanisms.展开更多
OBJECTIVE To identify the mechanisms by which the formyl peptide receptor 2(FPR2)mediates both inflammatory and anti-inflammatory signaling in an agonist-dependent manner.METHODS Cells expressing FPR2 were incubated w...OBJECTIVE To identify the mechanisms by which the formyl peptide receptor 2(FPR2)mediates both inflammatory and anti-inflammatory signaling in an agonist-dependent manner.METHODS Cells expressing FPR2 were incubated with weak agonists,Aβ42 and Ac2-26,before stimulation with a strong agonist,WKYMVm.Calcium mobilization,c AMP inhibition and MAP kinase activation were measured.Intramolecular FRET were determined using FPR2 constructs with an ECFP attached to the C-terminus and a Fl As H binding motif embedded in the first or third intracellular loop(IL1 or IL3,respectively).RESULTS Aβ42 did not induce significant Ca^(2+) mobilization,but positively modulated WKYMVm-induced Ca^(2+) mobilization and c AMP reduction in a dose-variable manner within a narrow range of ligand concentrations.Treating FPR2-expressing cells with Ac2-26,a peptide with anti-inflammatory activity,negatively modulated WKYMVm-induced Ca^(2+) mobilization and c AMP reduction.Intramolecular FRET assay showed that stimulation of the receptor constructs with Aβ42 brought the C-terminal domain closer to IL1 but away from IL3.An opposite conformational change was induced by Ac2-26.The FPR2 conformation induced by Aβ42 corresponded to enhanced ERK phosphorylation and attenuated p38 MAPK phosphorylation,whereas Ac2-26 induced FPR2 conformational change corresponding to elevated p38 MAPK phosphorylation and reduced ERK phosphorylation.CONCLUSION Aβ42 and Ac2-26 induce different conformational changes in FPR2.These findings provide a structural basis for FPR2 mediation of inflammatory vs anti-inflammatory functions and identify a type of receptor modulation that differs from the classic positive and negative allosteric modulation.展开更多
In this communication,sulfate dodecyl sodium(SDS)-induced thermodynamics and conformational changes of collagen were studied.We used ultrasensitive differential scanning calorimetry(US-DSC)to directly monitor the ther...In this communication,sulfate dodecyl sodium(SDS)-induced thermodynamics and conformational changes of collagen were studied.We used ultrasensitive differential scanning calorimetry(US-DSC)to directly monitor the thermal transition of collagen in the presence of SDS.The results show that SDS affects the conformation and thermal stability of collagen very differently depending on its concentrations.At C SDS≤0.05mM,the enhanced thermal stability of collagen indicates the stabilizing effect by SDS.However,a further increase of SDS leads to the denaturation of collagen,verifying the well-known ability of SDS to unfold proteins.This striking difference in thermodynamics and conformational changes of collagen caused by SDS concentrations can be explained in terms of their interactions.With increasing SDS,the binding of SDS to collagen can be dominated by electrostatic interaction shifting to hydrophobic interaction,and the latter plays a key role in loosening and unfolding the triple-helix structure of collagen.The important finding in the present study is the stabilizing effect of SDS on collagen molecules at extreme low concentration.展开更多
The structure and function of protein are strongly pH-dependence. However, the inonization state of the protein varied with the pH value also depends on the local structure
基金Supported by the National Natural Science Foundation of China under Grant Nos 11247010,11175055,11475053 and 11347017the Natural Science Foundation of Hebei Province under Grant Nos C2012202079 and C201400305
文摘We find that a conserved mutation residue Glu to residue Asp (E303D), which both have the same polar and charged properties, makes Kit2.1 protein lose its function. To understand the mechanism, we identify three interactions which control the conformation change and maintain the function of the Kit2.1 protein by combining homology modeling and molecular dynamics with targeted molecular dynamics. We find that the E303D mutation weakens these interactions and results in the loss of the related function. Our data indicate that not only the amino residues but also the interactions determine the function of proteins.
基金supported by the National Key R&D Program of China (Nos. 2016YFA0400900, 2017YFA0505300)the Instrument Developing Project of the Chinese Academy of Sciences (No. YZ201564)
文摘Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy(FLIM), which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin(FPN), a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on Cterminal of FPN(FPN-eGFP) and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn^(2+) treatment,suggesting a preference conformation of FPN in Mn^(2+) exposure. It is also observed that the lifetime(rather than intensity) measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn^(2+) treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.51622201,91733301,and 61571015)
文摘Proteins are crucial to most biological processes, such as enzymes, and in various catalytic processes a dynamic motion is required. The dynamics of protein are embodied as a conformational change, which is closely related to the flexibility of protein. Recently, nanopore sensors have become accepted as a low cost and high throughput method to study the features of proteins. In this article, we used a SiN nanopore device to study the flexibility of T7 RNA polymerase(RNAP) and its complex with DNA promoter. By calculating full-width at half-maximum(FWHM) of Gaussian fits to the blockade histograms, we found that T7 RNAP becomes more flexible after binding DNA promoter. Moreover, the distribution of fractional current blockade suggests that flexibility alters due to a breath-like change of the volume.
基金supported by US National Institutes of Health grant R01 AI44902 (to C Z )a Pre-doctoral Fellowship from the American Heart Association (to W C )
文摘Introduction Integrins are a large family of adhesion molecules broadly expressed on the surface of a wide variety of cells as heterodimers. Binding of integrins to ligands provides not only mechanical anchorage for the cell to another cell or
基金Supported by Shaan xi Provincial Scientific- Comm ittee( 96 H0 9)
文摘The thermostability of some proteins in weak cation-exchange chromatography was investigated at 20-80 ℃. The results show that there is a fixed thermal denaturation transition temperature for each protein. The appearance of the thermal transition temperature indicates that the conformations of the proteins are destroyed seriously. The thermal behavior of the proteins in weak cation-exchange and hydrophobic interaction chromatographies were compared in a wide temperature range. It was found that the proteins have a higher thermostability in a weak cation-exchange chromatography system. The thermodynamic parameters(Δ H 0, Δ S 0) of those proteins were determined by means of Vant Hoff relationship(ln k -1/ T ). According to standard entropy change(Δ S 0), the conformational change of the proteins was judged in the chromatographic process. The linear relationships between Δ H 0 and Δ S 0 can be used to evaluate 'compensation temperature'( β ) at the protein denaturation and identify the identity of the protein retention mechanism in weak cation-exchange chromatography.
基金Supported by the National Natural Science Foundation of China(No. 20771068)the Natural Science Foundation of Shanxi Province, China(No. 2007011024)
文摘It is known that urea and guanidine hydrochloride(GuHC1) induce conformational change of proteins in a certain range of molar ratios. In our research, a-naphthylamine(NA) above 10^-4 mol/L at pH 7.0 was discovered to perturb the conformation of CopC, a copper resistant protein with a Greek fl-barrel motif; this was reflected by the greater fluorescence quenching and red-shifted emission peak of CopC. The conformation change of CopC was also verified in acrylamide collision experiment by comparing quenching levels of CopC in the presence or the abscence of NA. Comparison of emission change of CopC-Cu^2+ with that of apo-CopC showed a consistent result with their denaturation in GuHCl. And decreasing fluorescence polarization of CopC with increasing NA concentration is consistent with a more extend conformation of CopC. Interaction mechanism was also explored.
基金supported by the National Natural Science Foundation of China(32001037,22176035)the National Key R&D Program of China(2020YFA0210800,2018YFA0902600)+1 种基金the Natural Science Foundation of Fujian Province(2020J01491,2020J05120)Fuzhou University Research Fund(GXRC-20033)。
文摘Small molecule aptamers discovered by traditional selection methods usually lack conformational changes upon target binding.This limits the use of aptamers as molecular probes for small molecule detection and regulatory elements of genetic circuits.Here,we report a new method called capture and in vitro transcription-systematic evolution of ligands by exponential enrichment(CIVT-SELEX)to select DNA aptamers that can not only bind to small molecule ligands but also undergo significant conformational changes.Through this method,we select a structure-switching aptamer of uridine-5′-diphosphate(UDP).Taking advantage of its conformational changes,we first construct a UDP-responsive transcriptional switch by inserting the aptamer in a genetic circuit and demonstrate that it can respond to the addition of UDP and regulate the transcription of downstream genes.We also build a UDP aptamer-based biosensor that can be used for active glycosyltransferase screening.We believe this method can provide a universal platform for selecting small molecule aptamers with conformational changes and expand the use of aptamers in small molecule detection and genetic regulation.
基金supported by the National Key R&D Program of China (Nos. 2016YFA0400900, 2017YFA0505300)the Instrument Developing Project of the Chinese Academy of Sciences (No. YZ201564)
文摘Fluorescence lifetime and anisotropy has become a prevalent tool to detect the structure change and motility property of proteins. YgaP is the only membrane-integrated rhodanese in E. coli. The sulfur transfer process has been characterized by various studies. However, the mechanism of the outward transportation of SCN-remains unclear. In this work, we examined the fluorescence lifetime and anisotropy of site-specific incorporated unnatural amino acid 7-HC to study the conformational change of YgaP upon SCN-binding. We also compared the fluorescence changes between detergent-wrapped environment in DPC and intact native membrane environment in SMA. Our results suggested the presence of at least two different conformations in YgaP protein. Both the residues in the middle of TMH2 and the residues near extracellular side play important roles in the binding and/or output of SCN-. SMA is a good material to reflect the in situ conformation changes of protein than micelles.
基金the Tsinghua U niversity- HongKong Baptist U niversity Joint Institute for Research ofChinese Medicine the" 985" Fund fromTsinghua U niversit
文摘Genistein can induce not only differentiation but also apoptosis in HL60 human leukemia cells. During the differentiation and apoptosis, the HL60 cells undergo some conformational changes. Fourier transform infrared (FT IR) was employed to detect those changes. The results showed that the contents of DNA/protein and glycoprotein/protein increased. The α helix of the membrane protein also increased. In addition, the CO (H) stretching mode of serine, threonine and tyrosine residues of the cell proteins also changed. Those conformational changes suggest some mechanisms for how genistein affects the HL60 cells.
文摘The refractive index increment, dynamic and static laser light scattering, intrinsic viscosity [η] and Huggins constant (KH) of nylon 12 have been measured in m-cresol and sulphuric acid/water system at 10-60℃. The intrinsic viscosity, Rn, Rg, A2, and (〈 S 〉2)^1/2 (calculated from viscosity data) and "a" values of nylon 12 are found to be higher in m-cresol than in sulphuric acid. All these parameters decrease with the increase in water contents in sulphuric acid. The refractive index increment, KH and activation energy show an opposite trend to that of [η]. The intrinsic viscosity, RH, Rg, A2, and (〈 S 〉2)^1/2 have maximum values around 30-40℃ in sulphuric acid/water system, whereas in m-cresol they fall at about 20℃. It has been concluded that the variation in size, interaction parameter (second virial coefficient), [η] and KH of the polymer solutions with the alteration in solvent composition and temperature are the out come of change in thermodynamic quality of solvents, selective adsorption, hydrogen bonding and conformational transitions. It has also been concluded that the increase in temperature first enhances the quality of the solvent, encourages hydrogen bonding and specific adsorption, and then deteriorates, bringing conformational transitions in the polymer molecules. However, the addition of water to sulphuric acid continuously deteriorates the solvent quality. This characteristic of the solvent system brings conformational changes in the polymer especially at low temperatures.
基金supported by the National Natural Science Foundation of China (Nos.51633001,51721002 and 51873040)National Key R&D Program of China (No.2016YFC1100300)。
文摘Negative thermal expansion(NTE) behavior has roused wide interest for the control of thermomechanical properties of functional materials.Although NTE behaviors have been found in kinds of compounds,it remains challenging for polymers to achieve intrinsic NTE property.In this work,we systematically studied the conformational change of dibenzocyclooctadiene(DBCOD) derivatives between chair(C) and twist-boat(TB) forms based on density-functional theo ry(DFT) calculations,and found clear evidence of the relationship between the structure of DBCOD units and the thermal contraction behavior of the related polymers.In order to obtain the polymer with NTE property,two conditions should be met for the thermal contracting DBCOD related units as follows:(i) the TB conformation can turn into C conformation as the temperature increases,and(ii) the volume of C conformation is smaller than that of TB conformation.This rule should offer a guidance to exploration of the new intrinsic NTE polymers in the future.
基金Supported by the National Basic Research Program of China(No.2006CB101706)the Hi-tech Research and DevelopmentProgram of China(No.2007AA100604)the National Natural Science Foundation of China(Nos.30170080and39770078).
文摘Phospholipid hydroperoxide glutathione peroxidase is an antioxidant enzyme that has the highest capability of reducing membrane-bound hydroperoxy lipids as compared to free organic and inorganic hydroperoxides amongst the glutathione peroxidases.In this study,urea-induced effects on the inactivation and unfolding of a recombinant phospholipid hydroperoxide glutathione peroxidase(PHGPx)from Oryza sativa were investigated by means of circular dichroism and fluorescence spectroscopy.With the increase of urea concentration,the residual activity of OsPHGPx decreases correspondingly.When the urea concentration is above 5.0 mol/L,there was no residual activity.In addition,the observed changes in intrinsic tryptophan fluorescence,the binding of the hydrophobic fluorescence probe ANS,and the far UV CD describe a common dependence on the concentration of urea suggesting that the conformational features of the native OsPHGPx are lost in a highly cooperative single transition.The unfolding process comprises of three zones:the native base-line zone between 0 and 2.5 mol/L urea,the transition zone between 2.5 and 5.5 mol/L urea,and the denatured base-line zone above 5.5 mol/L urea.The transition zone has a midpoint at about 4.0 mol/L urea.
文摘It has been reported that inactivation occurs before noticeable conformational changes can be detected during denaturation of creatine kinase and other enzymes. Therefore, Tsou suggested that enzyme active sites may display more conformational flexibility than the enzyme molecules as a whole. In the present investigation, the conformational changes of Penaeus penicillatus acid phosphatase during denaturation in urea solutions were studied by following changes in the intrinsic fluorescence, ultraviolet difference absorption, and circular dichroism spectra. Inactivation of the enzyme in urea solutions was compared with unfolding of the enzyme molecule. The results show that the extent of unfolding in guanidine solutions measured by several different methods closely coincides with each other and that slightly lower concentrations of guanidine are required to bring about inactivation than are required to produce significant conformational changes of the enzyme molecule. At the same concentrations, the inactivation rate constants are markedly faster than the rate constants for unfolding of the enzyme. The above results suggest that the active sites of this enzyme display more conformational flexibility than the enzyme molecule as a whole.
基金Project supported by the Natural Science Foundation of Shandong Province of China (Grant No. ZR2011AL010)the National Natural Science Foundation of China (Grant Nos. NSFC-10974078 and NSFC-11174117)
文摘Encapsulation of biomolecules inside a carbon nanotube (CNT) has attracted great interest because it could enable the delivery of nanoscale pharmaceutical drugs with CNT-based devices. Using a molecular dynamics simulation, we investigate the dynamic process by which human immunodeficiency virus (HIV) replication inhibitor peptides (HRIPs) are encapsulated in a water solution contained inside a CNT. The van der Waals attraction between the HRIPs and the CNT and the root-mean-square deviation are used to analyse the evolution of the encapsulation. It is found that the interaction between the HRIPs and the CNT is the main driving force for the encapsulation process, which does not cause an obvious conformational change to the HRIPs.
基金Supported by the National Natural Science Foundation of China(Nos.30400081 and 20772046)
文摘The gene of enzyme(Ape1547) was cloned from hyperthermophilic archaeon Aeropyrum pernix K1 and expressed in Escherichia coil.The effect of calcium cation on the properties of Ape1547 was studied.Ape1547 exhibits both peptidase activity and esterase activity.The fluorescence spectrum shows that calcium cation quenches the fluorescence of the enzyme through static quenching mechanism,indicating that calcium cation was bound to the enzyme.Based on the study of calcium cation on CD ellipticity of Ape1547 by circular dichroism,we concluded that the change of enzyme structure induced by calcium cation may be responsible for the change of enzyme activity.Calcium cation has dual effects on Ape1547:it could activate the enzyme activity when its concentration was 0.1 mol/L,and the enzyme had the highest activity;however,when its concentration was higher than 0.2 mol/L,the enzyme activity was inhibited.The results indicate that the activity center of peptidase activity might involve more amino acid residues than that of esterase activity.
文摘The effects of oligodeoxynucleotide (ODN) on the conformation of basic fibroblast growth factor (bFGF) were studied by spectral method. The results showed that ODN destabilized the protein.
基金supported by National Natural Science Foundation of China(31470856 to RDY)the Science and Technology Development Fund of Macao(FDCT 072/2015/A2)the University of Macao(SRG2015-00047-ICMS-QRCM)
文摘OBJECTIVE The chemokine-like receptor 1(CMKLR1,Chem R23) is a functional receptor for chemerin,the chemerin-derived nonapeptide(C9),and the amyloid β peptide 1-42(Aβ_(42)).Because these peptides share little sequence homology,studies were conducted to investigate their pharmacological properties and regulation at CMKLR1.METHODS Cells expressing CMKLR1 were incubated with Aβ_(42) before stimulation with a strong agonist,the C9 peptide.Calcium mobilization,c AMP inhibition and MAP kinase activation were measured.Intramolecular FRET were determined using CMKLR1 constructs with an ECFP attached to the C-terminus and a Fl As H binding motif embedded in the first intracellular loop(IL1).RESULTS Binding of both Aβ_(42) and the C9 peptide induced CMKLR1 internalization,but only the Aβ_(42)-induced receptor internalization involved clathrin-coated pits.Likewise,Aβ_(42) but not C9 stimulated β-arrestin 2 translocation to plasma membranes.A robust Ca^(2+)flux was observed following C9 stimulation,whereas Aβ_(42) was ineffective even at micromolar concentrations.Despite its low potency in calcium mobilization assay,Aβ_(42) was able to alter C9-induced Ca^(2+) flux in dose-dependent manner:a potentiation effect at 100 pmol·L^(-1) of Aβ_(42) was followed by a suppression at 10 nmol·L^(-1) and further potentiation at 1 μmol·L^(-1).This unusual and biphasic modulatory effect was also seen in the C9-induced ERK phosphorylation but the dose curve was opposite to that of Ca^(2+) flux and c AMP inhibition,suggesting a reciprocal regulatory mechanism.Intramolecular FRET assay confirmed that Aβ_(42) modulates CMKLR1 rather than its downstream signaling pathways.CONCLUSION These findings suggest Aβ_(42) as an allosteric modulator that can both positively and negatively regulate the activation state of CMKLR1 in a manner that differs from existing allosteric modulatory mechanisms.
基金supported by National Natural Science Foundation of China(31470856 to RDY)the Science and Technology Development Fund of Macao(FDCT 072/2015/A2)the University of Macao(SRG2015-00047-ICMS-QRCM)
文摘OBJECTIVE To identify the mechanisms by which the formyl peptide receptor 2(FPR2)mediates both inflammatory and anti-inflammatory signaling in an agonist-dependent manner.METHODS Cells expressing FPR2 were incubated with weak agonists,Aβ42 and Ac2-26,before stimulation with a strong agonist,WKYMVm.Calcium mobilization,c AMP inhibition and MAP kinase activation were measured.Intramolecular FRET were determined using FPR2 constructs with an ECFP attached to the C-terminus and a Fl As H binding motif embedded in the first or third intracellular loop(IL1 or IL3,respectively).RESULTS Aβ42 did not induce significant Ca^(2+) mobilization,but positively modulated WKYMVm-induced Ca^(2+) mobilization and c AMP reduction in a dose-variable manner within a narrow range of ligand concentrations.Treating FPR2-expressing cells with Ac2-26,a peptide with anti-inflammatory activity,negatively modulated WKYMVm-induced Ca^(2+) mobilization and c AMP reduction.Intramolecular FRET assay showed that stimulation of the receptor constructs with Aβ42 brought the C-terminal domain closer to IL1 but away from IL3.An opposite conformational change was induced by Ac2-26.The FPR2 conformation induced by Aβ42 corresponded to enhanced ERK phosphorylation and attenuated p38 MAPK phosphorylation,whereas Ac2-26 induced FPR2 conformational change corresponding to elevated p38 MAPK phosphorylation and reduced ERK phosphorylation.CONCLUSION Aβ42 and Ac2-26 induce different conformational changes in FPR2.These findings provide a structural basis for FPR2 mediation of inflammatory vs anti-inflammatory functions and identify a type of receptor modulation that differs from the classic positive and negative allosteric modulation.
基金The financial support of National Natural Science Foundation(NNSF)of China(21978177)the National Key R&D Program of China(2019YFC1904500)International Science and Technology Innovation Cooperation project of Sichuan Province(2020YFH0081)is gratefully acknowledged.
文摘In this communication,sulfate dodecyl sodium(SDS)-induced thermodynamics and conformational changes of collagen were studied.We used ultrasensitive differential scanning calorimetry(US-DSC)to directly monitor the thermal transition of collagen in the presence of SDS.The results show that SDS affects the conformation and thermal stability of collagen very differently depending on its concentrations.At C SDS≤0.05mM,the enhanced thermal stability of collagen indicates the stabilizing effect by SDS.However,a further increase of SDS leads to the denaturation of collagen,verifying the well-known ability of SDS to unfold proteins.This striking difference in thermodynamics and conformational changes of collagen caused by SDS concentrations can be explained in terms of their interactions.With increasing SDS,the binding of SDS to collagen can be dominated by electrostatic interaction shifting to hydrophobic interaction,and the latter plays a key role in loosening and unfolding the triple-helix structure of collagen.The important finding in the present study is the stabilizing effect of SDS on collagen molecules at extreme low concentration.
文摘The structure and function of protein are strongly pH-dependence. However, the inonization state of the protein varied with the pH value also depends on the local structure