The mechanism of urea's action in protein denaturation remains largely unknown.To provide an experimental basis for molecular dynamics(MD)simulations on urea-protein interactions,we investigated the effect of urea...The mechanism of urea's action in protein denaturation remains largely unknown.To provide an experimental basis for molecular dynamics(MD)simulations on urea-protein interactions,we investigated the effect of urea on human intestinal fatty acid binding protein(hIFABP)by nuclear magnetic resonance(NMR).Hydrogen-deuterium exchange(HDX)rates at2 M urea indicate that urea affects hIFABP in a residue-specific manner via direct urea-protein interactions and preferentially weakens hydrogen bonds between highly protected amides.Residue-specific effects of urea on NMR peak intensities and chemical shifts further support the presence of direct urea-protein interactions.Twodimensional(2D)water-rotating frame Overhauser enhancement(ROE)data shows one protein-bound water molecule in contact with Val66 and Trp82,one putative bound water molecule in interaction with Thr76 and E-F loop,and that urea at low concentrations cannot displace these protein-bound water molecules.Our urea-nuclear Overhauser effect(NOE)experiments using 15N-urea further show no tightly protein-bound urea molecules.Our results thus suggest specific,but weak or transient,urea-protein interactions,supporting the direct interaction model of urea denaturation.展开更多
Nickel,an important transi-tion metal element,is one of the trace elements for hu-man body and has a crucial impact on life and health.Some evidences show the excess exposure to metal ions might be associated with neu...Nickel,an important transi-tion metal element,is one of the trace elements for hu-man body and has a crucial impact on life and health.Some evidences show the excess exposure to metal ions might be associated with neurological diseases.Herein,we applied Raman spectroscopy to study the Ni(II)ion effect on kinetics of amyloid fibrillation of hen egg white lysozyme(HEWL)in thermal and acidic conditions.Using the well-known Raman indicators for protein tertiary and secondary structures,we monitored and analyzed the concentration effect of Ni(II)ions on the unfolding of tertiary structures and the transformation of sec-ondary structures.The experimental evidence validates the accelerator role of the metal ion in the kinetics.Notably,the additional analysis of the amide I band profile,combined with thioflavin-T fluorescence assays,clearly indicates the inhibitory effect of Ni(II)ions on the formation of amyloid fibrils with organizedβ-sheets structures.Instead,a more significant promotion influence is affirmed on the assembly into other aggregates with disordered struc-tures.The present results provide rich information about the specific metal-mediated protein fibrillation.展开更多
Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). ...Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). These protein samples were 99% pure HSA (termed HSA<sub>99</sub>) and 96% pure HSA (termed HSA<sub>96</sub>). According to the supplier, the 3% difference in purity between HSA<sub>96</sub> and HSA<sub>99</sub> is primarily attributed to the presence of globulins and fatty acids. Our primary aim was to investigate the utility of DSC in discerning changes in HSA that occur when the protein is specifically adducted, and determine how adduct formation manifests itself in HSA denaturation curves, or thermograms, measured by DSC. Effects of site specific covalent attachment of biotin (the adduct) on the thermodynamic stability of HSA were investigated. Each of the HSA preparations was modified by biotinylation targeting a single site, or multiple sites on the protein structure. Thermograms of both modified and unmodified HSA samples successfully demonstrated the ability of DSC to clearly discern the two HSA preparations and the presence or absence of covalent modifications. DSC thermogram analysis also provided thermodynamic characterization of the different HSA samples of the study, which provided insight into how the two forms of HSA respond to covalent modification with biotin. Consistent with published studies [1] HSA<sub>96</sub>, the preparation with contaminants that contain globulins and fatty acids seems to be comprised of two forms, HSA<sub>96-L</sub> and HSA<sub>96-H</sub>, with HSA<sub>96-L</sub> more stable than HSA<sub>99</sub>. The effect of multisite biotinylation is to stabilize HSA<sub>96-L</sub> and destabilize HSA<sub>96-H</sub>. Thermodynamic analysis suggests that the binding of ligands comprising the fatty acid and globulin-like contaminant contributes approximately 6.7 kcal/mol to the stability HSA<sub>96-L</sub>.展开更多
The hollow structure has long attracted great attention because of its excellent properties.However,this special structure is usually synthesized through some complex approaches.Herein,we discovered that denatured bov...The hollow structure has long attracted great attention because of its excellent properties.However,this special structure is usually synthesized through some complex approaches.Herein,we discovered that denatured bovine serum albumin(BSA)can trigger unusual biomineralization for the simple,green and shape-controllable synthesis of germanium oxide(GeOx)hollow microsphere(HMS).At high temperature(60℃),BSA was denatured,and a compact BSA layer was formed around the H2 bubbles.The denatured BSA layer was stable and suitable for anchoring and growing GeOx.By simply changing the BSA concentration and temperature,various morphologies of GeOx could be obtained.Due to the denatured protein skeletons and microenvironment-regulated collapse,GeOx HMS showed great potential for intelligently responsive pesticide delivery in the insect gut,showing superiority over traditional delivery systems,which early release pesticides in the mouth and stomach.Inspired by its large specific surface area,excellent biocompatibility,modifiable functional groups,and high electrocatalytic activity,GeOx HMS was also applied to versatile sensors for H_(2)O_(2) assays at physiological pH and rapid coronavirus COVID-19 detection.This work not only provides some evidence for understanding proteins in depth but also paves a new avenue for the biomineralization-inspired synthesis of hollow structures with versatile functions.展开更多
基金supported by a grant to D.Y.from Singapore Ministry of Education Academic Research Fund Tier 1(R154-000-C03e114 or A-0004464-00-00).
文摘The mechanism of urea's action in protein denaturation remains largely unknown.To provide an experimental basis for molecular dynamics(MD)simulations on urea-protein interactions,we investigated the effect of urea on human intestinal fatty acid binding protein(hIFABP)by nuclear magnetic resonance(NMR).Hydrogen-deuterium exchange(HDX)rates at2 M urea indicate that urea affects hIFABP in a residue-specific manner via direct urea-protein interactions and preferentially weakens hydrogen bonds between highly protected amides.Residue-specific effects of urea on NMR peak intensities and chemical shifts further support the presence of direct urea-protein interactions.Twodimensional(2D)water-rotating frame Overhauser enhancement(ROE)data shows one protein-bound water molecule in contact with Val66 and Trp82,one putative bound water molecule in interaction with Thr76 and E-F loop,and that urea at low concentrations cannot displace these protein-bound water molecules.Our urea-nuclear Overhauser effect(NOE)experiments using 15N-urea further show no tightly protein-bound urea molecules.Our results thus suggest specific,but weak or transient,urea-protein interactions,supporting the direct interaction model of urea denaturation.
基金supported by the National Natural Science Foundation of China(No.22073088,No.22027801 and No.21873089).
文摘Nickel,an important transi-tion metal element,is one of the trace elements for hu-man body and has a crucial impact on life and health.Some evidences show the excess exposure to metal ions might be associated with neurological diseases.Herein,we applied Raman spectroscopy to study the Ni(II)ion effect on kinetics of amyloid fibrillation of hen egg white lysozyme(HEWL)in thermal and acidic conditions.Using the well-known Raman indicators for protein tertiary and secondary structures,we monitored and analyzed the concentration effect of Ni(II)ions on the unfolding of tertiary structures and the transformation of sec-ondary structures.The experimental evidence validates the accelerator role of the metal ion in the kinetics.Notably,the additional analysis of the amide I band profile,combined with thioflavin-T fluorescence assays,clearly indicates the inhibitory effect of Ni(II)ions on the formation of amyloid fibrils with organizedβ-sheets structures.Instead,a more significant promotion influence is affirmed on the assembly into other aggregates with disordered struc-tures.The present results provide rich information about the specific metal-mediated protein fibrillation.
文摘Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). These protein samples were 99% pure HSA (termed HSA<sub>99</sub>) and 96% pure HSA (termed HSA<sub>96</sub>). According to the supplier, the 3% difference in purity between HSA<sub>96</sub> and HSA<sub>99</sub> is primarily attributed to the presence of globulins and fatty acids. Our primary aim was to investigate the utility of DSC in discerning changes in HSA that occur when the protein is specifically adducted, and determine how adduct formation manifests itself in HSA denaturation curves, or thermograms, measured by DSC. Effects of site specific covalent attachment of biotin (the adduct) on the thermodynamic stability of HSA were investigated. Each of the HSA preparations was modified by biotinylation targeting a single site, or multiple sites on the protein structure. Thermograms of both modified and unmodified HSA samples successfully demonstrated the ability of DSC to clearly discern the two HSA preparations and the presence or absence of covalent modifications. DSC thermogram analysis also provided thermodynamic characterization of the different HSA samples of the study, which provided insight into how the two forms of HSA respond to covalent modification with biotin. Consistent with published studies [1] HSA<sub>96</sub>, the preparation with contaminants that contain globulins and fatty acids seems to be comprised of two forms, HSA<sub>96-L</sub> and HSA<sub>96-H</sub>, with HSA<sub>96-L</sub> more stable than HSA<sub>99</sub>. The effect of multisite biotinylation is to stabilize HSA<sub>96-L</sub> and destabilize HSA<sub>96-H</sub>. Thermodynamic analysis suggests that the binding of ligands comprising the fatty acid and globulin-like contaminant contributes approximately 6.7 kcal/mol to the stability HSA<sub>96-L</sub>.
基金National Natural Science Foundation of China,Grant/Award Number:21705087Natural Science Foundation of Shandong Province,Grant/Award Number:ZR2021MB018+1 种基金Research Foundation for Distinguished Scholars of Qingdao Agricultural University,Grant/Award Number:663-1117015Introducing and Breeding Plan of Shandong Province Higher Educational Youth Innovation Talents(Shandong Provincial Department of Education)。
文摘The hollow structure has long attracted great attention because of its excellent properties.However,this special structure is usually synthesized through some complex approaches.Herein,we discovered that denatured bovine serum albumin(BSA)can trigger unusual biomineralization for the simple,green and shape-controllable synthesis of germanium oxide(GeOx)hollow microsphere(HMS).At high temperature(60℃),BSA was denatured,and a compact BSA layer was formed around the H2 bubbles.The denatured BSA layer was stable and suitable for anchoring and growing GeOx.By simply changing the BSA concentration and temperature,various morphologies of GeOx could be obtained.Due to the denatured protein skeletons and microenvironment-regulated collapse,GeOx HMS showed great potential for intelligently responsive pesticide delivery in the insect gut,showing superiority over traditional delivery systems,which early release pesticides in the mouth and stomach.Inspired by its large specific surface area,excellent biocompatibility,modifiable functional groups,and high electrocatalytic activity,GeOx HMS was also applied to versatile sensors for H_(2)O_(2) assays at physiological pH and rapid coronavirus COVID-19 detection.This work not only provides some evidence for understanding proteins in depth but also paves a new avenue for the biomineralization-inspired synthesis of hollow structures with versatile functions.
