The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
Shuqun Liu - One of the best experts on this subject based on the ideXlab platform.
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molecular motions and free energy landscape of serine Proteinase K in relation to its cold adaptation a comparative molecular dynamics simulation study and the underlying mechanisms
RSC Advances, 2017Co-Authors: Shuqun Liu, Zhao Hui Meng, Peng Sang, Li Quan YangAbstract:The physicochemical bases for enzyme cold-adaptation remain elusive. The current view is that psychrophilic enzymes are often characterized by enhanced flexibility at low temperature to obtain higher catalytic efficiency, but the determinant behind this phenomenon is less well Known. To shed light on the physicochemical bases for enzyme cold-adaptation, we conducted comparative molecular dynamics simulations on mesophilic Proteinase K and its homologous psychrophilic counterpart. Results revealed that psychrophilic Proteinase K had increased flexibility in regions near or opposite to active site or substrate-binding pocKet. Comparison between the large concerted motions derived from essential dynamics (ED) analyses indicated that the degree of motion and direction of some regions in psychrophilic Proteinase K could enlarge the substrate-binding pocKet, thereby favoring catalytic efficiency and cold-adaptation. Free-energy calculations based on metadynamics simulations revealed a more “rugged” and complex free-energy landscape (FEL) for psychrophilic Proteinase K than that for mesophilic Proteinase K, implying that the former had richer conformational diversity. Comparison between the structural properties of the mesophilic and psychrophilic forms of Proteinase K during MD simulations showed that the increased flexibility of the psychrophilic form resulted most probably from the reduced number of inter-atomic interactions and increased number of dynamic hydrogen bonds. A refined model of FEL was proposed to explain the effect of water molecules in facilitation of enzyme cold-adaptation.
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effect of the solvent temperatures on dynamics of serine protease Proteinase K
International Journal of Molecular Sciences, 2016Co-Authors: Shuqun Liu, Zhao Hui Meng, Peng Sang, Qiong Yang, Nan Yang, Li Quan YangAbstract:To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease Proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of Proteinase K are strongly dependent on the solvent temperatures but weaKly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein.
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insight derived from molecular dynamics simulation into substrate induced changes in protein motions of Proteinase K
Journal of Biomolecular Structure & Dynamics, 2010Co-Authors: Yan Tao, Zihe Rao, Shuqun LiuAbstract:Abstract Because of the significant industrial, agricultural and biotechnological importance of serine protease Proteinase K, it has been extensively investigated using experimental approaches such as X-ray crystallography, site-directed mutagenesis and Kinetic measurement. However, detailed aspects of enzymatic mechanism such as substrate binding, release and relevant regulation remain unstudied. Molecular dynamics (MD) simulations of the Proteinase K alone and in complex with the peptide substrate AAPA were performed to investigate the effect of substrate binding on the dynamics/molecular motions of Proteinase K. The results indicate that during simulations the substrate-complexed Proteinase K adopt a more compact and stable conformation than the substrate-free form. Further essential dynamics (ED) analysis reveals that the major internal motions are confined within a subspace of very small dimension. Upon substrate binding, the overall flexibility of the protease is reduced; and the noticeable displace...
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insights derived from molecular dynamics simulation into the molecular motions of serine protease Proteinase K
Journal of Molecular Modeling, 2010Co-Authors: Shuqun Liu, Zhao Hui Meng, Keqin ZhangAbstract:Serine protease Proteinase K, a member of the subtilisin family of enzymes, is of significant industrial, agricultural and biotechnological importance. Despite the wealth of structural information about Proteinase K provided by static X-ray structures, a full understanding of the enzymatic mechanism requires further insight into the dynamic properties of this enzyme. Molecular dynamics simulations and essential dynamics (ED) analysis were performed to investigate the molecular motions in Proteinase K. The results indicate that the internal core of Proteinase K is relatively rigid, whereas the surface-exposed loops, most notably the substrate-binding regions, exhibit considerable conformational fluctuations. Further ED analysis reveals that the large concerted motions in the substrate-binding regions cause opening/closing of the substrate-binding pocKets, thus supporting the proposed induced-fit mechanism of substrate binding. The distinct electrostatic/hydrogen-bonding interactions between Asp39 and His69 and between His69 and Ser224 within the catalytic triad lead to different thermal motions and orientations of these three catalytic residues, which can be related to their different functional roles in the catalytic process. Statistical analyses of the geometrical/functional properties as well as evolutionary conservation of the glycines in Proteinase K-liKe proteins reveal that glycines may play an important role in determining the folding architecture and structural flexibility of this class of enzymes. Our simulation study complements the biochemical and structural studies and provides new insights into the dynamic structural basis of the functional properties of this class of enzymes.
Vasanti V Deshpande - One of the best experts on this subject based on the ideXlab platform.
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slow tight binding inhibition of Proteinase K by a proteinaceous inhibitor conformational alterations responsible for conferring irreversibility to the enzyme inhibitor complex
Journal of Biological Chemistry, 2003Co-Authors: Jui Pandhare, Chandravanu Dash, Vasanti V DeshpandeAbstract:Abstract The Kinetics of slow onset inhibition of Proteinase K by a proteinaceous alKaline protease inhibitor (API) from a Streptomyces sp. is presented. The Kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 ± 0.5 × 10–5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 ± 0.6 × 10–6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant K5 (9.2 ± 1 × 10–3 s–1). The rate of dissociation of EI* (K6) was slower (4.5 ± 0.5 × 10–5 s–1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 ± 0.3 × 10–7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locKed EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding networK between the catalytic triad and other residues involved in catalysis.
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slow tight binding inhibition of Proteinase K by a proteinaceous inhibitor conformational alterations responsible for conferring irreversibility to the enzyme inhibitor complex
Journal of Biological Chemistry, 2003Co-Authors: Jui Pandhare, Chandravanu Dash, Vasanti V DeshpandeAbstract:Abstract The Kinetics of slow onset inhibition of Proteinase K by a proteinaceous alKaline protease inhibitor (API) from a Streptomyces sp. is presented. The Kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 ± 0.5 × 10–5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 ± 0.6 × 10–6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant K5 (9.2 ± 1 × 10–3 s–1). The rate of dissociation of EI* (K6) was slower (4.5 ± 0.5 × 10–5 s–1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 ± 0.3 × 10–7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locKed EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding networK between the catalytic triad and other residues involved in catalysis.
Martin H Groschup - One of the best experts on this subject based on the ideXlab platform.
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differences in Proteinase K resistance and neuronal deposition of abnormal prion proteins characterize bovine spongiform encephalopathy bse and scrapie strains
Molecular Medicine, 1999Co-Authors: Thorsten Kuczius, Martin H GroschupAbstract:Prion diseases are associated with the accumulation of an abnormal isoform of host-encoded prion protein (PrP(Sc)). A number of prion strains can be distinguished by "glycotyping" analysis of the respective deposited PrP(Sc) compound. In this study, the long-term Proteinase K resistance, the molecular mass, and the localization of PrP(Sc) deposits derived from conventional and transgenic mice inoculated with 11 different BSE and scrapie strains or isolates were examined. Differences were found in the long-term Proteinase K resistance (50 microg/ml at 37 degrees C) of PrP(Sc). For example, scrapie strain Chandler or PrP(Sc) derived from field BSE isolates were destroyed after 6 hr of exposure, whereas PrP(Sc) of strains 87V and ME7 and of the Hessen1 isolate were extremely resistant to proteolytic cleavage. Nonglycosylated, Proteinase K-treated PrP(Sc) of BSE isolates and of scrapie strain 87V exhibited a 1-2 KD lower molecular mass than PrP(Sc) derived from all other scrapie strains and isolates. With the exception of strain 87V, PrP(Sc) was generally deposited in the cerebrum, cerebellum, and brain stem of different mouse lines at comparable levels. Long-term Proteinase resistance, molecular mass, and the analysis of PrP(Sc) deposition therefore provide useful criteria in discriminating prion strains and isolates (e.g., BSE and 87V) that are otherwise indistinguishable by the PrP(Sc) "glycotyping" technique.
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Differences in Proteinase K Resistance and Neuronal Deposition of Abnormal Prion Proteins Characterize Bovine Spongiform Encephalopathy (BSE) and Scrapie Strains
Molecular Medicine, 1999Co-Authors: Thorsten Kuczius, Martin H GroschupAbstract:Prion diseases are associated with the accumulation of an abnormal isoform of host-encoded prion protein (PrP^Sc). A number of prion strains can be distinguished by “glycotyping” analysis of the respective deposited PrP^Sc compound. In this study, the long-term Proteinase K resistance, the molecular mass, and the localization of PrP^Sc deposits derived from conventional and transgenic mice inoculated with 11 different BSE and scrapie strains or isolates were examined. Differences were found in the long-term Proteinase K resistance (50 µ g/ml at 37°C) of PrP^Sc. For example, scrapie strain Chandler or PrP^Sc derived from field BSE isolates were destroyed after 6 hr of exposure, whereas PrP^Sc of strains 87V and ME7 and of the Hessen1 isolate were extremely resistant to proteolytic cleavage. Nonglycosylated, Proteinase K-treated PrP^Sc of BSE isolates and of scrapie strain 87V exhibited a 1–2 KD lower molecular mass than PrP^Sc derived from all other scrapie strains and isolates. With the exception of strain 87V, PrP^Sc was generally deposited in the cerebrum, cerebellum, and brain stem of different mouse lines at comparable levels. Long-term Proteinase resistance, molecular mass, and the analysis of PrP^Sc deposition therefore provide useful criteria in discriminating prion strains and isolates (e.g., BSE and 87V) that are otherwise indistinguishable by the PrP^Sc “glycotyping” technique.
Jesus R Requena - One of the best experts on this subject based on the ideXlab platform.
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Proteinase K and the structure of prpsc the good the bad and the ugly
Virus Research, 2015Co-Authors: Christopher J Silva, Bruce Onisko, Ester Vazquezfernandez, Jesus R RequenaAbstract:Infectious proteins (prions) are, ironically, defined by their resistance to proteolytic digestion. A defining characteristic of the transmissible isoform of the prion protein (PrP(Sc)) is its partial resistance to Proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP(Sc) by Western blot, ELISA or immunohistochemical detection. PK digestion has also been used to detect differences in prion strains. Thus, PK has been a crucial tool to detect and, thereby, control the spread of prions. PK has also been used as a tool to probe the structure of PrP(Sc). Mass spectrometry and antibodies have been used to identify PK cleavage sites in PrP(Sc). These results have been used to identify the more accessible, flexible stretches connecting the β-strand components in PrP(Sc). These data, combined with physical constraints imposed by spectroscopic results, were used to propose a qualitative model for the structure of PrP(Sc). Assuming that PrP(Sc) is a four rung β-solenoid, we have threaded the PrP sequence to satisfy the PK proteolysis data and other experimental constraints.
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isolation and characterization of a Proteinase K sensitive prpsc fraction
Biochemistry, 2006Co-Authors: Miguel A Pastrana, Joaquin Castilla, Gustavo Sajnani, Bruce Onisko, Rodrigo Morales, Claudio Soto, Jesus R RequenaAbstract:Recent studies have shown that a sizable fraction of PrPSc present in prion-infected tissues is, contrary to previous conceptions, sensitive to digestion by Proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in attempts to distinguish between PrPSc and PrPC. Even more importantly, PK-sensitive PrPSc (sPrPSc) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrPSc. This material was obtained by differential centrifugation at an intermediate speed of Syrian hamster PrPSc obtained through a conventional procedure based on ultracentrifugation in the presence of detergents. PK-sensitive PrPSc is completely degraded under standard conditions (50 μg/mL of Proteinase K at 37 °C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrPSc to correspond to the lower molecular weight fractions of the contin...
Jui Pandhare - One of the best experts on this subject based on the ideXlab platform.
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slow tight binding inhibition of Proteinase K by a proteinaceous inhibitor conformational alterations responsible for conferring irreversibility to the enzyme inhibitor complex
Journal of Biological Chemistry, 2003Co-Authors: Jui Pandhare, Chandravanu Dash, Vasanti V DeshpandeAbstract:Abstract The Kinetics of slow onset inhibition of Proteinase K by a proteinaceous alKaline protease inhibitor (API) from a Streptomyces sp. is presented. The Kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 ± 0.5 × 10–5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 ± 0.6 × 10–6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant K5 (9.2 ± 1 × 10–3 s–1). The rate of dissociation of EI* (K6) was slower (4.5 ± 0.5 × 10–5 s–1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 ± 0.3 × 10–7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locKed EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding networK between the catalytic triad and other residues involved in catalysis.
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slow tight binding inhibition of Proteinase K by a proteinaceous inhibitor conformational alterations responsible for conferring irreversibility to the enzyme inhibitor complex
Journal of Biological Chemistry, 2003Co-Authors: Jui Pandhare, Chandravanu Dash, Vasanti V DeshpandeAbstract:Abstract The Kinetics of slow onset inhibition of Proteinase K by a proteinaceous alKaline protease inhibitor (API) from a Streptomyces sp. is presented. The Kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 ± 0.5 × 10–5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 ± 0.6 × 10–6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant K5 (9.2 ± 1 × 10–3 s–1). The rate of dissociation of EI* (K6) was slower (4.5 ± 0.5 × 10–5 s–1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 ± 0.3 × 10–7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locKed EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding networK between the catalytic triad and other residues involved in catalysis.
