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BBA - Proteins and Proteomics (v.1824, #5)
Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase by Tatyana Belinskaya; Nagarajan Pattabiraman; Robert diTargiani; Moonsuk Choi; Ashima Saxena (pp. 701-710).
Senescence marker protein-30 (SMP-30) is a candidate enzyme that can function as a catalytic bioscavenger of organophosphorus (OP) nerve agents. We purified SMP-30 from mouse (Mo) liver and compared its hydrolytic activity towards various esters, lactones, and G-type nerve agents with that of human paraoxonase1 (Hu PON1) and squid diisopropylfluorophosphatase (DFPase). All three enzymes contain one or two metal ions in their active sites and fold into six-bladed β-propeller structures. While Hu PON1 hydrolyzed a variety of lactones, the only lactone that was a substrate for Mo SMP-30 wasd-(+)-gluconic acid δ-lactone. Squid DFPase was much more efficient at hydrolyzing DFP and G-type nerve agents as compared to Mo SMP-30 or Hu PON1. The Km values for DFP were in the following order: Mo SMP-30>Hu PON1>squid DFPase, suggesting that the efficiency of DFP hydrolysis may be related to its binding in the active sites of these enzymes. Thus, homology modeling and docking were used to simulate the binding of DFP and selected δ-lactones in the active sites of Hu SMP-30, Hu PON1, and squid DFPase. Results from molecular modeling studies suggest that differences in metal–ligand coordinations, the hydrophobicity of the binding pockets, and limited space in the binding pocket due to the presence of a loop, are responsible for substrate specificities of these enzymes.► Hydrolytic activities of Mo SMP-30, Hu PON1 and squid DFPase were compared. ► Mo SMP-30 hydrolyzed only D-(+)-gluconic acid δ-lactone. ► Hu PON1 hydrolyzed a variety of lactones; squid DFPase did not hydrolyze any lactones. ► Squid DFPase hydrolyzed OPs more efficiently as compared to Mo SMP-30 and Hu PON1. ► The observed substrate specificities were explained by molecular modeling studies.
Keywords: Mouse SMP-30; Squid DFPase; Human PON1; DFP; Catalytic bioscavenger; Molecular docking
Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase by Tatyana Belinskaya; Nagarajan Pattabiraman; Robert diTargiani; Moonsuk Choi; Ashima Saxena (pp. 701-710).
Senescence marker protein-30 (SMP-30) is a candidate enzyme that can function as a catalytic bioscavenger of organophosphorus (OP) nerve agents. We purified SMP-30 from mouse (Mo) liver and compared its hydrolytic activity towards various esters, lactones, and G-type nerve agents with that of human paraoxonase1 (Hu PON1) and squid diisopropylfluorophosphatase (DFPase). All three enzymes contain one or two metal ions in their active sites and fold into six-bladed β-propeller structures. While Hu PON1 hydrolyzed a variety of lactones, the only lactone that was a substrate for Mo SMP-30 wasd-(+)-gluconic acid δ-lactone. Squid DFPase was much more efficient at hydrolyzing DFP and G-type nerve agents as compared to Mo SMP-30 or Hu PON1. The Km values for DFP were in the following order: Mo SMP-30>Hu PON1>squid DFPase, suggesting that the efficiency of DFP hydrolysis may be related to its binding in the active sites of these enzymes. Thus, homology modeling and docking were used to simulate the binding of DFP and selected δ-lactones in the active sites of Hu SMP-30, Hu PON1, and squid DFPase. Results from molecular modeling studies suggest that differences in metal–ligand coordinations, the hydrophobicity of the binding pockets, and limited space in the binding pocket due to the presence of a loop, are responsible for substrate specificities of these enzymes.► Hydrolytic activities of Mo SMP-30, Hu PON1 and squid DFPase were compared. ► Mo SMP-30 hydrolyzed only D-(+)-gluconic acid δ-lactone. ► Hu PON1 hydrolyzed a variety of lactones; squid DFPase did not hydrolyze any lactones. ► Squid DFPase hydrolyzed OPs more efficiently as compared to Mo SMP-30 and Hu PON1. ► The observed substrate specificities were explained by molecular modeling studies.
Keywords: Mouse SMP-30; Squid DFPase; Human PON1; DFP; Catalytic bioscavenger; Molecular docking
Investigation of self-assembling proline- and glycine-rich recombinant proteins and peptides inspired by proteins from a symbiotic fungus using atomic force microscopy and circular dichroism spectroscopy by Rhiannon G. Creasey; Nicolas H. Voelcker; Carolyn J. Schultz (pp. 711-722).
Fiber-forming proteins and peptides are being scrutinized as a promising source of building blocks for new nanomaterials. Arabinogalactan-like (AGL) proteins expressed at the symbiotic interface between plant roots and arbuscular mycorrhizal fungi have novel sequences, hypothesized to form polyproline II (PPII) helix structures. The functional nature of these proteins is unknown but they may form structures for the establishment and maintenance of fungal hyphae. Here we show that recombinant AGL1 (rAGL1) and recombinant AGL3 (rAGL3) are extended proteins based upon secondary structural characteristics determined by electronic circular dichroism (CD) spectroscopy and can self-assemble into fibers and microtubes as observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). CD spectroscopy results of synthetic peptides based on repeat regions in AGL1, AGL2 and AGL3 suggest that the synthetic peptides contain significant amounts of extended PPII helices and that these structures are influenced by ionic strength and, at least in one case, by concentration. Point mutations of a single residue of the repeat region of AGL3 resulted in altered secondary structures. Self-assembly of these repeats was observed by means of AFM and optical microscopy. Peptide (APADGK)6 forms structures with similar morphology to rAGL1 suggesting that these repeats are crucial for the morphology of rAGL1 fibers. These novel self-assembling sequences may find applications as precursors for bioinspired nanomaterials.► Recombinant proteins based on genes from a symbiotic fungus self-assemble into fibers. ► Repetitive proline- and glycine-rich peptides with novel sequences inspired by fungal proteins. ► Synthetic repetitive peptides also form fibers and are affected by ionic conditions. ► Secondary structure of proteins and peptides is predominantly polyproline II. ► Secondary structure of synthetic repetitive peptides is sensitive to single residue mutations.
Keywords: Amphipathic peptide; Arabinogalactan-like protein; Atomic force microscopy; Circular dichroism spectroscopy; Self-assembly; Mycorrhizal symbiosis
Investigation of self-assembling proline- and glycine-rich recombinant proteins and peptides inspired by proteins from a symbiotic fungus using atomic force microscopy and circular dichroism spectroscopy by Rhiannon G. Creasey; Nicolas H. Voelcker; Carolyn J. Schultz (pp. 711-722).
Fiber-forming proteins and peptides are being scrutinized as a promising source of building blocks for new nanomaterials. Arabinogalactan-like (AGL) proteins expressed at the symbiotic interface between plant roots and arbuscular mycorrhizal fungi have novel sequences, hypothesized to form polyproline II (PPII) helix structures. The functional nature of these proteins is unknown but they may form structures for the establishment and maintenance of fungal hyphae. Here we show that recombinant AGL1 (rAGL1) and recombinant AGL3 (rAGL3) are extended proteins based upon secondary structural characteristics determined by electronic circular dichroism (CD) spectroscopy and can self-assemble into fibers and microtubes as observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). CD spectroscopy results of synthetic peptides based on repeat regions in AGL1, AGL2 and AGL3 suggest that the synthetic peptides contain significant amounts of extended PPII helices and that these structures are influenced by ionic strength and, at least in one case, by concentration. Point mutations of a single residue of the repeat region of AGL3 resulted in altered secondary structures. Self-assembly of these repeats was observed by means of AFM and optical microscopy. Peptide (APADGK)6 forms structures with similar morphology to rAGL1 suggesting that these repeats are crucial for the morphology of rAGL1 fibers. These novel self-assembling sequences may find applications as precursors for bioinspired nanomaterials.► Recombinant proteins based on genes from a symbiotic fungus self-assemble into fibers. ► Repetitive proline- and glycine-rich peptides with novel sequences inspired by fungal proteins. ► Synthetic repetitive peptides also form fibers and are affected by ionic conditions. ► Secondary structure of proteins and peptides is predominantly polyproline II. ► Secondary structure of synthetic repetitive peptides is sensitive to single residue mutations.
Keywords: Amphipathic peptide; Arabinogalactan-like protein; Atomic force microscopy; Circular dichroism spectroscopy; Self-assembly; Mycorrhizal symbiosis
Relaxation dynamics of a protein solution investigated by dielectric spectroscopy by M. Wolf; R. Gulich; P. Lunkenheimer; A. Loidl (pp. 723-730).
In the present work, we provide a dielectric study on two differently concentrated aqueous lysozyme solutions in the frequency range from 1MHz to 40GHz and for temperatures from 275 to 330K. We analyze the three dispersion regions, commonly found in protein solutions, usually termed β-, γ-, and δ-relaxations. The β-relaxation, occurring in the frequency range around 10MHz and the γ-relaxation around 20GHz (at room temperature) can be attributed to the rotation of the polar protein molecules in their aqueous medium and the reorientational motion of the free water molecules, respectively. The nature of the δ-relaxation, which is often ascribed to the motion of bound water molecules, is not yet fully understood. Here we provide data on the temperature dependence of the relaxation times and relaxation strengths of all three detected processes and on the dc conductivity arising from ionic charge transport. The temperature dependences of the β- and γ-relaxations are closely correlated. We found a significant temperature dependence of the dipole moment of the protein, indicating conformational changes. Moreover we find a breakdown of the Debye–Stokes–Einstein relation in this protein solution, i.e., the dc conductivity is not completely governed by the mobility of the solvent molecules. Instead it seems that the dc conductivity is closely connected to the hydration shell dynamics.Display Omitted► Temperature and concentration dependent dielectric spectra of a protein solution. ► Broad frequency range enabling detection of several dynamic processes. ► Calculation of effective dipole moment and hydrodynamic radius. ► Breakdown of the Debye-Stokes-Einstein relation. ► Temperature dependence of spectra covering all intrinsic relaxations.
Keywords: Protein dynamics; Protein solution; Dielectric spectroscopy; Lysozyme; Hydration shell; Debye–Stokes–Einstein relation
Relaxation dynamics of a protein solution investigated by dielectric spectroscopy by M. Wolf; R. Gulich; P. Lunkenheimer; A. Loidl (pp. 723-730).
In the present work, we provide a dielectric study on two differently concentrated aqueous lysozyme solutions in the frequency range from 1MHz to 40GHz and for temperatures from 275 to 330K. We analyze the three dispersion regions, commonly found in protein solutions, usually termed β-, γ-, and δ-relaxations. The β-relaxation, occurring in the frequency range around 10MHz and the γ-relaxation around 20GHz (at room temperature) can be attributed to the rotation of the polar protein molecules in their aqueous medium and the reorientational motion of the free water molecules, respectively. The nature of the δ-relaxation, which is often ascribed to the motion of bound water molecules, is not yet fully understood. Here we provide data on the temperature dependence of the relaxation times and relaxation strengths of all three detected processes and on the dc conductivity arising from ionic charge transport. The temperature dependences of the β- and γ-relaxations are closely correlated. We found a significant temperature dependence of the dipole moment of the protein, indicating conformational changes. Moreover we find a breakdown of the Debye–Stokes–Einstein relation in this protein solution, i.e., the dc conductivity is not completely governed by the mobility of the solvent molecules. Instead it seems that the dc conductivity is closely connected to the hydration shell dynamics.Display Omitted► Temperature and concentration dependent dielectric spectra of a protein solution. ► Broad frequency range enabling detection of several dynamic processes. ► Calculation of effective dipole moment and hydrodynamic radius. ► Breakdown of the Debye-Stokes-Einstein relation. ► Temperature dependence of spectra covering all intrinsic relaxations.
Keywords: Protein dynamics; Protein solution; Dielectric spectroscopy; Lysozyme; Hydration shell; Debye–Stokes–Einstein relation
Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan ( Rapana thomasiana) hemocyanin by Krassimira Idakieva; Filip Meersman; Constant Gielens (pp. 731-738).
Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV–vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60°C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80°C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50kDa functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal β-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90°C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc.Display Omitted►Thermal unfolding of hemocyanin, a type-3 copper protein, has been investigated. ►Loss of the copper–oxygen band at 344nm suggests local changes to the active site. ►This step is reversible up to ~71°C and without measurable heat absorption. ►It precedes overall loss of structure as seen by DSC and FTIR spectroscopy.
Keywords: Abbreviations; RtH; hemocyanin of; Rapana thomasiana; FTIR; Fourier transform infrared; DSC; differential scanning calorimetry; Tris–HCl; tris (hydroxymethyl) amino–methane hydrochloride; MES; 2-(; N; -morpholino) ethanesulfonic acidThermal stability; Differential scanning calorimetry; FTIR spectroscopy; Hemocyanin; Mollusca
Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan ( Rapana thomasiana) hemocyanin by Krassimira Idakieva; Filip Meersman; Constant Gielens (pp. 731-738).
Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV–vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60°C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80°C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50kDa functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal β-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90°C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc.Display Omitted►Thermal unfolding of hemocyanin, a type-3 copper protein, has been investigated. ►Loss of the copper–oxygen band at 344nm suggests local changes to the active site. ►This step is reversible up to ~71°C and without measurable heat absorption. ►It precedes overall loss of structure as seen by DSC and FTIR spectroscopy.
Keywords: Abbreviations; RtH; hemocyanin of; Rapana thomasiana; FTIR; Fourier transform infrared; DSC; differential scanning calorimetry; Tris–HCl; tris (hydroxymethyl) amino–methane hydrochloride; MES; 2-(; N; -morpholino) ethanesulfonic acidThermal stability; Differential scanning calorimetry; FTIR spectroscopy; Hemocyanin; Mollusca
Structural implication for the impaired binding of W150A mutant LOX-1 to oxidized low density lipoprotein, OxLDL by Shogo Nakano; Mamoru Sugihara; Risato Yamada; Katsuo Katayanagi; Shin-ichi Tate (pp. 739-749).
Lectin-like oxidized lipoprotein (OxLDL) receptor 1, LOX-1, is the major OxLDL receptor expressed on vascular endothelial cells. We have previously reported the ligand-recognition mode of LOX-1 based on the crystal structure of the ligand binding domain (C-type lectin-like domain, CTLD) and surface plasmon resonance analysis, which suggested that the functional significance of the CTLD dimer (the ‘canonical’ dimer) is to harbor the characteristic “basic spine” on its surface. In this study, we have identified the key inter-domain interactions in retaining the canonical CTLD dimer by X-ray structural analysis of the inactive mutant W150A CTLD. The canonical CTLD dimer forms through tight hydrophobic interactions, in which W150 engages in a lock-and-key manner and represents the main interaction. The loss of the Trp ring by mutation to Ala prevents the formation of the canonical dimer, as elucidated from docking calculations using the crystal structure of W150A CTLD. The results emphasize that the canonically formed CTLD dimer is essential for LOX-1 to bind to OxLDL, which supports our proposed view that the basic spine surface present in the correctly formed dimer plays a primal role in OxLDL recognition. This concept provides insight into the pathogenic pattern recognized by LOX-1 as a member of the pattern recognition receptors.Display Omitted► The key inter-domain interactions to make the active dimer were identified. ► The lack of W150 ring diminished the self-assembling feature of the CTLD. ► Basic spine on the CTLD dimer was suggested to be essential for OxLDL binding.
Keywords: C-type lectin-like domain; Oxidized low density lipoprotein; Atherosclerosis; Receptor structure; Pattern recognition receptor
Structural implication for the impaired binding of W150A mutant LOX-1 to oxidized low density lipoprotein, OxLDL by Shogo Nakano; Mamoru Sugihara; Risato Yamada; Katsuo Katayanagi; Shin-ichi Tate (pp. 739-749).
Lectin-like oxidized lipoprotein (OxLDL) receptor 1, LOX-1, is the major OxLDL receptor expressed on vascular endothelial cells. We have previously reported the ligand-recognition mode of LOX-1 based on the crystal structure of the ligand binding domain (C-type lectin-like domain, CTLD) and surface plasmon resonance analysis, which suggested that the functional significance of the CTLD dimer (the ‘canonical’ dimer) is to harbor the characteristic “basic spine” on its surface. In this study, we have identified the key inter-domain interactions in retaining the canonical CTLD dimer by X-ray structural analysis of the inactive mutant W150A CTLD. The canonical CTLD dimer forms through tight hydrophobic interactions, in which W150 engages in a lock-and-key manner and represents the main interaction. The loss of the Trp ring by mutation to Ala prevents the formation of the canonical dimer, as elucidated from docking calculations using the crystal structure of W150A CTLD. The results emphasize that the canonically formed CTLD dimer is essential for LOX-1 to bind to OxLDL, which supports our proposed view that the basic spine surface present in the correctly formed dimer plays a primal role in OxLDL recognition. This concept provides insight into the pathogenic pattern recognized by LOX-1 as a member of the pattern recognition receptors.Display Omitted► The key inter-domain interactions to make the active dimer were identified. ► The lack of W150 ring diminished the self-assembling feature of the CTLD. ► Basic spine on the CTLD dimer was suggested to be essential for OxLDL binding.
Keywords: C-type lectin-like domain; Oxidized low density lipoprotein; Atherosclerosis; Receptor structure; Pattern recognition receptor
QM/MM investigation on the catalytic mechanism of Bacteroides thetaiotaomicron α-glucosidase BtGH97a by Jinhu Wang; Xiang Sheng; Yi Zhao; Yongjun Liu; Chengbu Liu (pp. 750-758).
Bacteroides thetaiotaomicron α-glucosidase BtGH97a is an inverting enzyme. In this paper, the hydrolysis mechanism of p-nitro-phenyl α-d-glucopyranoside (pNP-Glc) catalyzed by BtGH97a was firstly studied by using quantum mechanical/molecular mechanical (QM/MM) approach. Two possible reaction pathways were considered. In the first pathway, a water molecule deprotonated by a nucleophilic base (here E439 or E508) attacks firstly on the anomeric carbon of pNP-Glc, then a proton from an acid residue (E532) attacks on the glycosidic oxygen to finish the hydrolysis reaction (named as nucleophilic attack-first pathway). In the second pathway, the proton from E532 attacks firstly on the glycosidic oxygen, then the water deprotonated by the nucleophilic base attacks on the anomeric carbon of pNP-Glc (named as proton attack-first pathway). Our calculation results indicate that the nucleophilic attack-first pathway is favorable in energy, in which the nucleophilic attack process is the rate-determining step with an energy barrier of 15.4kcal/mol in the case of residue E508 as nucleophilic base. In this rate-determining step, the deprotonation of water and the attack on the anomeric carbon are concerted. In the proton attack-first pathway, the proton attack on the glycosidic oxygen is the rate-determining step, and the energy barrier is 24.1kcal/mol. We conclude that the hydrolysis mechanism would follow nucleophilic attack-first pathway.Display Omitted► We carried out QM/MM calculations to elucidate the catalytic mechanism of BtGH97a. ► Two possible reaction pathways were considered. ► The nucleophilic attack-first pathway is favorable in energy. ► E508 is the possible nucleophilic base.
Keywords: Abbreviations; BtGH97a; Bacteroides thetaiotaomicron; α-glucosidase; pNP-Glc; p-nitro-phenyl α-; d; -glucopyranoside; dNP-Glc; dinitrophenyl α-; d; -glucopyranoside; QM/MM method; quantum mechanical/molecular mechanical method; Nu; nucleophilic; PT; proton transfer; R; reactant; IM; intermediate; TS; transition state; P; product; RMSD; root-mean-square deviation; MD; molecular dynamicBtGH97a; pNP-Glc; QM/MM; Reaction mechanism
QM/MM investigation on the catalytic mechanism of Bacteroides thetaiotaomicron α-glucosidase BtGH97a by Jinhu Wang; Xiang Sheng; Yi Zhao; Yongjun Liu; Chengbu Liu (pp. 750-758).
Bacteroides thetaiotaomicron α-glucosidase BtGH97a is an inverting enzyme. In this paper, the hydrolysis mechanism of p-nitro-phenyl α-d-glucopyranoside (pNP-Glc) catalyzed by BtGH97a was firstly studied by using quantum mechanical/molecular mechanical (QM/MM) approach. Two possible reaction pathways were considered. In the first pathway, a water molecule deprotonated by a nucleophilic base (here E439 or E508) attacks firstly on the anomeric carbon of pNP-Glc, then a proton from an acid residue (E532) attacks on the glycosidic oxygen to finish the hydrolysis reaction (named as nucleophilic attack-first pathway). In the second pathway, the proton from E532 attacks firstly on the glycosidic oxygen, then the water deprotonated by the nucleophilic base attacks on the anomeric carbon of pNP-Glc (named as proton attack-first pathway). Our calculation results indicate that the nucleophilic attack-first pathway is favorable in energy, in which the nucleophilic attack process is the rate-determining step with an energy barrier of 15.4kcal/mol in the case of residue E508 as nucleophilic base. In this rate-determining step, the deprotonation of water and the attack on the anomeric carbon are concerted. In the proton attack-first pathway, the proton attack on the glycosidic oxygen is the rate-determining step, and the energy barrier is 24.1kcal/mol. We conclude that the hydrolysis mechanism would follow nucleophilic attack-first pathway.Display Omitted► We carried out QM/MM calculations to elucidate the catalytic mechanism of BtGH97a. ► Two possible reaction pathways were considered. ► The nucleophilic attack-first pathway is favorable in energy. ► E508 is the possible nucleophilic base.
Keywords: Abbreviations; BtGH97a; Bacteroides thetaiotaomicron; α-glucosidase; pNP-Glc; p-nitro-phenyl α-; d; -glucopyranoside; dNP-Glc; dinitrophenyl α-; d; -glucopyranoside; QM/MM method; quantum mechanical/molecular mechanical method; Nu; nucleophilic; PT; proton transfer; R; reactant; IM; intermediate; TS; transition state; P; product; RMSD; root-mean-square deviation; MD; molecular dynamicBtGH97a; pNP-Glc; QM/MM; Reaction mechanism
Proteome analysis of a CTR9 deficient yeast strain suggests that Ctr9 has function(s) independent of the Paf1 complex by Aurélie Massoni-Laporte; Michel Perrot; Loïc Ponger; Hélian Boucherie; Anne-Laure Guieysse-Peugeot (pp. 759-768).
The Ctr9 protein is a member of the Paf1 complex implicated in multiple functions: transcription initiation and elongation by RNA pol II, RNA processing and histone modifications. It has also been described as a triple-helical DNA binding protein. Loss of Ctr9 results in severe phenotypes similar to the loss of Paf1p, a Paf1 complex subunit. However, the exact role of Ctr9 is not entirely established. To study the biological role of the protein Ctr9 in yeast, we used 2-D gel electrophoresis and characterized proteome alterations in a ctr9Δ mutant strain. Here we present results suggesting that Ctr9 has function distinct from its established role in the Paf1 complex. This role could be linked to its ability to bind to DNA complex structures as triplexes that may have function in regulation of gene expression.► Ctr9 is a yeast nuclear protein member of the Paf1 complex. ► The proteome of ctr9Δ and paf1Δ was analyzed. ► Ctr9 has distinct functions from its role in the Paf1 complex. ► Ctr9 binds to triple-helical DNA structures. ► Alterations in ctr9Δ are not directly correlated to unusual DNA structures.
Keywords: Yeast; Saccharomyces cerevisiae; Proteome; CTR9; PAF1
Proteome analysis of a CTR9 deficient yeast strain suggests that Ctr9 has function(s) independent of the Paf1 complex by Aurélie Massoni-Laporte; Michel Perrot; Loïc Ponger; Hélian Boucherie; Anne-Laure Guieysse-Peugeot (pp. 759-768).
The Ctr9 protein is a member of the Paf1 complex implicated in multiple functions: transcription initiation and elongation by RNA pol II, RNA processing and histone modifications. It has also been described as a triple-helical DNA binding protein. Loss of Ctr9 results in severe phenotypes similar to the loss of Paf1p, a Paf1 complex subunit. However, the exact role of Ctr9 is not entirely established. To study the biological role of the protein Ctr9 in yeast, we used 2-D gel electrophoresis and characterized proteome alterations in a ctr9Δ mutant strain. Here we present results suggesting that Ctr9 has function distinct from its established role in the Paf1 complex. This role could be linked to its ability to bind to DNA complex structures as triplexes that may have function in regulation of gene expression.► Ctr9 is a yeast nuclear protein member of the Paf1 complex. ► The proteome of ctr9Δ and paf1Δ was analyzed. ► Ctr9 has distinct functions from its role in the Paf1 complex. ► Ctr9 binds to triple-helical DNA structures. ► Alterations in ctr9Δ are not directly correlated to unusual DNA structures.
Keywords: Yeast; Saccharomyces cerevisiae; Proteome; CTR9; PAF1
Thermal stabilty of glucokinase (GK) as influenced by the substrate glucose, an allosteric glucokinase activator drug (GKA) and the osmolytes glycerol and urea by B. Zelent; C. Buettger; J. Grimsby; R. Sarabu; J.M. Vanderkooi; A.J. Wand; F.M. Matschinsky (pp. 769-784).
We investigated how glycerol, urea, glucose and a GKA influence kinetics and stability of wild-type and mutant GK. Glycerol and glucose stabilized GK additively. Glycerol barely affected the TF spectra of all GKs but decreased k cat, glucose S0.5 and KD values and ATP KM while leaving cooperativity unchanged. Glycerol sensitized all GKs to GKA as shown by TF. Glucose increased TF of GKs without influence of glycerol on the effect. Glycerol and GKA affected kinetics and binding additively. The activation energies for thermal denaturation of GK were a function of glucose with KDs of 3 and 1mM without and with glycerol, respectively. High urea denatured wild type GK reversibly at 20 and 60°C and urea treatment of irreversibly heat denatured GK allowed refolding as demonstrated by TF including glucose response. We concluded: Glycerol stabilizes GK indirectly without changing the folding structure of the apoenzyme, by restructuring the surface water of the protein, whereas glucose stabilizes GK directly by binding to its substrate site and inducing a compact conformation. Glucose or glycerol (alone or combined) is unable to prevent irreversible heat denaturation above 40°C. However, urea denatures GK reversibly even at 60°C by binding to the protein backbone and directly interacting with hydrophobic side chains. It prevents irreversible aggregation allowing complete refolding when urea is removed. This study establishes the foundation for exploring numerous instability mutants among the more than 600 variant GKs causing diabetes in animals and humans.► Structural and functional instability of glucokinase (GK) cause diabetes mellitus. ► Stability and structure of GK are studied by tryptophan fluorescence (TF) and DSC. ► Glucose influences solvent accessibility of GKs 3 tryptophans differentially. ► Glucose and glycerol affect GK kinetics and stability additively. ► Thermal inactivation of GK above 40°C and reactivation (8M urea).
Keywords: Diabetes; Glucokinase; Tryptophan fluorescence; Protein stability; Denaturation; Calorimetry
Thermal stabilty of glucokinase (GK) as influenced by the substrate glucose, an allosteric glucokinase activator drug (GKA) and the osmolytes glycerol and urea by B. Zelent; C. Buettger; J. Grimsby; R. Sarabu; J.M. Vanderkooi; A.J. Wand; F.M. Matschinsky (pp. 769-784).
We investigated how glycerol, urea, glucose and a GKA influence kinetics and stability of wild-type and mutant GK. Glycerol and glucose stabilized GK additively. Glycerol barely affected the TF spectra of all GKs but decreased k cat, glucose S0.5 and KD values and ATP KM while leaving cooperativity unchanged. Glycerol sensitized all GKs to GKA as shown by TF. Glucose increased TF of GKs without influence of glycerol on the effect. Glycerol and GKA affected kinetics and binding additively. The activation energies for thermal denaturation of GK were a function of glucose with KDs of 3 and 1mM without and with glycerol, respectively. High urea denatured wild type GK reversibly at 20 and 60°C and urea treatment of irreversibly heat denatured GK allowed refolding as demonstrated by TF including glucose response. We concluded: Glycerol stabilizes GK indirectly without changing the folding structure of the apoenzyme, by restructuring the surface water of the protein, whereas glucose stabilizes GK directly by binding to its substrate site and inducing a compact conformation. Glucose or glycerol (alone or combined) is unable to prevent irreversible heat denaturation above 40°C. However, urea denatures GK reversibly even at 60°C by binding to the protein backbone and directly interacting with hydrophobic side chains. It prevents irreversible aggregation allowing complete refolding when urea is removed. This study establishes the foundation for exploring numerous instability mutants among the more than 600 variant GKs causing diabetes in animals and humans.► Structural and functional instability of glucokinase (GK) cause diabetes mellitus. ► Stability and structure of GK are studied by tryptophan fluorescence (TF) and DSC. ► Glucose influences solvent accessibility of GKs 3 tryptophans differentially. ► Glucose and glycerol affect GK kinetics and stability additively. ► Thermal inactivation of GK above 40°C and reactivation (8M urea).
Keywords: Diabetes; Glucokinase; Tryptophan fluorescence; Protein stability; Denaturation; Calorimetry