Polish Journal of Food and Nutrition Sciences, 2013
Increasing the potency of antihypertensive food-derived peptides is a critical and important step... more Increasing the potency of antihypertensive food-derived peptides is a critical and important step in the development of natural drugs for cardiovascular diseases prevention. We have proposed the egg-white protein precipitate (EWPP) obtained as a byproduct of cystatin and lysozyme isolation as a potential source of ACE-inhibitory peptides derived by pepsin digestion. The results indicated that hydrolysis of EWPP with pepsin produced the ACE inhibitory activity. During 3-h hydrolysis (DH: 38.3%), the IC50 value of EWPP hydrolysate was significantly increased and finally reached IC50=643.1 μg/mL. This hydrolysate was further fractionated by RP-HPLC. The peptide fraction exhibiting the highest ACE inhibitory activity was rechromatographed. Three peptide subfractions exhibiting ACE-inhibitory activities of 69.0, 25.0, and 37.6 μg/mL were further characterised. In each of them, mixtures of peptides with different molecular masses were observed.
Numerous studies have shown that food proteins may be a source of bioactive peptides. Those pepti... more Numerous studies have shown that food proteins may be a source of bioactive peptides. Those peptides are encrypted in the protein sequence. They stay inactive within the parental protein until release by proteolytic enzymes (Mine and Kovacs-Nolan in Worlds
Conserved residues are often considered essential for function, and substitutions in such residue... more Conserved residues are often considered essential for function, and substitutions in such residues are expected to have a negative influence on the properties of a protein. However, mutations in a few highly conserved residues of the β‐lactamase from Mycobacterium tuberculosis, BlaC, were shown to have no or only limited negative effect on the enzyme. One such mutant, D179N, even conveyed increased ceftazidime resistance upon bacterial cells, while displaying good activity against penicillins. The crystal structures of BlaC D179N in resting state and in complex with sulbactam reveal subtle structural changes in the Ω‐loop as compared to the structure of wild‐type BlaC. Introducing this mutation in four other β‐lactamases, CTX‐M‐14, KPC‐2, NMC‐A and TEM‐1, resulted in decreased antibiotic resistance for penicillins and meropenem. The results demonstrate that the Asp in position 179 is generally essential for class A β‐lactamases but not for BlaC, which can be explained by the importa...
These data were obtained in studies on hypernucleosome formation, an apparently endless nucleosom... more These data were obtained in studies on hypernucleosome formation, an apparently endless nucleosome, by archaeal histones HMfA and HMfB from M. fervidus. Using tethered particle motion and magnetic tweezers the structural, mechanical as well as topological properties were investigated. In addition to the wild type proteins, derivatives mutated at residues proposed to be involved in 'stacking' interactions between layers of the hypernucleosome, were investigated; the data confirmed the importance of these residues.<br>
Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bi... more Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences.
Electrostatic interactions can strongly increase the efficiency of protein complex formation. The... more Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.
The application of double electron-electron resonance (DEER) with site-directedspin labeling (SDS... more The application of double electron-electron resonance (DEER) with site-directedspin labeling (SDSL)t om easure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed Gd III complexes,G d III-CLaNP13a/b/cw ere synthesized. Rather than the disulfide linkageo fm ost other CLaNP molecules, at hioether linkagew as used to avoid re-ductive dissociation of the linker.T he doubly Gd III labeled N55C/V57C/K147C/T151C variants of T4Lysozymew ere measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent withr esults from paramagnetic NMR. An arrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites.
Many archaea express histones, which organize the genome and play a key role in gene regulation. ... more Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone–DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an ‘endless’ histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB–DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque,...
The Mycobacterium tuberculosis β-lactamase BlaC is a broad-spectrum β-lactamase that can convert ... more The Mycobacterium tuberculosis β-lactamase BlaC is a broad-spectrum β-lactamase that can convert a range of β-lactam antibiotics. Enzymes with low specificity are expected to exhibit active-site flexibility. To probe the motions in BlaC, we studied the dynamic behavior in solution using nuclear magnetic resonance (NMR) spectroscopy. 15 N relaxation experiments show that BlaC is mostly rigid on the pico- to nanosecond timescale.
Synthetic metal complexes can be used as paramagnetic probes for the study of proteins and protei... more Synthetic metal complexes can be used as paramagnetic probes for the study of proteins and protein complexes.H erein, two transition metal NMR probes (TraNPs) are reported. TraNPs are attached through two arms to ap rotein to generate ap seudocontact shift (PCS) using cobalt(II), or paramagnetic relaxation enhancement (PRE) with manganese(II). The PCS analysis of TraNPs attached to three different proteins shows that the size of the anisotropic component of the magnetic susceptibility depends on the probe surroundings at the surface of the protein, contrary to what is observed for lanthanoid-based probes.T he observed PCS are relatively small, making cobalt-based probes suitable for localized studies,such as of an active site.The obtained PREs are stronger than those obtained with nitroxide spin labels and the possibility to generate both PCS and PRE offers advantages.T he properties of TraNPs in comparison with other cobalt-based probes are discussed.
Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands ... more Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1-3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.
remaining resonances the number of possible assignments is limited to two or three. With a single... more remaining resonances the number of possible assignments is limited to two or three. With a single tag, reliable assignments can be obtained for methyl groups with large PCS near the tag. It is concluded that assignment of methyl group resonances by paramagnetic tagging can be particularly useful in combination with some additional data, such as from mutagenesis or NOE-based experiments. Approaches to yield the best assignment results with PCS generating tags are discussed.
The rise of multi- and even totally antibiotic resistant forms of Mycobacterium tuberculosis unde... more The rise of multi- and even totally antibiotic resistant forms of Mycobacterium tuberculosis underlines the need for new antibiotics. The pathogen is resistant to β-lactam compounds due to its native serine β-lactamase, BlaC. This resistance can be circumvented by administration of a β-lactamase inhibitor. We studied the interaction between BlaC and the inhibitor clavulanic acid. Our data show hydrolysis of clavulanic acid and recovery of BlaC activity upon prolonged incubation. The rate of clavulanic acid hydrolysis is much higher in the presence of phosphate ions. A specific binding site for phosphate is identified in the active site pocket, both in the crystalline state and in solution. NMR spectroscopy experiments show that phosphate binds to this site with a dissociation constant of 30 mM in the free enzyme. We conclude that inhibition of BlaC by clavulanic acid is reversible and that phosphate ions can promote the hydrolysis of the inhibitor.
Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2015
The energy landscapes of proteins are highly complex and can be influenced by changes in physical... more The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction.
Paramagnetic NMR probes provide valuable long-range structural information on proteins and protei... more Paramagnetic NMR probes provide valuable long-range structural information on proteins and protein complexes. A new, stable, two-armed lanthanoid probe is reported that can be attached to a protein site-specifically via chemically inert thioether linkages.
Recent studies on the electron transfer complex formed by cytochrome f and plastocyanin from Nost... more Recent studies on the electron transfer complex formed by cytochrome f and plastocyanin from Nostoc revealed that both hydrophobic and electrostatic interactions play a role in the process of complex formation. To study the balance between these two types of interactions in the encounter and the final state, the complex between plastocyanin from Phormidium laminosum and cytochrome f from Nostoc sp. PCC 7119 was investigated using NMR spectroscopy and Monte Carlo docking. Cytochrome f has a highly negative charge. Phormidium plastocyanin is similar to that from Nostoc, but the net charge of the protein is negative rather than positive. NMR titrations of Zn-substituted Phormidium plastocyanin and Nostoc cytochrome f indicated that a complex with an affinity intermediate between those of the Nostoc and Phormidium complexes is formed. Plastocyanin was found in a head-on orientation, as determined using pseudocontact shifts, similar to that in the Phormidium complex, in which the hydrophobic patch represents the main site of interaction on plastocyanin. However, the interaction in the cross-complex is dependent on electrostatics, similar to that in the Nostoc complex. The negative charge of plastocyanin decreases, but not abolishes, the attraction to cytochrome f, resulting in the formation of a more diffuse encounter complex than in the Nostoc case, as could be determined using paramagnetic relaxation spectroscopy. This work illustrates the subtle interplay of electrostatic and hydrophobic interactions in the formation of transient protein complexes. The results are discussed in the context of a model for association on the basis of hydrophobic contacts in the encounter state.
The β-lactamase of Mycobacterium tuberculosis , BlaC, is susceptible to inhibition by clavulanic ... more The β-lactamase of Mycobacterium tuberculosis , BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli .
Polish Journal of Food and Nutrition Sciences, 2013
Increasing the potency of antihypertensive food-derived peptides is a critical and important step... more Increasing the potency of antihypertensive food-derived peptides is a critical and important step in the development of natural drugs for cardiovascular diseases prevention. We have proposed the egg-white protein precipitate (EWPP) obtained as a byproduct of cystatin and lysozyme isolation as a potential source of ACE-inhibitory peptides derived by pepsin digestion. The results indicated that hydrolysis of EWPP with pepsin produced the ACE inhibitory activity. During 3-h hydrolysis (DH: 38.3%), the IC50 value of EWPP hydrolysate was significantly increased and finally reached IC50=643.1 μg/mL. This hydrolysate was further fractionated by RP-HPLC. The peptide fraction exhibiting the highest ACE inhibitory activity was rechromatographed. Three peptide subfractions exhibiting ACE-inhibitory activities of 69.0, 25.0, and 37.6 μg/mL were further characterised. In each of them, mixtures of peptides with different molecular masses were observed.
Numerous studies have shown that food proteins may be a source of bioactive peptides. Those pepti... more Numerous studies have shown that food proteins may be a source of bioactive peptides. Those peptides are encrypted in the protein sequence. They stay inactive within the parental protein until release by proteolytic enzymes (Mine and Kovacs-Nolan in Worlds
Conserved residues are often considered essential for function, and substitutions in such residue... more Conserved residues are often considered essential for function, and substitutions in such residues are expected to have a negative influence on the properties of a protein. However, mutations in a few highly conserved residues of the β‐lactamase from Mycobacterium tuberculosis, BlaC, were shown to have no or only limited negative effect on the enzyme. One such mutant, D179N, even conveyed increased ceftazidime resistance upon bacterial cells, while displaying good activity against penicillins. The crystal structures of BlaC D179N in resting state and in complex with sulbactam reveal subtle structural changes in the Ω‐loop as compared to the structure of wild‐type BlaC. Introducing this mutation in four other β‐lactamases, CTX‐M‐14, KPC‐2, NMC‐A and TEM‐1, resulted in decreased antibiotic resistance for penicillins and meropenem. The results demonstrate that the Asp in position 179 is generally essential for class A β‐lactamases but not for BlaC, which can be explained by the importa...
These data were obtained in studies on hypernucleosome formation, an apparently endless nucleosom... more These data were obtained in studies on hypernucleosome formation, an apparently endless nucleosome, by archaeal histones HMfA and HMfB from M. fervidus. Using tethered particle motion and magnetic tweezers the structural, mechanical as well as topological properties were investigated. In addition to the wild type proteins, derivatives mutated at residues proposed to be involved in 'stacking' interactions between layers of the hypernucleosome, were investigated; the data confirmed the importance of these residues.<br>
Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bi... more Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences.
Electrostatic interactions can strongly increase the efficiency of protein complex formation. The... more Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.
The application of double electron-electron resonance (DEER) with site-directedspin labeling (SDS... more The application of double electron-electron resonance (DEER) with site-directedspin labeling (SDSL)t om easure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed Gd III complexes,G d III-CLaNP13a/b/cw ere synthesized. Rather than the disulfide linkageo fm ost other CLaNP molecules, at hioether linkagew as used to avoid re-ductive dissociation of the linker.T he doubly Gd III labeled N55C/V57C/K147C/T151C variants of T4Lysozymew ere measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent withr esults from paramagnetic NMR. An arrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites.
Many archaea express histones, which organize the genome and play a key role in gene regulation. ... more Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone–DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an ‘endless’ histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB–DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque,...
The Mycobacterium tuberculosis β-lactamase BlaC is a broad-spectrum β-lactamase that can convert ... more The Mycobacterium tuberculosis β-lactamase BlaC is a broad-spectrum β-lactamase that can convert a range of β-lactam antibiotics. Enzymes with low specificity are expected to exhibit active-site flexibility. To probe the motions in BlaC, we studied the dynamic behavior in solution using nuclear magnetic resonance (NMR) spectroscopy. 15 N relaxation experiments show that BlaC is mostly rigid on the pico- to nanosecond timescale.
Synthetic metal complexes can be used as paramagnetic probes for the study of proteins and protei... more Synthetic metal complexes can be used as paramagnetic probes for the study of proteins and protein complexes.H erein, two transition metal NMR probes (TraNPs) are reported. TraNPs are attached through two arms to ap rotein to generate ap seudocontact shift (PCS) using cobalt(II), or paramagnetic relaxation enhancement (PRE) with manganese(II). The PCS analysis of TraNPs attached to three different proteins shows that the size of the anisotropic component of the magnetic susceptibility depends on the probe surroundings at the surface of the protein, contrary to what is observed for lanthanoid-based probes.T he observed PCS are relatively small, making cobalt-based probes suitable for localized studies,such as of an active site.The obtained PREs are stronger than those obtained with nitroxide spin labels and the possibility to generate both PCS and PRE offers advantages.T he properties of TraNPs in comparison with other cobalt-based probes are discussed.
Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands ... more Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1-3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.
remaining resonances the number of possible assignments is limited to two or three. With a single... more remaining resonances the number of possible assignments is limited to two or three. With a single tag, reliable assignments can be obtained for methyl groups with large PCS near the tag. It is concluded that assignment of methyl group resonances by paramagnetic tagging can be particularly useful in combination with some additional data, such as from mutagenesis or NOE-based experiments. Approaches to yield the best assignment results with PCS generating tags are discussed.
The rise of multi- and even totally antibiotic resistant forms of Mycobacterium tuberculosis unde... more The rise of multi- and even totally antibiotic resistant forms of Mycobacterium tuberculosis underlines the need for new antibiotics. The pathogen is resistant to β-lactam compounds due to its native serine β-lactamase, BlaC. This resistance can be circumvented by administration of a β-lactamase inhibitor. We studied the interaction between BlaC and the inhibitor clavulanic acid. Our data show hydrolysis of clavulanic acid and recovery of BlaC activity upon prolonged incubation. The rate of clavulanic acid hydrolysis is much higher in the presence of phosphate ions. A specific binding site for phosphate is identified in the active site pocket, both in the crystalline state and in solution. NMR spectroscopy experiments show that phosphate binds to this site with a dissociation constant of 30 mM in the free enzyme. We conclude that inhibition of BlaC by clavulanic acid is reversible and that phosphate ions can promote the hydrolysis of the inhibitor.
Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2015
The energy landscapes of proteins are highly complex and can be influenced by changes in physical... more The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction.
Paramagnetic NMR probes provide valuable long-range structural information on proteins and protei... more Paramagnetic NMR probes provide valuable long-range structural information on proteins and protein complexes. A new, stable, two-armed lanthanoid probe is reported that can be attached to a protein site-specifically via chemically inert thioether linkages.
Recent studies on the electron transfer complex formed by cytochrome f and plastocyanin from Nost... more Recent studies on the electron transfer complex formed by cytochrome f and plastocyanin from Nostoc revealed that both hydrophobic and electrostatic interactions play a role in the process of complex formation. To study the balance between these two types of interactions in the encounter and the final state, the complex between plastocyanin from Phormidium laminosum and cytochrome f from Nostoc sp. PCC 7119 was investigated using NMR spectroscopy and Monte Carlo docking. Cytochrome f has a highly negative charge. Phormidium plastocyanin is similar to that from Nostoc, but the net charge of the protein is negative rather than positive. NMR titrations of Zn-substituted Phormidium plastocyanin and Nostoc cytochrome f indicated that a complex with an affinity intermediate between those of the Nostoc and Phormidium complexes is formed. Plastocyanin was found in a head-on orientation, as determined using pseudocontact shifts, similar to that in the Phormidium complex, in which the hydrophobic patch represents the main site of interaction on plastocyanin. However, the interaction in the cross-complex is dependent on electrostatics, similar to that in the Nostoc complex. The negative charge of plastocyanin decreases, but not abolishes, the attraction to cytochrome f, resulting in the formation of a more diffuse encounter complex than in the Nostoc case, as could be determined using paramagnetic relaxation spectroscopy. This work illustrates the subtle interplay of electrostatic and hydrophobic interactions in the formation of transient protein complexes. The results are discussed in the context of a model for association on the basis of hydrophobic contacts in the encounter state.
The β-lactamase of Mycobacterium tuberculosis , BlaC, is susceptible to inhibition by clavulanic ... more The β-lactamase of Mycobacterium tuberculosis , BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli .
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