Peter Borbat - Academia.edu (original) (raw)
Papers by Peter Borbat
Scientific reports, 2015
M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle... more M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle, hence it is a high-priority pharmacological target whose structure and functions require better understanding. We studied the mechanism of M2 transmembrane domain (M2TMD) assembly in lipid membranes by the powerful biophysical technique of double electron-electron resonance (DEER) spectroscopy. By varying the M2TMD-to-lipid molar ratio over a wide range from 1:18,800 to 1:160, we found that M2TMD exists as monomers, dimers, and tetramers whose relative populations shift to tetramers with the increase of peptide-to-lipid (P/L) molar ratio. Our results strongly support the tandem mechanism of M2 assembly that is monomers-to-dimer then dimers-to-tetramer, since tight dimers are abundant at small P/L's, and thereafter they assemble as dimers of dimers in weaker tetramers. The stepwise mechanism found for a single-pass membrane protein oligomeric assembly should contribute to the knowle...
Scientific reports, 2015
M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle... more M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle, hence it is a high-priority pharmacological target whose structure and functions require better understanding. We studied the mechanism of M2 transmembrane domain (M2TMD) assembly in lipid membranes by the powerful biophysical technique of double electron-electron resonance (DEER) spectroscopy. By varying the M2TMD-to-lipid molar ratio over a wide range from 1:18,800 to 1:160, we found that M2TMD exists as monomers, dimers, and tetramers whose relative populations shift to tetramers with the increase of peptide-to-lipid (P/L) molar ratio. Our results strongly support the tandem mechanism of M2 assembly that is monomers-to-dimer then dimers-to-tetramer, since tight dimers are abundant at small P/L's, and thereafter they assemble as dimers of dimers in weaker tetramers. The stepwise mechanism found for a single-pass membrane protein oligomeric assembly should contribute to the knowle...
Biochemistry, 2015
Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to fo... more Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (TarFO). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the TarFO modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The TarFO variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of TarFO to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of TarFO into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis methylation system (CheB/CheR). Overall, the TarFO modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation.
Biochemistry, 2015
Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to fo... more Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (TarFO). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the TarFO modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The TarFO variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of TarFO to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of TarFO into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis methylation system (CheB/CheR). Overall, the TarFO modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation.
Biophysical journal, Jan 17, 2015
Pulse dipolar electron-spin resonance in the form of double electron electron resonance was appli... more Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3'- and 5'-termini of the 27-base mini ...
Biophysical journal, Jan 17, 2015
Pulse dipolar electron-spin resonance in the form of double electron electron resonance was appli... more Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3'- and 5'-termini of the 27-base mini ...
Methods in enzymology, 2007
Applications of dipolar ESR spectroscopy to structural biology are rapidly expanding, and it has ... more Applications of dipolar ESR spectroscopy to structural biology are rapidly expanding, and it has become a useful method that is aimed at resolving protein structure and functional mechanisms. The method of pulsed dipolar ESR spectroscopy (PDS) is outlined in the first half of the chapter, and it illustrates the simplicity and potential of this developing technology with applications to various biological systems. A more detailed description is presented of the implementation of PDS to reconstruct the ternary structure of a large dimeric protein complex from Thermotoga maritima, formed by the histidine kinase CheA and the coupling protein CheW. This protein complex is a building block of an extensive array composed of coupled supramolecular structures assembled from CheA/CheW proteins and transmembrane signaling chemoreceptors, which make up a sensor that is key to controlling the motility in bacterial chemotaxis. The reconstruction of the CheA/CheW complex has employed several techn...
Methods in enzymology, 2007
Applications of dipolar ESR spectroscopy to structural biology are rapidly expanding, and it has ... more Applications of dipolar ESR spectroscopy to structural biology are rapidly expanding, and it has become a useful method that is aimed at resolving protein structure and functional mechanisms. The method of pulsed dipolar ESR spectroscopy (PDS) is outlined in the first half of the chapter, and it illustrates the simplicity and potential of this developing technology with applications to various biological systems. A more detailed description is presented of the implementation of PDS to reconstruct the ternary structure of a large dimeric protein complex from Thermotoga maritima, formed by the histidine kinase CheA and the coupling protein CheW. This protein complex is a building block of an extensive array composed of coupled supramolecular structures assembled from CheA/CheW proteins and transmembrane signaling chemoreceptors, which make up a sensor that is key to controlling the motility in bacterial chemotaxis. The reconstruction of the CheA/CheW complex has employed several techn...
Biophysical Journal, 2014
We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to ... more We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to report on the conformation of Cu-Zn superoxide dismutase (SOD1) through the sensitive measurement of dipolar interactions between inherent Cu 2þ ions. Although the extent and the anisotropy of the Cu ESR spectrum provides challenges for PDS, Ku-band (17.3 GHz) double electron-electron resonance and double-quantum coherence variants of PDS coupled with distance reconstruction methods recover Cu-Cu distances in good agreement with crystal structures. Moreover, Cu-PDS measurements expose distinct differences between the conformational properties of wild-type SOD1 and a single-residue variant (I149T) that leads to the disease amyotrophic lateral sclerosis (ALS). The I149T protein displays a broader Cu-Cu distance distribution within the SOD1 dimer compared to wild-type. In a nitroxide (NO)-labeled sample, distance distributions obtained from Cu-Cu, Cu-NO, and NO-NO separations reveal increased structural heterogeneity within the protein and a tendency for mutant dimers to associate. In contrast, perturbations caused by the ALS mutation are completely masked in the crystal structure of I149T. Thus, PDS readily detects alterations in metalloenzyme solution properties not easily deciphered by other methods and in doing so supports the notion that increased range of motion and associations of SOD1 ALS variants contribute to disease progression.
Biophysical Journal, 2014
We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to ... more We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to report on the conformation of Cu-Zn superoxide dismutase (SOD1) through the sensitive measurement of dipolar interactions between inherent Cu 2þ ions. Although the extent and the anisotropy of the Cu ESR spectrum provides challenges for PDS, Ku-band (17.3 GHz) double electron-electron resonance and double-quantum coherence variants of PDS coupled with distance reconstruction methods recover Cu-Cu distances in good agreement with crystal structures. Moreover, Cu-PDS measurements expose distinct differences between the conformational properties of wild-type SOD1 and a single-residue variant (I149T) that leads to the disease amyotrophic lateral sclerosis (ALS). The I149T protein displays a broader Cu-Cu distance distribution within the SOD1 dimer compared to wild-type. In a nitroxide (NO)-labeled sample, distance distributions obtained from Cu-Cu, Cu-NO, and NO-NO separations reveal increased structural heterogeneity within the protein and a tendency for mutant dimers to associate. In contrast, perturbations caused by the ALS mutation are completely masked in the crystal structure of I149T. Thus, PDS readily detects alterations in metalloenzyme solution properties not easily deciphered by other methods and in doing so supports the notion that increased range of motion and associations of SOD1 ALS variants contribute to disease progression.
Biophysical Journal, 2014
Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzh... more Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer's disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau's poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide insights into potential mechanisms for membrane-mediated tau aggregation. Furthermore, the results may have implications for the structural basis of tau-microtubule interactions and microtubule-mediated tau aggregation.
Biophysical Journal, 2014
Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzh... more Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer's disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau's poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide insights into potential mechanisms for membrane-mediated tau aggregation. Furthermore, the results may have implications for the structural basis of tau-microtubule interactions and microtubule-mediated tau aggregation.
Magnetic resonance force microscopy is a promising route to 3-dimensional nanoscale imaging of or... more Magnetic resonance force microscopy is a promising route to 3-dimensional nanoscale imaging of organic materials due to its high sensitivity and isotopic specificity. Labeling of proteins, DNA and biomolecular assemblies with free radical labels for inductive detection are well established techniques, although many of these radical's relaxation times are too short to support previously demonstrated techniques for single electron detection
Magnetic resonance force microscopy is a promising route to 3-dimensional nanoscale imaging of or... more Magnetic resonance force microscopy is a promising route to 3-dimensional nanoscale imaging of organic materials due to its high sensitivity and isotopic specificity. Labeling of proteins, DNA and biomolecular assemblies with free radical labels for inductive detection are well established techniques, although many of these radical's relaxation times are too short to support previously demonstrated techniques for single electron detection
Proceedings of the National Academy of Sciences of the United States of America, Jan 28, 2014
Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misass... more Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu(2+) binding site and defined cross-dimer copper-copper distance dis...
Proceedings of the National Academy of Sciences of the United States of America, Jan 28, 2014
Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misass... more Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu(2+) binding site and defined cross-dimer copper-copper distance dis...
Journal of molecular biology, Jan 20, 2014
At the base of the bacterial flagella, a cytoplasmic rotor (the C-ring) generates torque and reve... more At the base of the bacterial flagella, a cytoplasmic rotor (the C-ring) generates torque and reverses rotation sense in response to stimuli. The bulk of the C-ring forms from many copies of the proteins FliG, FliM, and FliN, which together constitute the switch complex. To help resolve outstanding issues regarding C-ring architecture, we have investigated interactions between FliM and FliG from Thermotoga maritima with X-ray crystallography and pulsed dipolar ESR spectroscopy (PDS). A new crystal structure of an 11-unit FliG:FliM complex produces a large arc with a curvature consistent with the dimensions of the C-ring. Previously determined structures along with this new structure provided a basis to test switch complex assembly models. PDS combined with mutational studies and targeted cross-linking reveal that FliM and FliG interact through their middle domains to form both parallel and antiparallel arrangements in solution. Residue substitutions at predicted interfaces disrupt hi...
Proceedings of the National Academy of Sciences, 2009
The sample was prepared by dissolving 0.0063 g of TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidin... more The sample was prepared by dissolving 0.0063 g of TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidine-1oxyl; Aldrich, 163945) in 1 mL of d 8 -toluene, 0.33 mL of which was further diluted to 1 mL and from that, a 0.1 mL alliquot was added to a solution of 0.039 g deuterated polystyrene (Polymer Source, P4179B-dPS, M n = 200 × 10 3 and M w /M n = 1.4) in 0.9 mL of d 8 -toluene. The resulting solution was spun at 2000 rpm for 30 s onto a 250 µm thick quartz chip (NOVA Electronics). Film thickness was determined by profilometry. A top coating of 20 nm of gold was electron-beam evaporated onto the chip at a rate of 0.2 nm/s. Samples for characterization by low temperature pulsed ESR were prepared identically, minus the gold, removed from the substrate and inserted into a 2 mm o.d. Suprasil tube.
Proceedings of the National Academy of Sciences, 2009
The sample was prepared by dissolving 0.0063 g of TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidin... more The sample was prepared by dissolving 0.0063 g of TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidine-1oxyl; Aldrich, 163945) in 1 mL of d 8 -toluene, 0.33 mL of which was further diluted to 1 mL and from that, a 0.1 mL alliquot was added to a solution of 0.039 g deuterated polystyrene (Polymer Source, P4179B-dPS, M n = 200 × 10 3 and M w /M n = 1.4) in 0.9 mL of d 8 -toluene. The resulting solution was spun at 2000 rpm for 30 s onto a 250 µm thick quartz chip (NOVA Electronics). Film thickness was determined by profilometry. A top coating of 20 nm of gold was electron-beam evaporated onto the chip at a rate of 0.2 nm/s. Samples for characterization by low temperature pulsed ESR were prepared identically, minus the gold, removed from the substrate and inserted into a 2 mm o.d. Suprasil tube.
Scientific reports, 2015
M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle... more M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle, hence it is a high-priority pharmacological target whose structure and functions require better understanding. We studied the mechanism of M2 transmembrane domain (M2TMD) assembly in lipid membranes by the powerful biophysical technique of double electron-electron resonance (DEER) spectroscopy. By varying the M2TMD-to-lipid molar ratio over a wide range from 1:18,800 to 1:160, we found that M2TMD exists as monomers, dimers, and tetramers whose relative populations shift to tetramers with the increase of peptide-to-lipid (P/L) molar ratio. Our results strongly support the tandem mechanism of M2 assembly that is monomers-to-dimer then dimers-to-tetramer, since tight dimers are abundant at small P/L's, and thereafter they assemble as dimers of dimers in weaker tetramers. The stepwise mechanism found for a single-pass membrane protein oligomeric assembly should contribute to the knowle...
Scientific reports, 2015
M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle... more M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle, hence it is a high-priority pharmacological target whose structure and functions require better understanding. We studied the mechanism of M2 transmembrane domain (M2TMD) assembly in lipid membranes by the powerful biophysical technique of double electron-electron resonance (DEER) spectroscopy. By varying the M2TMD-to-lipid molar ratio over a wide range from 1:18,800 to 1:160, we found that M2TMD exists as monomers, dimers, and tetramers whose relative populations shift to tetramers with the increase of peptide-to-lipid (P/L) molar ratio. Our results strongly support the tandem mechanism of M2 assembly that is monomers-to-dimer then dimers-to-tetramer, since tight dimers are abundant at small P/L's, and thereafter they assemble as dimers of dimers in weaker tetramers. The stepwise mechanism found for a single-pass membrane protein oligomeric assembly should contribute to the knowle...
Biochemistry, 2015
Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to fo... more Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (TarFO). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the TarFO modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The TarFO variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of TarFO to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of TarFO into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis methylation system (CheB/CheR). Overall, the TarFO modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation.
Biochemistry, 2015
Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to fo... more Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (TarFO). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the TarFO modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The TarFO variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of TarFO to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of TarFO into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis methylation system (CheB/CheR). Overall, the TarFO modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation.
Biophysical journal, Jan 17, 2015
Pulse dipolar electron-spin resonance in the form of double electron electron resonance was appli... more Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3'- and 5'-termini of the 27-base mini ...
Biophysical journal, Jan 17, 2015
Pulse dipolar electron-spin resonance in the form of double electron electron resonance was appli... more Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3'- and 5'-termini of the 27-base mini ...
Methods in enzymology, 2007
Applications of dipolar ESR spectroscopy to structural biology are rapidly expanding, and it has ... more Applications of dipolar ESR spectroscopy to structural biology are rapidly expanding, and it has become a useful method that is aimed at resolving protein structure and functional mechanisms. The method of pulsed dipolar ESR spectroscopy (PDS) is outlined in the first half of the chapter, and it illustrates the simplicity and potential of this developing technology with applications to various biological systems. A more detailed description is presented of the implementation of PDS to reconstruct the ternary structure of a large dimeric protein complex from Thermotoga maritima, formed by the histidine kinase CheA and the coupling protein CheW. This protein complex is a building block of an extensive array composed of coupled supramolecular structures assembled from CheA/CheW proteins and transmembrane signaling chemoreceptors, which make up a sensor that is key to controlling the motility in bacterial chemotaxis. The reconstruction of the CheA/CheW complex has employed several techn...
Methods in enzymology, 2007
Applications of dipolar ESR spectroscopy to structural biology are rapidly expanding, and it has ... more Applications of dipolar ESR spectroscopy to structural biology are rapidly expanding, and it has become a useful method that is aimed at resolving protein structure and functional mechanisms. The method of pulsed dipolar ESR spectroscopy (PDS) is outlined in the first half of the chapter, and it illustrates the simplicity and potential of this developing technology with applications to various biological systems. A more detailed description is presented of the implementation of PDS to reconstruct the ternary structure of a large dimeric protein complex from Thermotoga maritima, formed by the histidine kinase CheA and the coupling protein CheW. This protein complex is a building block of an extensive array composed of coupled supramolecular structures assembled from CheA/CheW proteins and transmembrane signaling chemoreceptors, which make up a sensor that is key to controlling the motility in bacterial chemotaxis. The reconstruction of the CheA/CheW complex has employed several techn...
Biophysical Journal, 2014
We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to ... more We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to report on the conformation of Cu-Zn superoxide dismutase (SOD1) through the sensitive measurement of dipolar interactions between inherent Cu 2þ ions. Although the extent and the anisotropy of the Cu ESR spectrum provides challenges for PDS, Ku-band (17.3 GHz) double electron-electron resonance and double-quantum coherence variants of PDS coupled with distance reconstruction methods recover Cu-Cu distances in good agreement with crystal structures. Moreover, Cu-PDS measurements expose distinct differences between the conformational properties of wild-type SOD1 and a single-residue variant (I149T) that leads to the disease amyotrophic lateral sclerosis (ALS). The I149T protein displays a broader Cu-Cu distance distribution within the SOD1 dimer compared to wild-type. In a nitroxide (NO)-labeled sample, distance distributions obtained from Cu-Cu, Cu-NO, and NO-NO separations reveal increased structural heterogeneity within the protein and a tendency for mutant dimers to associate. In contrast, perturbations caused by the ALS mutation are completely masked in the crystal structure of I149T. Thus, PDS readily detects alterations in metalloenzyme solution properties not easily deciphered by other methods and in doing so supports the notion that increased range of motion and associations of SOD1 ALS variants contribute to disease progression.
Biophysical Journal, 2014
We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to ... more We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to report on the conformation of Cu-Zn superoxide dismutase (SOD1) through the sensitive measurement of dipolar interactions between inherent Cu 2þ ions. Although the extent and the anisotropy of the Cu ESR spectrum provides challenges for PDS, Ku-band (17.3 GHz) double electron-electron resonance and double-quantum coherence variants of PDS coupled with distance reconstruction methods recover Cu-Cu distances in good agreement with crystal structures. Moreover, Cu-PDS measurements expose distinct differences between the conformational properties of wild-type SOD1 and a single-residue variant (I149T) that leads to the disease amyotrophic lateral sclerosis (ALS). The I149T protein displays a broader Cu-Cu distance distribution within the SOD1 dimer compared to wild-type. In a nitroxide (NO)-labeled sample, distance distributions obtained from Cu-Cu, Cu-NO, and NO-NO separations reveal increased structural heterogeneity within the protein and a tendency for mutant dimers to associate. In contrast, perturbations caused by the ALS mutation are completely masked in the crystal structure of I149T. Thus, PDS readily detects alterations in metalloenzyme solution properties not easily deciphered by other methods and in doing so supports the notion that increased range of motion and associations of SOD1 ALS variants contribute to disease progression.
Biophysical Journal, 2014
Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzh... more Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer's disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau's poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide insights into potential mechanisms for membrane-mediated tau aggregation. Furthermore, the results may have implications for the structural basis of tau-microtubule interactions and microtubule-mediated tau aggregation.
Biophysical Journal, 2014
Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzh... more Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer's disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau's poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide insights into potential mechanisms for membrane-mediated tau aggregation. Furthermore, the results may have implications for the structural basis of tau-microtubule interactions and microtubule-mediated tau aggregation.
Magnetic resonance force microscopy is a promising route to 3-dimensional nanoscale imaging of or... more Magnetic resonance force microscopy is a promising route to 3-dimensional nanoscale imaging of organic materials due to its high sensitivity and isotopic specificity. Labeling of proteins, DNA and biomolecular assemblies with free radical labels for inductive detection are well established techniques, although many of these radical's relaxation times are too short to support previously demonstrated techniques for single electron detection
Magnetic resonance force microscopy is a promising route to 3-dimensional nanoscale imaging of or... more Magnetic resonance force microscopy is a promising route to 3-dimensional nanoscale imaging of organic materials due to its high sensitivity and isotopic specificity. Labeling of proteins, DNA and biomolecular assemblies with free radical labels for inductive detection are well established techniques, although many of these radical's relaxation times are too short to support previously demonstrated techniques for single electron detection
Proceedings of the National Academy of Sciences of the United States of America, Jan 28, 2014
Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misass... more Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu(2+) binding site and defined cross-dimer copper-copper distance dis...
Proceedings of the National Academy of Sciences of the United States of America, Jan 28, 2014
Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misass... more Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu(2+) binding site and defined cross-dimer copper-copper distance dis...
Journal of molecular biology, Jan 20, 2014
At the base of the bacterial flagella, a cytoplasmic rotor (the C-ring) generates torque and reve... more At the base of the bacterial flagella, a cytoplasmic rotor (the C-ring) generates torque and reverses rotation sense in response to stimuli. The bulk of the C-ring forms from many copies of the proteins FliG, FliM, and FliN, which together constitute the switch complex. To help resolve outstanding issues regarding C-ring architecture, we have investigated interactions between FliM and FliG from Thermotoga maritima with X-ray crystallography and pulsed dipolar ESR spectroscopy (PDS). A new crystal structure of an 11-unit FliG:FliM complex produces a large arc with a curvature consistent with the dimensions of the C-ring. Previously determined structures along with this new structure provided a basis to test switch complex assembly models. PDS combined with mutational studies and targeted cross-linking reveal that FliM and FliG interact through their middle domains to form both parallel and antiparallel arrangements in solution. Residue substitutions at predicted interfaces disrupt hi...
Proceedings of the National Academy of Sciences, 2009
The sample was prepared by dissolving 0.0063 g of TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidin... more The sample was prepared by dissolving 0.0063 g of TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidine-1oxyl; Aldrich, 163945) in 1 mL of d 8 -toluene, 0.33 mL of which was further diluted to 1 mL and from that, a 0.1 mL alliquot was added to a solution of 0.039 g deuterated polystyrene (Polymer Source, P4179B-dPS, M n = 200 × 10 3 and M w /M n = 1.4) in 0.9 mL of d 8 -toluene. The resulting solution was spun at 2000 rpm for 30 s onto a 250 µm thick quartz chip (NOVA Electronics). Film thickness was determined by profilometry. A top coating of 20 nm of gold was electron-beam evaporated onto the chip at a rate of 0.2 nm/s. Samples for characterization by low temperature pulsed ESR were prepared identically, minus the gold, removed from the substrate and inserted into a 2 mm o.d. Suprasil tube.
Proceedings of the National Academy of Sciences, 2009
The sample was prepared by dissolving 0.0063 g of TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidin... more The sample was prepared by dissolving 0.0063 g of TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidine-1oxyl; Aldrich, 163945) in 1 mL of d 8 -toluene, 0.33 mL of which was further diluted to 1 mL and from that, a 0.1 mL alliquot was added to a solution of 0.039 g deuterated polystyrene (Polymer Source, P4179B-dPS, M n = 200 × 10 3 and M w /M n = 1.4) in 0.9 mL of d 8 -toluene. The resulting solution was spun at 2000 rpm for 30 s onto a 250 µm thick quartz chip (NOVA Electronics). Film thickness was determined by profilometry. A top coating of 20 nm of gold was electron-beam evaporated onto the chip at a rate of 0.2 nm/s. Samples for characterization by low temperature pulsed ESR were prepared identically, minus the gold, removed from the substrate and inserted into a 2 mm o.d. Suprasil tube.