Peptide-polymer complementary pairs can provide useful tools for isolating, organizing and separa... more Peptide-polymer complementary pairs can provide useful tools for isolating, organizing and separating biomacromolecules. We describe a procedure for selecting a high affinity complementary peptide-polymer nanoparticle (NP) pair using phage display. A hydrogel copolymer nanoparticle containing a statistical distribution of negatively charged and hydrophobic groups was used to select a peptide sequence from a phage displayed library of >10 10 peptides. The NP has low nanomolar affinity for the selected cyclic peptide and exhibited low affinity for a panel of diverse proteins and peptide variants. Affinity arises from the complementary physiochemical properties of both NP and peptide as well as the specific peptide sequence. Comparison of linear and cyclic variants of the peptide established that peptide structure also contributes to affinity. These findings offer a general method for identifying polymer-peptide complementary pairs. Significantly, precise polymer sequences (proteins) are not a requirement, a low information statistical copolymer can be used to select for a specific peptide sequence with affinity and selectivity comparable to that of an antibody. The data also provides evidence for the physiochemical and structural contributions to binding. The results confirm the utility of abiotic, statistical, synthetic copolymers as selective, high affinity peptide affinity reagents.
Single-walled carbon nanotube-based circuits, developed by the Weiss and Collins laboratories (UC... more Single-walled carbon nanotube-based circuits, developed by the Weiss and Collins laboratories (UCI), allows for the detection of minute changes in circuit conductance caused by attached single molecules. Chemical reactions involving the attached biomolecule and the surrounding environment can be monitored in real-time. This approach is a new method to investigate enzyme catalysis and obviates limitations associated with other single molecule techniques (e.g. photobleaching inherent to FRET). Data generated by lysozyme-functionalized nanocircuits identifies electronic signal purturbations as signatures of enzyme catalysis. These signals have been correlated to individual catalytic events: substrate binding, hydrolysis, and release. The electronic measurements distinguish seven independent time scales from a single molecule, including periods of chemically ineffective lysozyme hinge motion at 330 Hz and processive enzymatic turnover occurring around 15 Hz. Employing this novel platfor...
The enormous sensitivity of carbon nanotubes to their chemical environment prompted substantial e... more The enormous sensitivity of carbon nanotubes to their chemical environment prompted substantial excitement about their promise for commercial sensors and detectors. Subsequent research, using both nanotube and graphene electrodes, has generally demonstrated high sensitivity but poor stability and selectivity, as the electrodes are famously sensitive to most analytes including air, water, and noble gases. Much ongoing research has focused on improving selectivity and reducing unwanted responses, though the direct physiochemical mechanisms responsible for sensing remain poorly understood. In fact, the combination of possible interactions is daunting and remains under investigation on an analyte-by-analyte basis, as described in a recent review.[1] Part of the difficulty is that very small amounts of disorder, such as from atomic defects, chemical adducts, or contaminants, enormously enhance electron transfer between the environment and the otherwise inert graphitic surfaces. This enha...
Single-molecule studies of enzymes open a window into their dynamics and kinetics. A single molec... more Single-molecule studies of enzymes open a window into their dynamics and kinetics. A single molecule of the catalytic domain of cAMP-dependent protein kinase A (PKA) was attached to a single-walled carbon nanotube device for long-duration monitoring. The electronic recording clearly resolves substrate binding, ATP binding, and cooperative formation of PKA's catalytically functional, ternary complex. Using recordings of a single PKA molecule extending over 10 min and tens of thousands of binding events, we determine the full transition probability matrix and conversion rates governing formation of the apo, intermediate, and closed enzyme configurations. We also observe kinetic rates varying over 2 orders of magnitude from one second to another. Anti-correlation of the on and off rates for PKA binding to the peptide substrate, but not ATP, demonstrates that regulation of enzyme activity results from altering the stability of the PKA−substrate complex, not its binding to ATP. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute useful for an enzyme with crucial roles in cell signaling.
The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of eit... more The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single-molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field-effect transistors through pyrene linker molecules. The substrate-driven hinge-bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibited processive low turnover rates of 20-50 s(-1) and rapid (200-400 s(-1)) nonproductive motions. The latter nonproductive binding events occupied 43% of the enzyme's time in the presence of the cross-linked peptidoglycan but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate b...
Tethering a single lysozyme molecule to a carbon nanotube field-effect transistor produced a stab... more Tethering a single lysozyme molecule to a carbon nanotube field-effect transistor produced a stable, high-bandwidth transducer for protein motion. Electronic monitoring during 10-minute periods extended well beyond the limitations of fluorescence techniques to uncover dynamic disorder within a single molecule and establish lysozyme as a processive enzyme. On average, 100 chemical bonds are processively hydrolyzed, at 15-hertz rates, before lysozyme returns to its nonproductive, 330-hertz hinge motion. Statistical analysis differentiated single-step hinge closure from enzyme opening, which requires two steps. Seven independent time scales governing lysozyme's activity were observed. The pH dependence of lysozyme activity arises not from changes to its processive kinetics but rather from increasing time spent in either nonproductive rapid motions or an inactive, closed conformation.
DNA 'breathing' is a thermally driven process in which nucleotide residues near single-strand (ss... more DNA 'breathing' is a thermally driven process in which nucleotide residues near single-strand (ss)-double-strand (ds) DNA forks and junctions temporarily adopt local conformations that depart from their most stable structures. It is thought that the transient occurrence of these 'open' conformations is centrally involved in the proper function of DNA-protein complexes responsible for replication, transcription, and many other reactions that involve the manipulation
A protein without natural binding functions was engineered to bind HIV-1 integrase. Phage display... more A protein without natural binding functions was engineered to bind HIV-1 integrase. Phage display selections applied a library of variants based on the C-terminal domain of the eye lens protein human γS-crystallin. Multiple loop regions were altered to encode libraries with ≈3.6×10 11 different variants. A crystallin variant, termed Integrase-Binding Protein-10 (IBP-10), inhibits integrase catalysis with nanomolar K i values. IBP-10 interacts with the integrase Cterminal domain and inhibits integrase substrate affinity. This allosteric mechanism allows IBP-10 to inhibit drug resistant integrase variants. The results demonstrate the applicability of the crystallin scaffold for the discovery of binding partners and enzyme inhibitors. Engineered protein scaffolds can offer affinity reagents to solve biomedical challenges. 1,2 Anti-HIV therapies, for example, require new binding partners, such as protein therapeutics, in response to the very high rate of viral mutagenesis and consequent acquisition of drug resistance by the virus. 3,4 Thus, developing alternative agents to target HIV proteins, such as integrase, could supplement and accelerate treatments for this ongoing pandemic. 5 Globally, >33 million people are infected with HIV, and ≈2 million people are killed each year by the virus. 6
Outside of fluorescence measurements, there are currently few means of observing characteristic t... more Outside of fluorescence measurements, there are currently few means of observing characteristic time constants of individual molecules. We describe the development of a single molecule technique utilizing a point-functionalized SWCNT electronic circuit [1]. Time-dependent components of the SWCNT conductance reveal real-time interactions between a covalently attached protein and the immediate electrolytic environment. We will demonstrate electronic transduction of protein-substrate interactions with single molecule resolution. On-line analysis based on normalization of the power spectrum helps to enhance the resulting signals, even to the extent of providing the user with real time feedback regarding the experiment status. [1] B. Goldsmith et al, Science 315 77 (2007)
When a SWCNT conductor contains a defect, its electronic fluctuations are sensitive indicators of... more When a SWCNT conductor contains a defect, its electronic fluctuations are sensitive indicators of the surrounding chemical environment and of the chemical state of the defect itself. We demonstrate this effect by fabricating single SWCNT devices and then engineering their defect condition through the method of electrochemical point-functionalization. By characterizing the same SWCNT before and after the introduction of a point defect, we clearly establish the defect's contribution to the overall device noise. Carboxylate defects are particularly interesting because they have a deprotonated state that is sensitive to pH, electrolyte, and electrochemical potential. Large amplitude, two level fluctuations are observed from carboxylate sites when probed under conditions near the dissociation constant pKa, and the occupation statistics can be reversibly tuned by either pH or potential. We interpret the fluctuation in terms of the controlled protonation and deprotonation of the defect site, and describe a simple electrostatic gating model that supports this conclusion.
resolution, real-time monitoring of a single lysozyme molecule is demonstrated by fabricating nan... more resolution, real-time monitoring of a single lysozyme molecule is demonstrated by fabricating nanoscale electronic devices based on single-walled carbon nanotubes (SWCNT). In this sensor platform, a biomolecule of interest is attached to a single SWCNT device. The electrical conductance transduces chemical events with single molecule sensitivity and 10 microsecond resolution. In this work, enzymatic turnover by lysozyme is investigated, because the mechanistic details for its processivity and dynamics remain incompletely understood. Stochastically distributed binding events between a lysozyme and its binding substrate, peptidoglycan, are monitored via the sensor conductance. Furthermore, the magnitude and repetition rate of these events varies with pH and the presence of inhibitors or denaturation agents. Changes in the conductance signal are analyzed in terms of lysozyme's internal hinge motion, binding events, and enzymatic processing.
The dynamic processivity of individual lysozyme molecules was monitored in the presence of either... more The dynamic processivity of individual lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates using a single-walled carbon nanotube transistor. The substrate-driven, hinge bending motions of lysozyme induce dynamic electronic signals in the underlying transistor to allow long-term monitoring of the same molecule, all without the limitations of fluorophore quenching or bleaching. For both types of substrates, lysozyme exhibits slow, processive turnover at 20 Hz and also rapid, nonproductive motions at 300 Hz. However, the latter type of motion nearly vanishes with the linear substrate, which lacks cross-links. Specifically, the nonproductive binding fills 43% of the enzyme's total activity when the substrate has cross-links, but only 7% with the cross-links are absent. The continuous, uninterrupted processing indicates that lysozyme can catalytically hydrolyze glycosidic bonds all the way to the end of a linear substrate, and that the motion attributed to nonproductive binding may be the lysozyme sidestepping the peptide cross-links.
electronic devices like field-effect transistors (FETs) have long promised to provide sensitive, ... more electronic devices like field-effect transistors (FETs) have long promised to provide sensitive, label-free detection of biomolecules. In particular, single-walled carbon nanotubes (SWNTs) have the requisite sensitivity to detect single molecule events, and have sufficient bandwidth to directly monitor single molecule dynamics in real time. Recent measurements have demonstrated this premise by monitoring the dynamic, single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of polymerase I. Initial successes in each case indicate the generality and attractiveness of SWNT FETs as a new tool to complement other single molecule techniques. Furthermore, our focused research on transduction mechanisms provides the design rules necessary to further generalize this SWNT FET technique. This presentation will summarize these rules, and demonstrate how the purposeful incorporation of just one amino acid is sufficient to fabricate effective, single molecule nanocircuits from a wide range of enzymes or proteins.
Using single-walled carbon nanotube (SWNT) transistors, we monitored the processivity and dynamic... more Using single-walled carbon nanotube (SWNT) transistors, we monitored the processivity and dynamics of single molecules of cAMP-dependent protein kinase (PKA). As PKA enzymatically phosphorylates its peptide substrate, it generates an electronic signal in the transistor that can be monitored continuously and with 20 µs resolution. The electronic recording directly resolves substrate binding, ATP binding, and cooperative formation of PKA's catalytically functional, ternary complex. Statistical analysis of many events determines on-and off-rates for each of these events, as well as the full transistion probability matrix between them. Long duration monitoring further revealed minute-to-minute rate variability for a single molecule, and different mechanistic statistics for ATP binding than for substrate. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute that is useful for an enzyme that plays crucial roles in cell signaling.
experiments, a total of 199 junctional SR and 191 free SR proteins were identified from 16,344 MS... more experiments, a total of 199 junctional SR and 191 free SR proteins were identified from 16,344 MSMS spectra with protein false discovery rate (FDR) at 0.8% and peptide FDR at 0.0%. SERCA2a was the most abundant protein in both fractions, as expected, and virtually all of the known cardiac SR proteins were also identified. In jSR, the major 4 components of the Ca 2þ -release complex were found exclusively, including calsequestrin-2, ryanodine receptor 2 and 3, triadin, and junctin. Another 57 proteins were also identified as specific to this subcompartment. In fSR, 53 proteins were exclusively identified. Relative enrichments in one of the SR subcompartments were found for an additional 86 proteins, and 52 additional proteins were identified in the cardiac SR, without obvious subcompartment origin. SR proteins that showed less specificity were mitochondrial proteins (more enriched in the lower-density jSR), ER chaperones (roughly evenly distributed), lipid-metabolizing enzymes, and filamentous proteins. Proteomic analyses of classical cardiac SR subfractions extends our understanding of the cardiac secretory compartments, and serves as foundation for future exploration and understanding of cardiac cell biology.
Peptide-polymer complementary pairs can provide useful tools for isolating, organizing and separa... more Peptide-polymer complementary pairs can provide useful tools for isolating, organizing and separating biomacromolecules. We describe a procedure for selecting a high affinity complementary peptide-polymer nanoparticle (NP) pair using phage display. A hydrogel copolymer nanoparticle containing a statistical distribution of negatively charged and hydrophobic groups was used to select a peptide sequence from a phage displayed library of >10 10 peptides. The NP has low nanomolar affinity for the selected cyclic peptide and exhibited low affinity for a panel of diverse proteins and peptide variants. Affinity arises from the complementary physiochemical properties of both NP and peptide as well as the specific peptide sequence. Comparison of linear and cyclic variants of the peptide established that peptide structure also contributes to affinity. These findings offer a general method for identifying polymer-peptide complementary pairs. Significantly, precise polymer sequences (proteins) are not a requirement, a low information statistical copolymer can be used to select for a specific peptide sequence with affinity and selectivity comparable to that of an antibody. The data also provides evidence for the physiochemical and structural contributions to binding. The results confirm the utility of abiotic, statistical, synthetic copolymers as selective, high affinity peptide affinity reagents.
Single-walled carbon nanotube-based circuits, developed by the Weiss and Collins laboratories (UC... more Single-walled carbon nanotube-based circuits, developed by the Weiss and Collins laboratories (UCI), allows for the detection of minute changes in circuit conductance caused by attached single molecules. Chemical reactions involving the attached biomolecule and the surrounding environment can be monitored in real-time. This approach is a new method to investigate enzyme catalysis and obviates limitations associated with other single molecule techniques (e.g. photobleaching inherent to FRET). Data generated by lysozyme-functionalized nanocircuits identifies electronic signal purturbations as signatures of enzyme catalysis. These signals have been correlated to individual catalytic events: substrate binding, hydrolysis, and release. The electronic measurements distinguish seven independent time scales from a single molecule, including periods of chemically ineffective lysozyme hinge motion at 330 Hz and processive enzymatic turnover occurring around 15 Hz. Employing this novel platfor...
The enormous sensitivity of carbon nanotubes to their chemical environment prompted substantial e... more The enormous sensitivity of carbon nanotubes to their chemical environment prompted substantial excitement about their promise for commercial sensors and detectors. Subsequent research, using both nanotube and graphene electrodes, has generally demonstrated high sensitivity but poor stability and selectivity, as the electrodes are famously sensitive to most analytes including air, water, and noble gases. Much ongoing research has focused on improving selectivity and reducing unwanted responses, though the direct physiochemical mechanisms responsible for sensing remain poorly understood. In fact, the combination of possible interactions is daunting and remains under investigation on an analyte-by-analyte basis, as described in a recent review.[1] Part of the difficulty is that very small amounts of disorder, such as from atomic defects, chemical adducts, or contaminants, enormously enhance electron transfer between the environment and the otherwise inert graphitic surfaces. This enha...
Single-molecule studies of enzymes open a window into their dynamics and kinetics. A single molec... more Single-molecule studies of enzymes open a window into their dynamics and kinetics. A single molecule of the catalytic domain of cAMP-dependent protein kinase A (PKA) was attached to a single-walled carbon nanotube device for long-duration monitoring. The electronic recording clearly resolves substrate binding, ATP binding, and cooperative formation of PKA's catalytically functional, ternary complex. Using recordings of a single PKA molecule extending over 10 min and tens of thousands of binding events, we determine the full transition probability matrix and conversion rates governing formation of the apo, intermediate, and closed enzyme configurations. We also observe kinetic rates varying over 2 orders of magnitude from one second to another. Anti-correlation of the on and off rates for PKA binding to the peptide substrate, but not ATP, demonstrates that regulation of enzyme activity results from altering the stability of the PKA−substrate complex, not its binding to ATP. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute useful for an enzyme with crucial roles in cell signaling.
The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of eit... more The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single-molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field-effect transistors through pyrene linker molecules. The substrate-driven hinge-bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibited processive low turnover rates of 20-50 s(-1) and rapid (200-400 s(-1)) nonproductive motions. The latter nonproductive binding events occupied 43% of the enzyme's time in the presence of the cross-linked peptidoglycan but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate b...
Tethering a single lysozyme molecule to a carbon nanotube field-effect transistor produced a stab... more Tethering a single lysozyme molecule to a carbon nanotube field-effect transistor produced a stable, high-bandwidth transducer for protein motion. Electronic monitoring during 10-minute periods extended well beyond the limitations of fluorescence techniques to uncover dynamic disorder within a single molecule and establish lysozyme as a processive enzyme. On average, 100 chemical bonds are processively hydrolyzed, at 15-hertz rates, before lysozyme returns to its nonproductive, 330-hertz hinge motion. Statistical analysis differentiated single-step hinge closure from enzyme opening, which requires two steps. Seven independent time scales governing lysozyme's activity were observed. The pH dependence of lysozyme activity arises not from changes to its processive kinetics but rather from increasing time spent in either nonproductive rapid motions or an inactive, closed conformation.
DNA 'breathing' is a thermally driven process in which nucleotide residues near single-strand (ss... more DNA 'breathing' is a thermally driven process in which nucleotide residues near single-strand (ss)-double-strand (ds) DNA forks and junctions temporarily adopt local conformations that depart from their most stable structures. It is thought that the transient occurrence of these 'open' conformations is centrally involved in the proper function of DNA-protein complexes responsible for replication, transcription, and many other reactions that involve the manipulation
A protein without natural binding functions was engineered to bind HIV-1 integrase. Phage display... more A protein without natural binding functions was engineered to bind HIV-1 integrase. Phage display selections applied a library of variants based on the C-terminal domain of the eye lens protein human γS-crystallin. Multiple loop regions were altered to encode libraries with ≈3.6×10 11 different variants. A crystallin variant, termed Integrase-Binding Protein-10 (IBP-10), inhibits integrase catalysis with nanomolar K i values. IBP-10 interacts with the integrase Cterminal domain and inhibits integrase substrate affinity. This allosteric mechanism allows IBP-10 to inhibit drug resistant integrase variants. The results demonstrate the applicability of the crystallin scaffold for the discovery of binding partners and enzyme inhibitors. Engineered protein scaffolds can offer affinity reagents to solve biomedical challenges. 1,2 Anti-HIV therapies, for example, require new binding partners, such as protein therapeutics, in response to the very high rate of viral mutagenesis and consequent acquisition of drug resistance by the virus. 3,4 Thus, developing alternative agents to target HIV proteins, such as integrase, could supplement and accelerate treatments for this ongoing pandemic. 5 Globally, >33 million people are infected with HIV, and ≈2 million people are killed each year by the virus. 6
Outside of fluorescence measurements, there are currently few means of observing characteristic t... more Outside of fluorescence measurements, there are currently few means of observing characteristic time constants of individual molecules. We describe the development of a single molecule technique utilizing a point-functionalized SWCNT electronic circuit [1]. Time-dependent components of the SWCNT conductance reveal real-time interactions between a covalently attached protein and the immediate electrolytic environment. We will demonstrate electronic transduction of protein-substrate interactions with single molecule resolution. On-line analysis based on normalization of the power spectrum helps to enhance the resulting signals, even to the extent of providing the user with real time feedback regarding the experiment status. [1] B. Goldsmith et al, Science 315 77 (2007)
When a SWCNT conductor contains a defect, its electronic fluctuations are sensitive indicators of... more When a SWCNT conductor contains a defect, its electronic fluctuations are sensitive indicators of the surrounding chemical environment and of the chemical state of the defect itself. We demonstrate this effect by fabricating single SWCNT devices and then engineering their defect condition through the method of electrochemical point-functionalization. By characterizing the same SWCNT before and after the introduction of a point defect, we clearly establish the defect's contribution to the overall device noise. Carboxylate defects are particularly interesting because they have a deprotonated state that is sensitive to pH, electrolyte, and electrochemical potential. Large amplitude, two level fluctuations are observed from carboxylate sites when probed under conditions near the dissociation constant pKa, and the occupation statistics can be reversibly tuned by either pH or potential. We interpret the fluctuation in terms of the controlled protonation and deprotonation of the defect site, and describe a simple electrostatic gating model that supports this conclusion.
resolution, real-time monitoring of a single lysozyme molecule is demonstrated by fabricating nan... more resolution, real-time monitoring of a single lysozyme molecule is demonstrated by fabricating nanoscale electronic devices based on single-walled carbon nanotubes (SWCNT). In this sensor platform, a biomolecule of interest is attached to a single SWCNT device. The electrical conductance transduces chemical events with single molecule sensitivity and 10 microsecond resolution. In this work, enzymatic turnover by lysozyme is investigated, because the mechanistic details for its processivity and dynamics remain incompletely understood. Stochastically distributed binding events between a lysozyme and its binding substrate, peptidoglycan, are monitored via the sensor conductance. Furthermore, the magnitude and repetition rate of these events varies with pH and the presence of inhibitors or denaturation agents. Changes in the conductance signal are analyzed in terms of lysozyme's internal hinge motion, binding events, and enzymatic processing.
The dynamic processivity of individual lysozyme molecules was monitored in the presence of either... more The dynamic processivity of individual lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates using a single-walled carbon nanotube transistor. The substrate-driven, hinge bending motions of lysozyme induce dynamic electronic signals in the underlying transistor to allow long-term monitoring of the same molecule, all without the limitations of fluorophore quenching or bleaching. For both types of substrates, lysozyme exhibits slow, processive turnover at 20 Hz and also rapid, nonproductive motions at 300 Hz. However, the latter type of motion nearly vanishes with the linear substrate, which lacks cross-links. Specifically, the nonproductive binding fills 43% of the enzyme's total activity when the substrate has cross-links, but only 7% with the cross-links are absent. The continuous, uninterrupted processing indicates that lysozyme can catalytically hydrolyze glycosidic bonds all the way to the end of a linear substrate, and that the motion attributed to nonproductive binding may be the lysozyme sidestepping the peptide cross-links.
electronic devices like field-effect transistors (FETs) have long promised to provide sensitive, ... more electronic devices like field-effect transistors (FETs) have long promised to provide sensitive, label-free detection of biomolecules. In particular, single-walled carbon nanotubes (SWNTs) have the requisite sensitivity to detect single molecule events, and have sufficient bandwidth to directly monitor single molecule dynamics in real time. Recent measurements have demonstrated this premise by monitoring the dynamic, single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of polymerase I. Initial successes in each case indicate the generality and attractiveness of SWNT FETs as a new tool to complement other single molecule techniques. Furthermore, our focused research on transduction mechanisms provides the design rules necessary to further generalize this SWNT FET technique. This presentation will summarize these rules, and demonstrate how the purposeful incorporation of just one amino acid is sufficient to fabricate effective, single molecule nanocircuits from a wide range of enzymes or proteins.
Using single-walled carbon nanotube (SWNT) transistors, we monitored the processivity and dynamic... more Using single-walled carbon nanotube (SWNT) transistors, we monitored the processivity and dynamics of single molecules of cAMP-dependent protein kinase (PKA). As PKA enzymatically phosphorylates its peptide substrate, it generates an electronic signal in the transistor that can be monitored continuously and with 20 µs resolution. The electronic recording directly resolves substrate binding, ATP binding, and cooperative formation of PKA's catalytically functional, ternary complex. Statistical analysis of many events determines on-and off-rates for each of these events, as well as the full transistion probability matrix between them. Long duration monitoring further revealed minute-to-minute rate variability for a single molecule, and different mechanistic statistics for ATP binding than for substrate. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute that is useful for an enzyme that plays crucial roles in cell signaling.
experiments, a total of 199 junctional SR and 191 free SR proteins were identified from 16,344 MS... more experiments, a total of 199 junctional SR and 191 free SR proteins were identified from 16,344 MSMS spectra with protein false discovery rate (FDR) at 0.8% and peptide FDR at 0.0%. SERCA2a was the most abundant protein in both fractions, as expected, and virtually all of the known cardiac SR proteins were also identified. In jSR, the major 4 components of the Ca 2þ -release complex were found exclusively, including calsequestrin-2, ryanodine receptor 2 and 3, triadin, and junctin. Another 57 proteins were also identified as specific to this subcompartment. In fSR, 53 proteins were exclusively identified. Relative enrichments in one of the SR subcompartments were found for an additional 86 proteins, and 52 additional proteins were identified in the cardiac SR, without obvious subcompartment origin. SR proteins that showed less specificity were mitochondrial proteins (more enriched in the lower-density jSR), ER chaperones (roughly evenly distributed), lipid-metabolizing enzymes, and filamentous proteins. Proteomic analyses of classical cardiac SR subfractions extends our understanding of the cardiac secretory compartments, and serves as foundation for future exploration and understanding of cardiac cell biology.
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Papers by Issa Moody