Scientific grounds for submission to Neuron: Prion diseases are transmissible fatal neurodegenera... more Scientific grounds for submission to Neuron: Prion diseases are transmissible fatal neurodegenerative diseases affecting humans and animals. The agents believed to cause these diseases are prions, infectious misfolded forms of the host-encoded PrP protein. Although supporting evidence exists, the crucial prediction that familial mutations can cause PrP to misfold into an infectious conformation has not been demonstrated. To test this prediction we generated mice to model fatal familial insomnia. They develop a disease which is very similar to the human form, and which is directly transmissible to mice expressing wild-type PrP. We provide the first unequivocal evidence that a protein carrying a familial mutation is sufficient to spontaneously generate infectious prion particles, providing long-sought critical support for the protein-only hypothesis. The countervailing theory that familial mutations simply sensitize carriers to exogenous sources appears incorrect.
The folding and stability of globular proteins are determined by a variety of chemical mechanisms... more The folding and stability of globular proteins are determined by a variety of chemical mechanisms, including hydrogen bonds, salt bridges and the hydrophobic effect. Of particular interest are weakly polar interactions involving aromatic rings, which are proposed to regulate the geometry of closely packed protein interiors. Such interactions reflect the electrostatic contribution of pi-electrons and, unlike van der Waals' interactions and the hydrophobic effect, may, in principle, introduce a directional force in a protein's hydrophobic core. Although the weakly polar hypothesis is supported by a statistical analysis of protein structures, the general importance of such contributions to protein folding and stability is unclear. Here, we show the presence of alternative aromatic-aromatic interactions in the two-dimensional nuclear magnetic resonance structure of a mutant Zn finger. Changes in aromatic packing lead in turn to local and non-local differences between the structures of a wild-type and mutant domain. The results provide insight into the evolution of Zn finger sequences and have implications for understanding how geometric relationships may be chemically encoded in a simple sequence template.
Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit... more Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magnetofluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close-packed magnetic nanoparticle 'core', which is fully surrounded by a 'shell' of fluorescent quantum dots. A thin layer of silica coating provides high colloidal stability and biocompatibility, and a versatile surface functionality. We demonstrate that after surface pegylation, these silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe.
Citation Faas, Henryk et al. "Context-dependent perturbation of neural systems in transgenic mice... more Citation Faas, Henryk et al. "Context-dependent perturbation of neural systems in transgenic mice expressing a cytosolic prion protein." NeuroImage 49.3 (2010): 2607-2617.
New tools that allow dynamic visualization of molecular neural events are important for studying ... more New tools that allow dynamic visualization of molecular neural events are important for studying the basis of brain activity and disease. Sensors that permit ligand-sensitive magnetic resonance imaging (MRI) are useful reagents due to the noninvasive nature and good temporal and spatial resolution of MR methods. Paramagnetic metalloproteins can be effective MRI sensors due to the selectivity imparted by the protein active site and the ability to tune protein properties using techniques such as directed evolution. Here, we show that structure-guided directed evolution of the active site of the cytochrome P450-BM3 heme domain produces highly selective MRI probes with submicromolar affinities for small molecules. We report a new, high-affinity dopamine sensor as well as the first MRI reporter for serotonin, with which we demonstrate quantification of neurotransmitter release in vitro. We also present a detailed structural analysis of evolved cytochrome P450-BM3 heme domain lineages to systematically dissect the molecular basis of neurotransmitter binding affinity, selectivity, and enhanced MRI contrast activity in these engineered proteins.
The ability to map patterns of gene expression noninvasively in living animals could have impact ... more The ability to map patterns of gene expression noninvasively in living animals could have impact in many areas of biology. Reporter systems compatible with MRI could be particularly valuable, but existing strategies tend to lack sensitivity or specificity. Here we address the challenge of MRI-based gene mapping using the reporter enzyme secreted alkaline phosphatase (SEAP), in conjunction with a water-soluble metalloporphyrin contrast agent. SEAP cleaves the porphyrin into an insoluble product that accumulates at sites of enzyme expression and can be visualized by MRI and optical absorbance. The contrast mechanism functions in vitro, in brain slices, and in animals. The system also provides the possibility of readout both in the living animal and by postmortem histology, and it notably does not require intracellular delivery of the contrast agent. The solubility switch mechanism used to detect SEAP could be adapted for imaging of additional reporter enzymes or endogenous targets.
Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of ta... more Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of target proteins in most cells. This promiscuity underlies CaM's role as a shared participant in calciumdependent signal transduction pathways, but imposes a handicap on popular CaM-based calcium biosensors, which display an undesired tendency to cross-react with cellular proteins. Designed CaM/ target pairs that retain high affinity for one another, but lack affinity for wild-type CaM and its natural interaction partners, would therefore be useful as sensor components, and possibly also as elements of "synthetic" cellular signaling networks. Here we have adopted a rational approach to creating suitably modified CaM/target complexes by using computational design methods to guide parallel site-directed mutagenesis of both binding partners. A hierarchical design procedure was applied to suggest a small number of complementary mutations on CaM and on a peptide ligand derived from skeletal muscle light chain kinase (M13). Experimental analysis showed that the procedure was successful in identifying CaM and M13 mutants with novel specificity for one another. Importantly, the designed complexes retained affinity comparable to the wild-type CaM/M13 complex. These results represent a step toward the creation of CaM and M13 derivatives with specificity fully orthogonal to the wild-type proteins, and show that qualitatively accurate predictions may be obtained from computational methods applied simultaneously to two proteins involved in multiple linked binding equilibria.
An important goal in modern biology is to understand how molecular processes commonly studied at ... more An important goal in modern biology is to understand how molecular processes commonly studied at the cellular level give rise to physiological functions in complex tissues and organisms. Non-invasive imaging of gene-expression patterns in whole animals could provide information critical to this end, but current methods lack sensitivity and spatiotemporal precision. Enzymatic reporter systems detectable by magnetic resonance imaging (MRI) address these limitations by combining the relatively high spatial and temporal resolution of MRI with the ability of each genetically expressed enzyme to generate many MRI-detectable product molecules. A challenge with the imaging-based detection of some of the most popular reporter enzymes is the need to deliver MRI probes to their sites of action within cells. Herein we describe a new reporter-gene system for MRI that relieves this problem by harnessing an extracellular enzyme, the mammalian secreted alkaline phosphatase (SEAP).
Scientific grounds for submission to Neuron: Prion diseases are transmissible fatal neurodegenera... more Scientific grounds for submission to Neuron: Prion diseases are transmissible fatal neurodegenerative diseases affecting humans and animals. The agents believed to cause these diseases are prions, infectious misfolded forms of the host-encoded PrP protein. Although supporting evidence exists, the crucial prediction that familial mutations can cause PrP to misfold into an infectious conformation has not been demonstrated. To test this prediction we generated mice to model fatal familial insomnia. They develop a disease which is very similar to the human form, and which is directly transmissible to mice expressing wild-type PrP. We provide the first unequivocal evidence that a protein carrying a familial mutation is sufficient to spontaneously generate infectious prion particles, providing long-sought critical support for the protein-only hypothesis. The countervailing theory that familial mutations simply sensitize carriers to exogenous sources appears incorrect.
The folding and stability of globular proteins are determined by a variety of chemical mechanisms... more The folding and stability of globular proteins are determined by a variety of chemical mechanisms, including hydrogen bonds, salt bridges and the hydrophobic effect. Of particular interest are weakly polar interactions involving aromatic rings, which are proposed to regulate the geometry of closely packed protein interiors. Such interactions reflect the electrostatic contribution of pi-electrons and, unlike van der Waals' interactions and the hydrophobic effect, may, in principle, introduce a directional force in a protein's hydrophobic core. Although the weakly polar hypothesis is supported by a statistical analysis of protein structures, the general importance of such contributions to protein folding and stability is unclear. Here, we show the presence of alternative aromatic-aromatic interactions in the two-dimensional nuclear magnetic resonance structure of a mutant Zn finger. Changes in aromatic packing lead in turn to local and non-local differences between the structures of a wild-type and mutant domain. The results provide insight into the evolution of Zn finger sequences and have implications for understanding how geometric relationships may be chemically encoded in a simple sequence template.
Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit... more Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magnetofluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close-packed magnetic nanoparticle 'core', which is fully surrounded by a 'shell' of fluorescent quantum dots. A thin layer of silica coating provides high colloidal stability and biocompatibility, and a versatile surface functionality. We demonstrate that after surface pegylation, these silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe.
Citation Faas, Henryk et al. "Context-dependent perturbation of neural systems in transgenic mice... more Citation Faas, Henryk et al. "Context-dependent perturbation of neural systems in transgenic mice expressing a cytosolic prion protein." NeuroImage 49.3 (2010): 2607-2617.
New tools that allow dynamic visualization of molecular neural events are important for studying ... more New tools that allow dynamic visualization of molecular neural events are important for studying the basis of brain activity and disease. Sensors that permit ligand-sensitive magnetic resonance imaging (MRI) are useful reagents due to the noninvasive nature and good temporal and spatial resolution of MR methods. Paramagnetic metalloproteins can be effective MRI sensors due to the selectivity imparted by the protein active site and the ability to tune protein properties using techniques such as directed evolution. Here, we show that structure-guided directed evolution of the active site of the cytochrome P450-BM3 heme domain produces highly selective MRI probes with submicromolar affinities for small molecules. We report a new, high-affinity dopamine sensor as well as the first MRI reporter for serotonin, with which we demonstrate quantification of neurotransmitter release in vitro. We also present a detailed structural analysis of evolved cytochrome P450-BM3 heme domain lineages to systematically dissect the molecular basis of neurotransmitter binding affinity, selectivity, and enhanced MRI contrast activity in these engineered proteins.
The ability to map patterns of gene expression noninvasively in living animals could have impact ... more The ability to map patterns of gene expression noninvasively in living animals could have impact in many areas of biology. Reporter systems compatible with MRI could be particularly valuable, but existing strategies tend to lack sensitivity or specificity. Here we address the challenge of MRI-based gene mapping using the reporter enzyme secreted alkaline phosphatase (SEAP), in conjunction with a water-soluble metalloporphyrin contrast agent. SEAP cleaves the porphyrin into an insoluble product that accumulates at sites of enzyme expression and can be visualized by MRI and optical absorbance. The contrast mechanism functions in vitro, in brain slices, and in animals. The system also provides the possibility of readout both in the living animal and by postmortem histology, and it notably does not require intracellular delivery of the contrast agent. The solubility switch mechanism used to detect SEAP could be adapted for imaging of additional reporter enzymes or endogenous targets.
Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of ta... more Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of target proteins in most cells. This promiscuity underlies CaM's role as a shared participant in calciumdependent signal transduction pathways, but imposes a handicap on popular CaM-based calcium biosensors, which display an undesired tendency to cross-react with cellular proteins. Designed CaM/ target pairs that retain high affinity for one another, but lack affinity for wild-type CaM and its natural interaction partners, would therefore be useful as sensor components, and possibly also as elements of "synthetic" cellular signaling networks. Here we have adopted a rational approach to creating suitably modified CaM/target complexes by using computational design methods to guide parallel site-directed mutagenesis of both binding partners. A hierarchical design procedure was applied to suggest a small number of complementary mutations on CaM and on a peptide ligand derived from skeletal muscle light chain kinase (M13). Experimental analysis showed that the procedure was successful in identifying CaM and M13 mutants with novel specificity for one another. Importantly, the designed complexes retained affinity comparable to the wild-type CaM/M13 complex. These results represent a step toward the creation of CaM and M13 derivatives with specificity fully orthogonal to the wild-type proteins, and show that qualitatively accurate predictions may be obtained from computational methods applied simultaneously to two proteins involved in multiple linked binding equilibria.
An important goal in modern biology is to understand how molecular processes commonly studied at ... more An important goal in modern biology is to understand how molecular processes commonly studied at the cellular level give rise to physiological functions in complex tissues and organisms. Non-invasive imaging of gene-expression patterns in whole animals could provide information critical to this end, but current methods lack sensitivity and spatiotemporal precision. Enzymatic reporter systems detectable by magnetic resonance imaging (MRI) address these limitations by combining the relatively high spatial and temporal resolution of MRI with the ability of each genetically expressed enzyme to generate many MRI-detectable product molecules. A challenge with the imaging-based detection of some of the most popular reporter enzymes is the need to deliver MRI probes to their sites of action within cells. Herein we describe a new reporter-gene system for MRI that relieves this problem by harnessing an extracellular enzyme, the mammalian secreted alkaline phosphatase (SEAP).
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Papers by Alan Jasanoff