We explore the use of preparative size-exclusion chromatography (SEC) and high-performance liquid... more We explore the use of preparative size-exclusion chromatography (SEC) and high-performance liquid chromatography (HPLC) to purify quantum dots (QDs) after surface modification. In one example, in which Bio-Beads (S-X1) were used as the packing material for the preparative SEC column, CdSe QDs treated with a functional coumarin dye could be separated from the excess free dye by using tetrahydrofuran (THF)
This review paper describes a new technology, mass cytometry, that addresses applications typical... more This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not
Rapid, sensitive, and quantitative assays for proteases are important for drug development and in... more Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma–mass spectrometry (ICP–MS) detection is described. Peptidic α-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to
... Langdon J. Martin, Bianca R. Sculimbrene, Mark Nitz and Barbara Imperiali* Department of Chem... more ... Langdon J. Martin, Bianca R. Sculimbrene, Mark Nitz and Barbara Imperiali* Department of Chemistry and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA ... b) R. Barbieri, I. Bertini, G. Cavallaro, YM Lee, C. Lunchinat, A. Rosato, J. Am. ...
This paper describes the development and application of new metal-tagging reagents and a novel ma... more This paper describes the development and application of new metal-tagging reagents and a novel mass spectrometer (MS) detector for a flow cytometer that enables highly multiplexed measurement of many biomarkers in individual cells. A new class of tag- ging reagents, based on an acrylic polymer backbone that incorporates a reproducible num- ber of lanthanide elements, has been developed. When linked
Synthetic oligomers of the antigenic Candida albicans (1-->2)-beta-mannopyranans adopt a compa... more Synthetic oligomers of the antigenic Candida albicans (1-->2)-beta-mannopyranans adopt a compact solution conformation that leads to numerous inter-residue nuclear Overhauser effects, including unprecedented nuclear Overhauser effects between n and n + 3 residues. In excellent agreement with experimentally determined distances, unrestrained molecular dynamics point to a single family of conformations that approximate a compact helical motif with a three-residue repeat for this unique homopolymer. When the synthetic di- to hexasaccharides were employed as inhibitors of monoclonal antibodies, which protect mice against a lethal dose of the yeast pathogen, a novel pattern of inhibitor activity was observed. Instead of the paradigm first reported by Kabat (Kabat, E. A. (1962) Fed. Proc. 21, 694-701; Kabat, E. A. (1966) J. Immunol. 97, 1-11), wherein homo-oligosaccharides exhibit increasing inhibitory activity with increasing size, here the maximum activity is reached for di- and tris...
Lanthanide-binding tags (LBTs) are peptide sequences of up to 20 encoded amino acids that tightly... more Lanthanide-binding tags (LBTs) are peptide sequences of up to 20 encoded amino acids that tightly and selectively complex lanthanide ions and can sensitize terbium (Tb3+) luminescence. On the basis of these properties, it was predicted that increasing the number of bound lanthanides would improve the capabilities of these tags. Therefore, using a structurally well-characterized single-LBT sequence as a starting point, a "double-LBT" (dLBT), which concatenates two lanthanide-binding motifs, was designed. Herein we report the generation of dLBT peptides and luminescence and NMR studies on a dLBT-tagged ubiquitin fusion protein. These lanthanide-bound constructs are shown to be improved luminescent tags with avid lanthanide binding and up to 3-fold greater luminescence intensity. NMR experiments were conducted on the ubiquitin construct, wherein bound paramagnetic lanthanides were used as alignment-inducing agents to gain residual dipolar couplings, which are valuable restraints for macromolecular structure determination. Together, these results indicate that dLBTs will be valuable chemical tools for biophysical applications leading to new approaches for studying the structure, function, and dynamics of proteins.
Proceedings of the National Academy of Sciences of the United States of America, Jan 29, 2014
Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of me... more Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de-N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB's N- and C-terminal domains forms a cleft required for the binding and de-N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C...
The catalytic mechanism of 'retaining' beta-glycosidases has been the subject of ... more The catalytic mechanism of 'retaining' beta-glycosidases has been the subject of considerable interest and debate for many years. The visualization of a covalent glycosyl enzyme intermediate by X-ray crystallography was first accomplished with a saccharide substrate substituted with fluorine at its 2-position. The structure implicated major roles for residue His 205 and for the 2-hydroxyl position of the proximal saccharide in binding and catalysis. Here we have studied the kinetic behavior of various His 205 mutants. One of these mutants, a double mutant H205N/E127A, has been used to stabilize a covalent glycosyl-enzyme intermediate involving an unsubstituted sugar, permitting crystallographic analysis of the interactions between its 2-hydroxyl group and the enzyme.
IntroductionThe aim of the study was to evaluate the uptake of [18F]-1-deoxy-1-fluoro-scyllo-inos... more IntroductionThe aim of the study was to evaluate the uptake of [18F]-1-deoxy-1-fluoro-scyllo-inositol ([18F]-scyllo-inositol) in human breast cancer (BC) and glioma xenografts, as well as in inflammatory tissue, in immunocompromised mice. Studies of [18F]-2-fluoro-2-deoxy-d-glucose ([18F]-FDG) under the same conditions were also performed.
We explore the use of preparative size-exclusion chromatography (SEC) and high-performance liquid... more We explore the use of preparative size-exclusion chromatography (SEC) and high-performance liquid chromatography (HPLC) to purify quantum dots (QDs) after surface modification. In one example, in which Bio-Beads (S-X1) were used as the packing material for the preparative SEC column, CdSe QDs treated with a functional coumarin dye could be separated from the excess free dye by using tetrahydrofuran (THF)
This review paper describes a new technology, mass cytometry, that addresses applications typical... more This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not
Rapid, sensitive, and quantitative assays for proteases are important for drug development and in... more Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma–mass spectrometry (ICP–MS) detection is described. Peptidic α-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to
... Langdon J. Martin, Bianca R. Sculimbrene, Mark Nitz and Barbara Imperiali* Department of Chem... more ... Langdon J. Martin, Bianca R. Sculimbrene, Mark Nitz and Barbara Imperiali* Department of Chemistry and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA ... b) R. Barbieri, I. Bertini, G. Cavallaro, YM Lee, C. Lunchinat, A. Rosato, J. Am. ...
This paper describes the development and application of new metal-tagging reagents and a novel ma... more This paper describes the development and application of new metal-tagging reagents and a novel mass spectrometer (MS) detector for a flow cytometer that enables highly multiplexed measurement of many biomarkers in individual cells. A new class of tag- ging reagents, based on an acrylic polymer backbone that incorporates a reproducible num- ber of lanthanide elements, has been developed. When linked
Synthetic oligomers of the antigenic Candida albicans (1-->2)-beta-mannopyranans adopt a compa... more Synthetic oligomers of the antigenic Candida albicans (1-->2)-beta-mannopyranans adopt a compact solution conformation that leads to numerous inter-residue nuclear Overhauser effects, including unprecedented nuclear Overhauser effects between n and n + 3 residues. In excellent agreement with experimentally determined distances, unrestrained molecular dynamics point to a single family of conformations that approximate a compact helical motif with a three-residue repeat for this unique homopolymer. When the synthetic di- to hexasaccharides were employed as inhibitors of monoclonal antibodies, which protect mice against a lethal dose of the yeast pathogen, a novel pattern of inhibitor activity was observed. Instead of the paradigm first reported by Kabat (Kabat, E. A. (1962) Fed. Proc. 21, 694-701; Kabat, E. A. (1966) J. Immunol. 97, 1-11), wherein homo-oligosaccharides exhibit increasing inhibitory activity with increasing size, here the maximum activity is reached for di- and tris...
Lanthanide-binding tags (LBTs) are peptide sequences of up to 20 encoded amino acids that tightly... more Lanthanide-binding tags (LBTs) are peptide sequences of up to 20 encoded amino acids that tightly and selectively complex lanthanide ions and can sensitize terbium (Tb3+) luminescence. On the basis of these properties, it was predicted that increasing the number of bound lanthanides would improve the capabilities of these tags. Therefore, using a structurally well-characterized single-LBT sequence as a starting point, a "double-LBT" (dLBT), which concatenates two lanthanide-binding motifs, was designed. Herein we report the generation of dLBT peptides and luminescence and NMR studies on a dLBT-tagged ubiquitin fusion protein. These lanthanide-bound constructs are shown to be improved luminescent tags with avid lanthanide binding and up to 3-fold greater luminescence intensity. NMR experiments were conducted on the ubiquitin construct, wherein bound paramagnetic lanthanides were used as alignment-inducing agents to gain residual dipolar couplings, which are valuable restraints for macromolecular structure determination. Together, these results indicate that dLBTs will be valuable chemical tools for biophysical applications leading to new approaches for studying the structure, function, and dynamics of proteins.
Proceedings of the National Academy of Sciences of the United States of America, Jan 29, 2014
Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of me... more Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de-N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB's N- and C-terminal domains forms a cleft required for the binding and de-N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C...
The catalytic mechanism of 'retaining' beta-glycosidases has been the subject of ... more The catalytic mechanism of 'retaining' beta-glycosidases has been the subject of considerable interest and debate for many years. The visualization of a covalent glycosyl enzyme intermediate by X-ray crystallography was first accomplished with a saccharide substrate substituted with fluorine at its 2-position. The structure implicated major roles for residue His 205 and for the 2-hydroxyl position of the proximal saccharide in binding and catalysis. Here we have studied the kinetic behavior of various His 205 mutants. One of these mutants, a double mutant H205N/E127A, has been used to stabilize a covalent glycosyl-enzyme intermediate involving an unsubstituted sugar, permitting crystallographic analysis of the interactions between its 2-hydroxyl group and the enzyme.
IntroductionThe aim of the study was to evaluate the uptake of [18F]-1-deoxy-1-fluoro-scyllo-inos... more IntroductionThe aim of the study was to evaluate the uptake of [18F]-1-deoxy-1-fluoro-scyllo-inositol ([18F]-scyllo-inositol) in human breast cancer (BC) and glioma xenografts, as well as in inflammatory tissue, in immunocompromised mice. Studies of [18F]-2-fluoro-2-deoxy-d-glucose ([18F]-FDG) under the same conditions were also performed.
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