Parminder Kaur - Profile on Academia.edu (original) (raw)
Papers by Parminder Kaur
Nucleic Acids Research, 2018
Supplementary Figure S1. Stability of mtSSB as determined by differential scanning fluorimetry. T... more Supplementary Figure S1. Stability of mtSSB as determined by differential scanning fluorimetry. The thermal stability of mtSSB was determined by differential scanning fluorimetry, as described in Material and Methods. Triplicate samples (red, blue, green) contained 30 mM HEPES-KOH (pH 7.5), 1 mM 2mercaptoethanol, 50 mM NaCl, 4x SYPRO Orange, and 5.0 µM mtSSB tetramers. SYPRO Orange fluorescence (470nm ex/574nm em) was determined at the indicated temperatures. Progressive exposure of hydrophobic protein surfaces during thermal denaturation increased binding and fluorescence of the hydrophobic dye probe. The thermal unfolding transition midpoints (Tm) were identified as peaks in the first derivative of the fluorescence signal as a function of temperature. The average Tm for mtSSB was 72.5±1.1˚C.
Many processes in the cell, such as DNA replication, repair, and transcription, require the assem... more Many processes in the cell, such as DNA replication, repair, and transcription, require the assembly of multiple proteins on DNA. Single-molecule, high-resolution imaging techniques such as electron microscopy (EM) and, more recently, atomic force microscopy (AFM) have provided invaluable information about the conformations, structures, and function of protein-DNA complexes. However, these techniques cannot reveal essential information regarding DNA paths in protein-DNA complexes. In this work, we developed an exquisitely sensitive Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) that permits high-resolution imaging of weak electrostatic potentials and resolves DNA paths within proteins-DNA complexes.. We simultaneously collect AFM topographic and DREEM images by mechanically vibrating the cantilever at its fundamental resonance while applying a modulated bias voltage at the first overtone. To illustrate the power of DREEM, we imaged nucleosomes and DNA mismatch repair complexes. The DREEM images of nucleosomes reveal the path of the DNA, and in some cases, it is possible to resolve two double strands of DNA wrapping around the histones. The DREEM images of the DNA mismatch repair complexes demonstrate the path of the DNA through both single proteins and multiprotein complexes. Finally, DREEM imaging requires only minor modifications of many of the existing commercial AFMs, making the technique readily available.
Molecular cell, Jan 6, 2016
Many cellular functions require the assembly of multiprotein-DNA complexes. A growing area of str... more Many cellular functions require the assembly of multiprotein-DNA complexes. A growing area of structural biology aims to characterize these dynamic structures by combining atomic-resolution crystal structures with lower-resolution data from techniques that provide distributions of species, such as small-angle X-ray scattering, electron microscopy, and atomic force microscopy (AFM). A significant limitation in these combinatorial methods is localization of the DNA within the multiprotein complex. Here, we combine AFM with an electrostatic force microscopy (EFM) method to develop an exquisitely sensitive dual-resonance-frequency-enhanced EFM (DREEM) capable of resolving DNA within protein-DNA complexes. Imaging of nucleosomes and DNA mismatch repair complexes demonstrates that DREEM can reveal both the path of the DNA wrapping around histones and the path of DNA as it passes through both single proteins and multiprotein complexes. Finally, DREEM imaging requires only minor modificatio...
Molecular Cell, 2016
Highlights d TRF2 modifies DNA topology by wrapping 90 base pairs of DNA around its TRFH domain d... more Highlights d TRF2 modifies DNA topology by wrapping 90 base pairs of DNA around its TRFH domain d A mutant deficient in DNA wrapping, Topless , causes relaxation of telomeric DNA d Topless telomeres are deprotected and harbor fewer t-loops but are not fused by NHEJ d RAP1 protects Topless telomeres against fusions
Journal of pharmaceutical sciences, Jan 7, 2015
Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit... more Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb...
AFM images show that chromatin reconstituted on methylated DNA (meDNA) is compacted when imaged u... more AFM images show that chromatin reconstituted on methylated DNA (meDNA) is compacted when imaged under water. Chromatin reconstituted on unmethylated DNA is less compacted and less sensitive to hydration. These differences must reflect changes in the physical properties of DNA on methylation, but prior studies have not revealed large differences between methylated and unmethylated DNA. Quasi-elastic light scattering studies of solutions of methylated and unmethylated DNA support this view. In contrast, AFM images of molecules at a water/solid interface yield a persistence length that nearly doubles (to 92.5 ± 4 nm) when 9% of the total DNA is methylated. This increase in persistence length is accompanied by a decrease in contour length, suggesting that a significant fraction of the meDNA changes into the stiffer A form as the more hydrophobic meDNA is dehydrated at the interface. This suggests a simple mechanism for gene silencing as the stiffer meDNA is more difficult to remove from nucleosomes.
Force spectroscopy and recognition imaging are important techniques for characterizing and mappin... more Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance-possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics.
Nucleic Acids Research, 2018
Supplementary Figure S1. Stability of mtSSB as determined by differential scanning fluorimetry. T... more Supplementary Figure S1. Stability of mtSSB as determined by differential scanning fluorimetry. The thermal stability of mtSSB was determined by differential scanning fluorimetry, as described in Material and Methods. Triplicate samples (red, blue, green) contained 30 mM HEPES-KOH (pH 7.5), 1 mM 2mercaptoethanol, 50 mM NaCl, 4x SYPRO Orange, and 5.0 µM mtSSB tetramers. SYPRO Orange fluorescence (470nm ex/574nm em) was determined at the indicated temperatures. Progressive exposure of hydrophobic protein surfaces during thermal denaturation increased binding and fluorescence of the hydrophobic dye probe. The thermal unfolding transition midpoints (Tm) were identified as peaks in the first derivative of the fluorescence signal as a function of temperature. The average Tm for mtSSB was 72.5±1.1˚C.
Many processes in the cell, such as DNA replication, repair, and transcription, require the assem... more Many processes in the cell, such as DNA replication, repair, and transcription, require the assembly of multiple proteins on DNA. Single-molecule, high-resolution imaging techniques such as electron microscopy (EM) and, more recently, atomic force microscopy (AFM) have provided invaluable information about the conformations, structures, and function of protein-DNA complexes. However, these techniques cannot reveal essential information regarding DNA paths in protein-DNA complexes. In this work, we developed an exquisitely sensitive Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) that permits high-resolution imaging of weak electrostatic potentials and resolves DNA paths within proteins-DNA complexes.. We simultaneously collect AFM topographic and DREEM images by mechanically vibrating the cantilever at its fundamental resonance while applying a modulated bias voltage at the first overtone. To illustrate the power of DREEM, we imaged nucleosomes and DNA mismatch repair complexes. The DREEM images of nucleosomes reveal the path of the DNA, and in some cases, it is possible to resolve two double strands of DNA wrapping around the histones. The DREEM images of the DNA mismatch repair complexes demonstrate the path of the DNA through both single proteins and multiprotein complexes. Finally, DREEM imaging requires only minor modifications of many of the existing commercial AFMs, making the technique readily available.
Molecular cell, Jan 6, 2016
Many cellular functions require the assembly of multiprotein-DNA complexes. A growing area of str... more Many cellular functions require the assembly of multiprotein-DNA complexes. A growing area of structural biology aims to characterize these dynamic structures by combining atomic-resolution crystal structures with lower-resolution data from techniques that provide distributions of species, such as small-angle X-ray scattering, electron microscopy, and atomic force microscopy (AFM). A significant limitation in these combinatorial methods is localization of the DNA within the multiprotein complex. Here, we combine AFM with an electrostatic force microscopy (EFM) method to develop an exquisitely sensitive dual-resonance-frequency-enhanced EFM (DREEM) capable of resolving DNA within protein-DNA complexes. Imaging of nucleosomes and DNA mismatch repair complexes demonstrates that DREEM can reveal both the path of the DNA wrapping around histones and the path of DNA as it passes through both single proteins and multiprotein complexes. Finally, DREEM imaging requires only minor modificatio...
Molecular Cell, 2016
Highlights d TRF2 modifies DNA topology by wrapping 90 base pairs of DNA around its TRFH domain d... more Highlights d TRF2 modifies DNA topology by wrapping 90 base pairs of DNA around its TRFH domain d A mutant deficient in DNA wrapping, Topless , causes relaxation of telomeric DNA d Topless telomeres are deprotected and harbor fewer t-loops but are not fused by NHEJ d RAP1 protects Topless telomeres against fusions
Journal of pharmaceutical sciences, Jan 7, 2015
Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit... more Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb...
AFM images show that chromatin reconstituted on methylated DNA (meDNA) is compacted when imaged u... more AFM images show that chromatin reconstituted on methylated DNA (meDNA) is compacted when imaged under water. Chromatin reconstituted on unmethylated DNA is less compacted and less sensitive to hydration. These differences must reflect changes in the physical properties of DNA on methylation, but prior studies have not revealed large differences between methylated and unmethylated DNA. Quasi-elastic light scattering studies of solutions of methylated and unmethylated DNA support this view. In contrast, AFM images of molecules at a water/solid interface yield a persistence length that nearly doubles (to 92.5 ± 4 nm) when 9% of the total DNA is methylated. This increase in persistence length is accompanied by a decrease in contour length, suggesting that a significant fraction of the meDNA changes into the stiffer A form as the more hydrophobic meDNA is dehydrated at the interface. This suggests a simple mechanism for gene silencing as the stiffer meDNA is more difficult to remove from nucleosomes.
Force spectroscopy and recognition imaging are important techniques for characterizing and mappin... more Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance-possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics.