Jennifer C Flanagan - Academia.edu (original) (raw)

Papers by Jennifer C Flanagan

Biophysical Journal, May 1, 2019

Transmembrane peptides contain polar residues in the interior of the membrane, which may alter th... more Transmembrane peptides contain polar residues in the interior of the membrane, which may alter the electrostatic environment and favor hydration in the otherwise nonpolar environment of the membrane core. Here, we demonstrate a general, nonperturbative strategy to probe hydration of the peptide backbone at specific depths within the bilayer using a combination of site-specific isotope labels, ultrafast two-dimensional infrared spectroscopy, and spectral modeling based on molecular dynamics simulations. Our results show that the amphiphilic pH-low insertion peptide supports a highly heterogeneous environment, with significant backbone hydration of nonpolar residues neighboring charged residues. For example, a leucine residue located as far as 1 nm into the hydrophobic bulk reports hydrogen-bonded populations as high as $20%. These findings indicate that the polar nature of these residues may facilitate the transport of water molecules into the hydrophobic core of the membrane.

Single molecule localization microscopy (SMLM) permits the visualization of cellular structures a... more Single molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Here we present a simple method that uses the pair autocorrelation function evaluated both in space and time to measure the time-interval dependent point-spread function of SMLM images of static samples. Using this approach, we demonstrate that experimentally obtained images typically have effective point spread functions that are broader than expected from the localization precision alone, due to additional uncertainty arising from drift and drift correction algorithms. This resolution metric reports on how precisely one can measure pairwise distances between labeled objects and is complementary to the commonly used Fourier Ring Correlation metric that also considers spat...

ABSTRACTSingle molecule localization microscopy (SMLM) permits the visualization of cellular stru... more ABSTRACTSingle molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Here we present a simple method that uses the pair autocorrelation function evaluated both in space and time to measure the time-interval dependent effective point spread function of SMLM images of static samples. Using this approach, we demonstrate that experimentally obtained images typically have effective point spread functions that are broader than expected from the localization precision alone, due to additional uncertainty arising from factors such as drift and drift correction algorithms. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labelled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion ...

Plasma membranes are the main liaisons between the intercellular and extracellular environment, p... more Plasma membranes are the main liaisons between the intercellular and extracellular environment, playing a critical role in numerous biological processes. Recent research has challenged the long-standing "fluid mosaic model," representing membranes as densely packed, heterogeneous environments. Within these complex membranes are transmembrane proteins which comprise up to 50% of the membrane mass, and are themselves diverse in sequence, structure, and function. Combining two-dimensional infrared spectroscopy (2D IR) and molecular dynamics simulation (MD), this thesis explores membrane complexity from two perspectives: first, it addresses the sequence heterogeneity in transmembrane peptides; and second, it explores the effect this crowded environment has on the lipids themselves and the implications this has on future membrane studies. Site-specific hydration of transmembrane peptides was probed using singly isotope-labeled pH (Low) Insertion Peptides, or pHLIPs. These peptides are a class of small transmembrane helices containing ~30% polar residues. By including a single-residue ¹³C=¹⁸O isotope label on the pHLIP backbone, IR experiments effectively produce a single-residue spectrum separate from the main peptide peak. With computational models to connect atomistic structure from MD to infrared frequency shifts, these site-specific spectra reveal local hydration as far as 1 nm into the hydrophobic membrane core. Crowding experiments probed dynamics at the lipid-water interface of model membranes as a function of transmembrane peptide concentration. These dynamics, drawn from time-dependent 2D IR, trend non-monotonically with peptide concentration, revealing three dynamical regimes: a pure lipid-like, a bulk-like, and a crowded regime. Through similar computational methods, these dynamics were linked to water structure at the lipid-water interface, which is perturbed by peptide insertion. Finally, preliminary work has been carried out in developing transient 2D IR methods for applications to protein folding. The f [...]

Biophysical Journal, 2022

ConspectusLipid membranes are more than just barriers between cell compartments; they provide mol... more ConspectusLipid membranes are more than just barriers between cell compartments; they provide molecular environments with a finely tuned balance between hydrophilic and hydrophobic interactions that enable proteins to dynamically fold and self-assemble to regulate biological function. Characterizing dynamics at the lipid-water interface is essential to understanding molecular complexities from the thermodynamics of liquid-liquid phase separation down to picosecond-scale reorganization of interfacial hydrogen-bond networks.Ultrafast vibrational spectroscopy, including two-dimensional infrared (2D IR) and vibrational sum-frequency generation (VSFG) spectroscopies, is a powerful tool to examine picosecond interfacial dynamics. Two-dimensional IR spectroscopy provides a bond-centered view of dynamics with subpicosecond time resolutions, as vibrational frequencies are highly sensitive to the local environment. Recently, 2D IR spectroscopy has been applied to carbonyl and phosphate vibrations intrinsically located at the lipid-water interface. Interface-specific VSFG spectroscopy probes the water vibrational modes directly, accessing H-bond strength and water organization at lipid headgroup positions. Signals in VSFG arise from the interfacial dipole contributions, directly probing headgroup ordering and water orientation to provide a structural view of the interface.In this Account we discuss novel applications of ultrafast spectroscopy to lipid membranes, a field that has experienced significant growth over the past decade. In particular, ultrafast experiments now offer a molecular perspective on increasingly complex membranes. The powerful combination of ultrafast, interface-selective spectroscopy and simulations opens up new routes to understanding multicomponent membranes and their function. This Account highlights key prevailing views that have emerged from recent experiments: (1) Water dynamics at the lipid-water interface are slow compared to those of bulk water as a result of disrupted H-bond networks near the headgroups. (2) Peptides, ions, osmolytes, and cosolvents perturb interfacial dynamics, indicating that dynamics at the interface are affected by bulk solvent dynamics and vice versa. (3) The interfacial environment is generally dictated by the headgroup structure and orientation, but hydrophobic interactions within the acyl chains also modulate interfacial dynamics. Ultrafast spectroscopy has been essential to characterizing the biophysical chemistry of the lipid-water interface; however, challenges remain in interpreting congested spectra as well as designing appropriate model systems to capture the complexity of a membrane environment.

Biology takes place in crowded, heterogeneous environments, and it is therefore essential to acco... more Biology takes place in crowded, heterogeneous environments, and it is therefore essential to account for crowding effects in our under-standing of biophysical processes at the molecular level. Comparable to the cytosol, proteins occupy approximately 30% of the plasma membrane surface, thus crowding should have an effect on the local structure and dynamics at the lipid-water interface. Using a combi-nation of ultrafast two-dimensional infrared spectroscopy and molecular dynamics simulations, we quantify the effects of membrane peptide concentration on the picosecond interfacial H-bond dynamics. The measurements reveal nonmonotonic dependence of water orientation and dynamics as a function of transmembrane peptide-to-lipid ratio. We observe three dynamical regimes: a pure lipid-like regime at low peptide concentration; a bulk-like region at intermediate peptide concentration where dynamics are faster by ~20% com-pared to the pure lipid bilayer; and a crowded regime where high peptide concentration slows dynamics by ~50%.

The Journal of Physical Chemistry Letters

Liquid-liquid phase separation is common in complex mixtures, but the behavior of nanoconfined li... more Liquid-liquid phase separation is common in complex mixtures, but the behavior of nanoconfined liquids is poorly understood from a physical perspective. Dimethyl sulfoxide (DMSO) is an amphiphilic molecule with unique concentration-dependent bulk properties in mixtures with water. Here, we use ultrafast two-dimensional infrared (2D IR) spectroscopy to measure the H-bond dynamics of two probe molecules with different polarities: formamide (FA) and dimethyl formamide (DMF). Picosecond H-bond dynamics are fastest in the intermediate concentration regime (20-50 mol% DMSO); as such confined water exhibits bulk-like dynamics. Each vibrational probe experiences a unique microscopic environment as a result of nanoscale phase separation. Molecular dynamics simulations show that the dynamics span multiple timescales from femtoseconds to nanoseconds. Our studies suggest a previously unknown liquid environment, which we label "local bulk", in which despite the local heterogeneity, the ultrafast H-bond dynamics are similar to bulk water.

Optics Letters

We present a pH-jump two-dimensional infrared (2D IR) spectrometer to probe pH-dependent conforma... more We present a pH-jump two-dimensional infrared (2D IR) spectrometer to probe pH-dependent conformational changes from nanoseconds to milliseconds. The design incorporates a nanosecond 355 nm source into a pulse-shaper-based 2D IR spectrometer to trigger dissociation of a caged proton prior to probing subsequent conformational changes with femtosecond 2D IR spectroscopy. We observe a blue shift in the amide I mode (C═O stretch) of diglycine induced by protonation of the terminal amine. This method combines the bond-specific structural sensitivity of ultrafast 2D IR with triggered conformational dynamics, providing structural access to multiscale biomolecular transformations such as protein folding.

The Journal of Physical Chemistry A, 2016

Hydrogen bonding (HB) populations were computed for the terminal oxygen atom, OT, and the solvent... more Hydrogen bonding (HB) populations were computed for the terminal oxygen atom, OT, and the solvent H atoms of interest for HB-donating solvents used to optimize the map: hexanol, ethanol, methanol, and D2O, as well as the HB-donating solvents used to assess the map capabilities: isopropanol, butanol, diethylene glycol, and dioxane. All HB analysis used the analysis tools in GROMACS 4.5.3. 1 The HB definition consists of a 30° cutoff H-donor-acceptor angle and a 0.35 nm cutoff donor-acceptor radius. Hydrogen bond analyses were performed for each stored snapshot of the 10 ns trajectory. The trajectories were then split into 0, 1, and 2 HB sub-trajectories for analysis of the electrostatic parameters and fitting of the corresponding peaks in the experimental FTIR spectra as discussed in the main text.

Biophysical Journal

Transmembrane peptides contain polar residues in the interior of the membrane, which may alter th... more Transmembrane peptides contain polar residues in the interior of the membrane, which may alter the electrostatic environment and favor hydration in the otherwise nonpolar environment of the membrane core. Here, we demonstrate a general, nonperturbative strategy to probe hydration of the peptide backbone at specific depths within the bilayer using a combination of site-specific isotope labels, ultrafast two-dimensional infrared spectroscopy, and spectral modeling based on molecular dynamics simulations. Our results show that the amphiphilic pH-low insertion peptide supports a highly heterogeneous environment, with significant backbone hydration of nonpolar residues neighboring charged residues. For example, a leucine residue located as far as 1 nm into the hydrophobic bulk reports hydrogen-bonded populations as high as ∼20%. These findings indicate that the polar nature of these residues may facilitate the transport of water molecules into the hydrophobic core of the membrane.

Biophysical Journal, May 1, 2019

Transmembrane peptides contain polar residues in the interior of the membrane, which may alter th... more Transmembrane peptides contain polar residues in the interior of the membrane, which may alter the electrostatic environment and favor hydration in the otherwise nonpolar environment of the membrane core. Here, we demonstrate a general, nonperturbative strategy to probe hydration of the peptide backbone at specific depths within the bilayer using a combination of site-specific isotope labels, ultrafast two-dimensional infrared spectroscopy, and spectral modeling based on molecular dynamics simulations. Our results show that the amphiphilic pH-low insertion peptide supports a highly heterogeneous environment, with significant backbone hydration of nonpolar residues neighboring charged residues. For example, a leucine residue located as far as 1 nm into the hydrophobic bulk reports hydrogen-bonded populations as high as $20%. These findings indicate that the polar nature of these residues may facilitate the transport of water molecules into the hydrophobic core of the membrane.

Single molecule localization microscopy (SMLM) permits the visualization of cellular structures a... more Single molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Here we present a simple method that uses the pair autocorrelation function evaluated both in space and time to measure the time-interval dependent point-spread function of SMLM images of static samples. Using this approach, we demonstrate that experimentally obtained images typically have effective point spread functions that are broader than expected from the localization precision alone, due to additional uncertainty arising from drift and drift correction algorithms. This resolution metric reports on how precisely one can measure pairwise distances between labeled objects and is complementary to the commonly used Fourier Ring Correlation metric that also considers spat...

ABSTRACTSingle molecule localization microscopy (SMLM) permits the visualization of cellular stru... more ABSTRACTSingle molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Here we present a simple method that uses the pair autocorrelation function evaluated both in space and time to measure the time-interval dependent effective point spread function of SMLM images of static samples. Using this approach, we demonstrate that experimentally obtained images typically have effective point spread functions that are broader than expected from the localization precision alone, due to additional uncertainty arising from factors such as drift and drift correction algorithms. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labelled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion ...

Plasma membranes are the main liaisons between the intercellular and extracellular environment, p... more Plasma membranes are the main liaisons between the intercellular and extracellular environment, playing a critical role in numerous biological processes. Recent research has challenged the long-standing "fluid mosaic model," representing membranes as densely packed, heterogeneous environments. Within these complex membranes are transmembrane proteins which comprise up to 50% of the membrane mass, and are themselves diverse in sequence, structure, and function. Combining two-dimensional infrared spectroscopy (2D IR) and molecular dynamics simulation (MD), this thesis explores membrane complexity from two perspectives: first, it addresses the sequence heterogeneity in transmembrane peptides; and second, it explores the effect this crowded environment has on the lipids themselves and the implications this has on future membrane studies. Site-specific hydration of transmembrane peptides was probed using singly isotope-labeled pH (Low) Insertion Peptides, or pHLIPs. These peptides are a class of small transmembrane helices containing ~30% polar residues. By including a single-residue ¹³C=¹⁸O isotope label on the pHLIP backbone, IR experiments effectively produce a single-residue spectrum separate from the main peptide peak. With computational models to connect atomistic structure from MD to infrared frequency shifts, these site-specific spectra reveal local hydration as far as 1 nm into the hydrophobic membrane core. Crowding experiments probed dynamics at the lipid-water interface of model membranes as a function of transmembrane peptide concentration. These dynamics, drawn from time-dependent 2D IR, trend non-monotonically with peptide concentration, revealing three dynamical regimes: a pure lipid-like, a bulk-like, and a crowded regime. Through similar computational methods, these dynamics were linked to water structure at the lipid-water interface, which is perturbed by peptide insertion. Finally, preliminary work has been carried out in developing transient 2D IR methods for applications to protein folding. The f [...]

Biophysical Journal, 2022

ConspectusLipid membranes are more than just barriers between cell compartments; they provide mol... more ConspectusLipid membranes are more than just barriers between cell compartments; they provide molecular environments with a finely tuned balance between hydrophilic and hydrophobic interactions that enable proteins to dynamically fold and self-assemble to regulate biological function. Characterizing dynamics at the lipid-water interface is essential to understanding molecular complexities from the thermodynamics of liquid-liquid phase separation down to picosecond-scale reorganization of interfacial hydrogen-bond networks.Ultrafast vibrational spectroscopy, including two-dimensional infrared (2D IR) and vibrational sum-frequency generation (VSFG) spectroscopies, is a powerful tool to examine picosecond interfacial dynamics. Two-dimensional IR spectroscopy provides a bond-centered view of dynamics with subpicosecond time resolutions, as vibrational frequencies are highly sensitive to the local environment. Recently, 2D IR spectroscopy has been applied to carbonyl and phosphate vibrations intrinsically located at the lipid-water interface. Interface-specific VSFG spectroscopy probes the water vibrational modes directly, accessing H-bond strength and water organization at lipid headgroup positions. Signals in VSFG arise from the interfacial dipole contributions, directly probing headgroup ordering and water orientation to provide a structural view of the interface.In this Account we discuss novel applications of ultrafast spectroscopy to lipid membranes, a field that has experienced significant growth over the past decade. In particular, ultrafast experiments now offer a molecular perspective on increasingly complex membranes. The powerful combination of ultrafast, interface-selective spectroscopy and simulations opens up new routes to understanding multicomponent membranes and their function. This Account highlights key prevailing views that have emerged from recent experiments: (1) Water dynamics at the lipid-water interface are slow compared to those of bulk water as a result of disrupted H-bond networks near the headgroups. (2) Peptides, ions, osmolytes, and cosolvents perturb interfacial dynamics, indicating that dynamics at the interface are affected by bulk solvent dynamics and vice versa. (3) The interfacial environment is generally dictated by the headgroup structure and orientation, but hydrophobic interactions within the acyl chains also modulate interfacial dynamics. Ultrafast spectroscopy has been essential to characterizing the biophysical chemistry of the lipid-water interface; however, challenges remain in interpreting congested spectra as well as designing appropriate model systems to capture the complexity of a membrane environment.

Biology takes place in crowded, heterogeneous environments, and it is therefore essential to acco... more Biology takes place in crowded, heterogeneous environments, and it is therefore essential to account for crowding effects in our under-standing of biophysical processes at the molecular level. Comparable to the cytosol, proteins occupy approximately 30% of the plasma membrane surface, thus crowding should have an effect on the local structure and dynamics at the lipid-water interface. Using a combi-nation of ultrafast two-dimensional infrared spectroscopy and molecular dynamics simulations, we quantify the effects of membrane peptide concentration on the picosecond interfacial H-bond dynamics. The measurements reveal nonmonotonic dependence of water orientation and dynamics as a function of transmembrane peptide-to-lipid ratio. We observe three dynamical regimes: a pure lipid-like regime at low peptide concentration; a bulk-like region at intermediate peptide concentration where dynamics are faster by ~20% com-pared to the pure lipid bilayer; and a crowded regime where high peptide concentration slows dynamics by ~50%.

The Journal of Physical Chemistry Letters

Liquid-liquid phase separation is common in complex mixtures, but the behavior of nanoconfined li... more Liquid-liquid phase separation is common in complex mixtures, but the behavior of nanoconfined liquids is poorly understood from a physical perspective. Dimethyl sulfoxide (DMSO) is an amphiphilic molecule with unique concentration-dependent bulk properties in mixtures with water. Here, we use ultrafast two-dimensional infrared (2D IR) spectroscopy to measure the H-bond dynamics of two probe molecules with different polarities: formamide (FA) and dimethyl formamide (DMF). Picosecond H-bond dynamics are fastest in the intermediate concentration regime (20-50 mol% DMSO); as such confined water exhibits bulk-like dynamics. Each vibrational probe experiences a unique microscopic environment as a result of nanoscale phase separation. Molecular dynamics simulations show that the dynamics span multiple timescales from femtoseconds to nanoseconds. Our studies suggest a previously unknown liquid environment, which we label "local bulk", in which despite the local heterogeneity, the ultrafast H-bond dynamics are similar to bulk water.

Optics Letters

We present a pH-jump two-dimensional infrared (2D IR) spectrometer to probe pH-dependent conforma... more We present a pH-jump two-dimensional infrared (2D IR) spectrometer to probe pH-dependent conformational changes from nanoseconds to milliseconds. The design incorporates a nanosecond 355 nm source into a pulse-shaper-based 2D IR spectrometer to trigger dissociation of a caged proton prior to probing subsequent conformational changes with femtosecond 2D IR spectroscopy. We observe a blue shift in the amide I mode (C═O stretch) of diglycine induced by protonation of the terminal amine. This method combines the bond-specific structural sensitivity of ultrafast 2D IR with triggered conformational dynamics, providing structural access to multiscale biomolecular transformations such as protein folding.

The Journal of Physical Chemistry A, 2016

Hydrogen bonding (HB) populations were computed for the terminal oxygen atom, OT, and the solvent... more Hydrogen bonding (HB) populations were computed for the terminal oxygen atom, OT, and the solvent H atoms of interest for HB-donating solvents used to optimize the map: hexanol, ethanol, methanol, and D2O, as well as the HB-donating solvents used to assess the map capabilities: isopropanol, butanol, diethylene glycol, and dioxane. All HB analysis used the analysis tools in GROMACS 4.5.3. 1 The HB definition consists of a 30° cutoff H-donor-acceptor angle and a 0.35 nm cutoff donor-acceptor radius. Hydrogen bond analyses were performed for each stored snapshot of the 10 ns trajectory. The trajectories were then split into 0, 1, and 2 HB sub-trajectories for analysis of the electrostatic parameters and fitting of the corresponding peaks in the experimental FTIR spectra as discussed in the main text.

Biophysical Journal

Transmembrane peptides contain polar residues in the interior of the membrane, which may alter th... more Transmembrane peptides contain polar residues in the interior of the membrane, which may alter the electrostatic environment and favor hydration in the otherwise nonpolar environment of the membrane core. Here, we demonstrate a general, nonperturbative strategy to probe hydration of the peptide backbone at specific depths within the bilayer using a combination of site-specific isotope labels, ultrafast two-dimensional infrared spectroscopy, and spectral modeling based on molecular dynamics simulations. Our results show that the amphiphilic pH-low insertion peptide supports a highly heterogeneous environment, with significant backbone hydration of nonpolar residues neighboring charged residues. For example, a leucine residue located as far as 1 nm into the hydrophobic bulk reports hydrogen-bonded populations as high as ∼20%. These findings indicate that the polar nature of these residues may facilitate the transport of water molecules into the hydrophobic core of the membrane.