Steven Roeters | University of Amsterdam (original) (raw)

Papers by Steven Roeters

Biointerphases, 2022

In this Tutorial series, we aim to provide an accessible introduction to vibrational sum frequenc... more In this Tutorial series, we aim to provide an accessible introduction to vibrational sum frequency generation (VSFG) spectroscopy, targeted toward people entering the VSFG world without a rigorous formal background in optical physics or nonlinear spectroscopy. In this article, we describe in depth how a broadband VSFG spectrometer is designed and constructed, using the instrument in SurfLab, Aarhus University, as an illustrative case. Detailed information about specific instrumentation (together with reasons why things are the way they are) is given throughout. This information is often omitted in other descriptions of such instrumentation and so will be invaluable to people new to the field.

Journal of Biological Chemistry, Sep 1, 2016

Human ␣-synuclein (␣S) has been shown to be N terminally acetylated in its physiological state. T... more Human ␣-synuclein (␣S) has been shown to be N terminally acetylated in its physiological state. This modification is proposed to modulate the function and aggregation of ␣S into amyloid fibrils. Using bacterially expressed acetylated-␣S (NTAc-␣S) and endogenous ␣S (Endo-␣S) from human erythrocytes, we show that N-terminal acetylation has little impact on ␣S binding to anionic membranes and thus likely not relevant for regulating membrane affinity. N-terminal acetylation does have an effect on ␣S aggregation, resulting in a narrower distribution of the aggregation lag times and rates. 2D-IR spectra show that acetylation changes the secondary structure of ␣S in fibrils. This difference may arise from the slightly higher helical propensity of acetylated-␣S in solution leading to a more homogenous fibril population with different fibril structure than non-acetylated ␣S. We speculate that N-terminal acetylation imposes conformational restraints on N-terminal residues in ␣S, thus predisposing ␣S toward specific interactions with other binding partners or alternatively decrease nonspecific interactions.

The misfolding of ⍺-synuclein (aS) into amyloid aggregates is associated with severe brain disord... more The misfolding of ⍺-synuclein (aS) into amyloid aggregates is associated with severe brain disorders. Aggregated copies of aS are found in the amyloid aggregates observed in brain tissues from Parkinson's patients. Surfaces are known to catalyze the formation of amyloid aS aggregates. Despite the importance of the role of interfaces and several decades of structural studies, the 3D structure of aS when bound to interfaces is still not completely clear. Hydrophobic interfaces are particularly important here. We report interface-specific sum-frequency generation (SFG) experiments to determine how monomeric aS binds to the air-water interface, a model system for hydrophobic surfaces in general. We model the SFG data by combining the experimental data directly to theoretical spectra calculations from molecular dynamics simulations. We find that aS, which is an intrinsically disordered protein in solution, folds into a defined, mostly helical, secondary structure at the air-water interface. The binding pose is reminiscent of an umbrella-shape, where the C-terminus represents the 'pole' and protrudes into the water phase, while the N-terminus and the NAC region span the canopy at the interface. In this binding pose, aS is prone to aggregate, which could explain the catalytic effect of hydrophobic interfaces and air bubbles on aS fibrillation.

Journal of Physical Chemistry B, Jan 6, 2023

Langmuir, Nov 3, 2021

High-resolution structural information on membrane proteins is essential for understanding cell b... more High-resolution structural information on membrane proteins is essential for understanding cell biology and for the structure-based design of new medical drugs and drug delivery strategies. X-ray diffraction (XRD) can provide angstrom-level information about the structure of membrane proteins, yet for XRD experiments, proteins are removed from their native membrane environment, chemically stabilized, and crystallized, all of which can compromise the conformation. Here, we describe how a combination of surface-sensitive vibrational spectroscopy and molecular dynamics simulations can account for the native membrane environment. We observe the structure of a glycerol facilitator channel (GlpF), an aquaporin membrane channel finely tuned to selectively transport water and glycerol molecules across the membrane barrier. We find subtle but significant differences between the XRD structure and the inferred in situ structure of GlpF.

Actin, a key component of the cytoskeleton in eukaryotic cells, plays a crucial role in regulatin... more Actin, a key component of the cytoskeleton in eukaryotic cells, plays a crucial role in regulating cell morphology and transport. The morphology, mechanical, and biochemical properties of these filaments and bundles are determined by their monomer structure and by protein-protein contacts. Crowded environments are known to organize filaments into bundles. However, less is known how crowding and bundling affect the structure of F-actin. Here, we employed two-dimensional infrared (2D IR) spectroscopy and structure-based spectral calculations to investigate the morphology-dependent secondary-structure and local environments in filaments and weakly or strongly bundled networks. The results indicate that actin undergo secondary structural changes upon bundling, resulting in a decrease in beta-sheet and increase in the loop conformations. Moreover, strongly bundled networks experience a decrease in backbone solvent exposure, with relatively low perturbation of alpha-helix and the beta-she...

The misfolding of ⍺-synuclein (aS) into amyloid aggregates is associated with severe brain disord... more The misfolding of ⍺-synuclein (aS) into amyloid aggregates is associated with severe brain disorders. Aggregat-ed copies of aS are found in the amyloid aggregates observed in brain tissues from Parkinson’s patients. Surfaces are known to catalyze the formation of amyloid aS aggregates. Despite the importance of the role of inter-faces and several decades of structural studies, the 3D structure of aS when bound to interfaces is still not completely clear. Hydrophobic interfaces are particularly important here. We report interface-specific sum-frequency generation (SFG) experiments to determine how monomeric aS binds to the air-water interface, a model system for hydrophobic surfaces in general. We model the SFG data by combining the experimental data directly to theoretical spectra calculations from molecular dynamics simulations. We find that aS, which is an intrinsically disordered protein in solution, folds into a defined, mostly helical, secondary structure at the air-water inter...

The Journal of Physical Chemistry B

The aberrant folding of proteins into amyloid aggregates is associated with over 50 diseases. The... more The aberrant folding of proteins into amyloid aggregates is associated with over 50 diseases. The amyloid aggregation of α-synuclein (αS), related to Parkinson’s disease, can be catalyzed by lipid-membrane surfaces. Despite the importance of lipid surfaces and several decades of structural studies, the 3D-structure of lipid-membrane bound αS is still not known in detail. In particular, there is little information about the self-assembly and orientation of αS when interacting with lipid surfaces under physiologically relevant conditions. Here, we report interface-specific vibrational sum-frequency generation (VSFG) experiments revealing how monomeric αS binds, folds and orients at anionic lipid membranes. Since VSFG is inherently surface specific, the experiments can be performed at high αS–lipid ratios, far beyond previous structural studies. To interpret the experimental VSFG data, we present an analysis method in which out-of-equilibrium molecular-dynamics simulations are used to ...

SFG and 2D IR Spectra of the ice nucleation protein construct InaZ9R.

Proteins are the molecular machines of life. Shaped by billions of years of evolution, they perfo... more Proteins are the molecular machines of life. Shaped by billions of years of evolution, they perform tasks that vary from DNA transcription to storage of energy from sunlight to extracellular protection - with an efficiency, versatility and durability that synthetic chemists can only dream of. Various physical techniques such as X-ray crystallography and nuclear magnetic resonance spectroscopy, have helped to unravel the structure and dynamics of a large number of proteins. However, in some cases it is challenging to study proteins with conventional techniques. Notable examples are proteins active at interfaces (e.g. cell membranes or the air-water interface), and protein species (oligomers) that are crucial in the formation of amyloid structures, which are thought to be related to diseases like Alzheimer's and Parkinson's. In this thesis, we show how linear and non-linear vibrational spectroscopy (in particular: vibrational sum-frequency generation spectroscopy, one- and two...

The Journal of Physical Chemistry Letters, 2021

The conversion of biomass into green fuels and chemicals is of great societal interest. Engineers... more The conversion of biomass into green fuels and chemicals is of great societal interest. Engineers have been designing new cellulase enzymes for the breakdown of otherwise insoluble cellulose materials. A barrier to the rational design of new enzymes has been our lack of a molecular picture of how cellulase binding occurs. A critical factor is the attachment via the enzyme's carbohydrate binding module (CBM). To elucidate the structural and mechanistic details of cellulase adsorption, we have combined experimental data from sum frequency generation spectroscopy with molecular dynamics simulations to probe the equilibrium structure and surface alignment of a 14-residue peptide mimicking the CBM. The data show that binding is driven by hydrogen bonding and that tyrosine side chains within the CBM align the cellulase with the registry of the cellulose surface. Such an alignment is favorable for the translocation and effective cellulose breakdown and is therefore likely an important parameter for the design of novel enzymes.

The Journal of Physical Chemistry B, 2021

In this study, we use surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy ... more In this study, we use surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy to investigate the interaction between model lipid monolayers and Aβ(1-42) in its monomeric and aggregated states. Combining VSFG with atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence measurements, we found that only small aggregates with probably a β-hairpin-like structure adsorbed to the zwitterionic lipid monolayer (DOPC). In contrast, larger aggregates with an extended β-sheet structure adsorbed to a negatively charged lipid monolayer (DOPG). The adsorption of small, initially formed aggregates strongly destabilized both monolayers, but only the DOPC monolayer was completely disrupted. We showed that the intensity of the amide-II' band in achiral (SSP) and chiral (SPP) polarization combinations increased in time when Aβ(1-42) aggregates accumulated at the DOPG monolayer. Nevertheless, almost no adsorption of preformed mature fibrils to DOPG monolayers was detected. By performing spectral VSFG calculations, we revealed a clear correlation between the amide-II' signal and the degree of amyloid aggregates (e.g., oligomers or (proto)fibrils) of various Aβ(1-42) structures. The calculations showed that only structures with a significant amyloid β-sheet content have a strong amide-II' intensity, in line with previous Raman studies. The combination of the presented results substantiates the amide-II(') band as a legitimate amyloid marker.

Silaffin peptide R5 is key for the biogenesis of silica cell walls of diatoms. Biosilification by... more Silaffin peptide R5 is key for the biogenesis of silica cell walls of diatoms. Biosilification by the R5 peptide has potential in biotechnology, drug development, and materials science due to its ability to precipitate stable, high fidelity silica sheets and particles. A true barrier for the design of novel peptide-based architectures for wider applications has been the limited understanding of the interfacial structure of R5 when precipitating silica nanoparticles. While R5-silica interactions have been studied in detail at flat surfaces, the structure within nanophase particles is still being debated. We herein elucidate the conformation of R5 in its active form within silica particles by combining interface-specific vibrational spectroscopy data with solid-state NMR torsion angles using theoretical spectra. Our calculations show that R5 is structured and undergoes a conformational transition from a strand-type motif in solution to a more curved, contracted structure when interacting with silica precursors.

We propose a novel numerical method able to determine efficiently and effectively the relationshi... more We propose a novel numerical method able to determine efficiently and effectively the relationship of complementarity between portions of protein surfaces. This innovative and general procedure, based on the representation of the molecular iso-electron density surface in terms of 2D Zernike polynomials, allows the rapid and quantitative assessment of the geometrical shape complementarity between interacting proteins, that was unfeasible with previous methods. We first tested the method with a large dataset of known protein complexes obtaining an overall area under the ROC curve of 0.76 in the blind recognition of binding sites and then applied it to investigate the features of the interaction between the Spike protein of SARS-CoV-2 and human cellular receptors. Our results indicate that SARS-CoV-2 uses a dual strategy: its spike protein could also interact with sialic acid receptors of the cells in the upper airways, in addition to the known interaction with Angiotensin-converting e...

The Journal of Physical Chemistry B, 2020

Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sa... more Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sarcolemma resides the multidomain dysferlin protein. Mutations in this protein render it unable to repair the sarcolemma and have been linked to muscular dystrophy. A key step in dysferlin-regulated repair is the binding of the C2A domain to the lipid membrane upon increased intracellular calcium. Mutations mapped to this domain cause loss of binding ability of the C2A domain. There is a crucial need to understand the geometry of dysferlin C2A at a membrane interface as well as cell membrane lipid reorientation when compared to that of a mutant. Here, we describe a comparison between the wild-type dysferlin C2A and a mutation to the conserved aspartic acids in the domain binding loops. To identify both the geometry and the cell membrane lipid reorientation, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the interaction with a model cell membrane composed of phosphotidylserine and phosphotidylcholine. Observed changes in surface pressure demonstrate that calcium-bridged electrostatic interactions govern the initial interaction of the C2A domains docking with a lipid membrane. SFG spectra taken from the amide-I region for the wild type and variant contain features near 1642, 1663, and 1675 cm-1 related to the C2A domain β-sandwich secondary structure, indicating that the domain binds in a specific orientation. Mapping simulated SFG spectra to the experimentally collected spectra indicated that both wild-type and variant domains have nearly the same orientation to the membrane surface. However, examining the ordering of the lipids that make up a model membrane using SFG, we find that the wild type clusters the lipids as seen by the increase in the ratio of the CD3 and CD2 symmetric intensities by 170% for the wild type and by 120% for the variant. This study highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.

Understanding the assembly of proteins at the air water interface (AWI) informs the formation of ... more Understanding the assembly of proteins at the air water interface (AWI) informs the formation of protein films, emulsion properties, and protein aggregation. Determination of protein conformation and orientation at an interface is difficult to resolve with a single experimental or simulation technique alone. To date, the interfacial structure of even one of the most widely studied proteins, lysozyme, at the AWI remains unresolved. In this study, MD simulations, are used to determine if the protein adopts a side-on, head-on, or axial orientation at the AWI with two different forcefields GROMOS-53a6+SPC/E and a99SB-disp+TIP4P-D. Vibrational SFG spectroscopy experiments and spectral SFG calculations validate consistency between the structure determined from MD and experiments. Overall, we show with strong agreement, that lysozyme adopts an axial conformation at pH 7. Further, we provide molecular-level insight as to how pH influences the binding domains of lysozyme resulting in side-on adsorption near the isoelectric point of lysozyme.

Biointerphases, 2022

In this Tutorial series, we aim to provide an accessible introduction to vibrational sum frequenc... more In this Tutorial series, we aim to provide an accessible introduction to vibrational sum frequency generation (VSFG) spectroscopy, targeted toward people entering the VSFG world without a rigorous formal background in optical physics or nonlinear spectroscopy. In this article, we describe in depth how a broadband VSFG spectrometer is designed and constructed, using the instrument in SurfLab, Aarhus University, as an illustrative case. Detailed information about specific instrumentation (together with reasons why things are the way they are) is given throughout. This information is often omitted in other descriptions of such instrumentation and so will be invaluable to people new to the field.

Journal of Biological Chemistry, Sep 1, 2016

Human ␣-synuclein (␣S) has been shown to be N terminally acetylated in its physiological state. T... more Human ␣-synuclein (␣S) has been shown to be N terminally acetylated in its physiological state. This modification is proposed to modulate the function and aggregation of ␣S into amyloid fibrils. Using bacterially expressed acetylated-␣S (NTAc-␣S) and endogenous ␣S (Endo-␣S) from human erythrocytes, we show that N-terminal acetylation has little impact on ␣S binding to anionic membranes and thus likely not relevant for regulating membrane affinity. N-terminal acetylation does have an effect on ␣S aggregation, resulting in a narrower distribution of the aggregation lag times and rates. 2D-IR spectra show that acetylation changes the secondary structure of ␣S in fibrils. This difference may arise from the slightly higher helical propensity of acetylated-␣S in solution leading to a more homogenous fibril population with different fibril structure than non-acetylated ␣S. We speculate that N-terminal acetylation imposes conformational restraints on N-terminal residues in ␣S, thus predisposing ␣S toward specific interactions with other binding partners or alternatively decrease nonspecific interactions.

The misfolding of ⍺-synuclein (aS) into amyloid aggregates is associated with severe brain disord... more The misfolding of ⍺-synuclein (aS) into amyloid aggregates is associated with severe brain disorders. Aggregated copies of aS are found in the amyloid aggregates observed in brain tissues from Parkinson's patients. Surfaces are known to catalyze the formation of amyloid aS aggregates. Despite the importance of the role of interfaces and several decades of structural studies, the 3D structure of aS when bound to interfaces is still not completely clear. Hydrophobic interfaces are particularly important here. We report interface-specific sum-frequency generation (SFG) experiments to determine how monomeric aS binds to the air-water interface, a model system for hydrophobic surfaces in general. We model the SFG data by combining the experimental data directly to theoretical spectra calculations from molecular dynamics simulations. We find that aS, which is an intrinsically disordered protein in solution, folds into a defined, mostly helical, secondary structure at the air-water interface. The binding pose is reminiscent of an umbrella-shape, where the C-terminus represents the 'pole' and protrudes into the water phase, while the N-terminus and the NAC region span the canopy at the interface. In this binding pose, aS is prone to aggregate, which could explain the catalytic effect of hydrophobic interfaces and air bubbles on aS fibrillation.

Journal of Physical Chemistry B, Jan 6, 2023

Langmuir, Nov 3, 2021

High-resolution structural information on membrane proteins is essential for understanding cell b... more High-resolution structural information on membrane proteins is essential for understanding cell biology and for the structure-based design of new medical drugs and drug delivery strategies. X-ray diffraction (XRD) can provide angstrom-level information about the structure of membrane proteins, yet for XRD experiments, proteins are removed from their native membrane environment, chemically stabilized, and crystallized, all of which can compromise the conformation. Here, we describe how a combination of surface-sensitive vibrational spectroscopy and molecular dynamics simulations can account for the native membrane environment. We observe the structure of a glycerol facilitator channel (GlpF), an aquaporin membrane channel finely tuned to selectively transport water and glycerol molecules across the membrane barrier. We find subtle but significant differences between the XRD structure and the inferred in situ structure of GlpF.

Actin, a key component of the cytoskeleton in eukaryotic cells, plays a crucial role in regulatin... more Actin, a key component of the cytoskeleton in eukaryotic cells, plays a crucial role in regulating cell morphology and transport. The morphology, mechanical, and biochemical properties of these filaments and bundles are determined by their monomer structure and by protein-protein contacts. Crowded environments are known to organize filaments into bundles. However, less is known how crowding and bundling affect the structure of F-actin. Here, we employed two-dimensional infrared (2D IR) spectroscopy and structure-based spectral calculations to investigate the morphology-dependent secondary-structure and local environments in filaments and weakly or strongly bundled networks. The results indicate that actin undergo secondary structural changes upon bundling, resulting in a decrease in beta-sheet and increase in the loop conformations. Moreover, strongly bundled networks experience a decrease in backbone solvent exposure, with relatively low perturbation of alpha-helix and the beta-she...

The misfolding of ⍺-synuclein (aS) into amyloid aggregates is associated with severe brain disord... more The misfolding of ⍺-synuclein (aS) into amyloid aggregates is associated with severe brain disorders. Aggregat-ed copies of aS are found in the amyloid aggregates observed in brain tissues from Parkinson’s patients. Surfaces are known to catalyze the formation of amyloid aS aggregates. Despite the importance of the role of inter-faces and several decades of structural studies, the 3D structure of aS when bound to interfaces is still not completely clear. Hydrophobic interfaces are particularly important here. We report interface-specific sum-frequency generation (SFG) experiments to determine how monomeric aS binds to the air-water interface, a model system for hydrophobic surfaces in general. We model the SFG data by combining the experimental data directly to theoretical spectra calculations from molecular dynamics simulations. We find that aS, which is an intrinsically disordered protein in solution, folds into a defined, mostly helical, secondary structure at the air-water inter...

The Journal of Physical Chemistry B

The aberrant folding of proteins into amyloid aggregates is associated with over 50 diseases. The... more The aberrant folding of proteins into amyloid aggregates is associated with over 50 diseases. The amyloid aggregation of α-synuclein (αS), related to Parkinson’s disease, can be catalyzed by lipid-membrane surfaces. Despite the importance of lipid surfaces and several decades of structural studies, the 3D-structure of lipid-membrane bound αS is still not known in detail. In particular, there is little information about the self-assembly and orientation of αS when interacting with lipid surfaces under physiologically relevant conditions. Here, we report interface-specific vibrational sum-frequency generation (VSFG) experiments revealing how monomeric αS binds, folds and orients at anionic lipid membranes. Since VSFG is inherently surface specific, the experiments can be performed at high αS–lipid ratios, far beyond previous structural studies. To interpret the experimental VSFG data, we present an analysis method in which out-of-equilibrium molecular-dynamics simulations are used to ...

SFG and 2D IR Spectra of the ice nucleation protein construct InaZ9R.

Proteins are the molecular machines of life. Shaped by billions of years of evolution, they perfo... more Proteins are the molecular machines of life. Shaped by billions of years of evolution, they perform tasks that vary from DNA transcription to storage of energy from sunlight to extracellular protection - with an efficiency, versatility and durability that synthetic chemists can only dream of. Various physical techniques such as X-ray crystallography and nuclear magnetic resonance spectroscopy, have helped to unravel the structure and dynamics of a large number of proteins. However, in some cases it is challenging to study proteins with conventional techniques. Notable examples are proteins active at interfaces (e.g. cell membranes or the air-water interface), and protein species (oligomers) that are crucial in the formation of amyloid structures, which are thought to be related to diseases like Alzheimer's and Parkinson's. In this thesis, we show how linear and non-linear vibrational spectroscopy (in particular: vibrational sum-frequency generation spectroscopy, one- and two...

The Journal of Physical Chemistry Letters, 2021

The conversion of biomass into green fuels and chemicals is of great societal interest. Engineers... more The conversion of biomass into green fuels and chemicals is of great societal interest. Engineers have been designing new cellulase enzymes for the breakdown of otherwise insoluble cellulose materials. A barrier to the rational design of new enzymes has been our lack of a molecular picture of how cellulase binding occurs. A critical factor is the attachment via the enzyme's carbohydrate binding module (CBM). To elucidate the structural and mechanistic details of cellulase adsorption, we have combined experimental data from sum frequency generation spectroscopy with molecular dynamics simulations to probe the equilibrium structure and surface alignment of a 14-residue peptide mimicking the CBM. The data show that binding is driven by hydrogen bonding and that tyrosine side chains within the CBM align the cellulase with the registry of the cellulose surface. Such an alignment is favorable for the translocation and effective cellulose breakdown and is therefore likely an important parameter for the design of novel enzymes.

The Journal of Physical Chemistry B, 2021

In this study, we use surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy ... more In this study, we use surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy to investigate the interaction between model lipid monolayers and Aβ(1-42) in its monomeric and aggregated states. Combining VSFG with atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence measurements, we found that only small aggregates with probably a β-hairpin-like structure adsorbed to the zwitterionic lipid monolayer (DOPC). In contrast, larger aggregates with an extended β-sheet structure adsorbed to a negatively charged lipid monolayer (DOPG). The adsorption of small, initially formed aggregates strongly destabilized both monolayers, but only the DOPC monolayer was completely disrupted. We showed that the intensity of the amide-II' band in achiral (SSP) and chiral (SPP) polarization combinations increased in time when Aβ(1-42) aggregates accumulated at the DOPG monolayer. Nevertheless, almost no adsorption of preformed mature fibrils to DOPG monolayers was detected. By performing spectral VSFG calculations, we revealed a clear correlation between the amide-II' signal and the degree of amyloid aggregates (e.g., oligomers or (proto)fibrils) of various Aβ(1-42) structures. The calculations showed that only structures with a significant amyloid β-sheet content have a strong amide-II' intensity, in line with previous Raman studies. The combination of the presented results substantiates the amide-II(') band as a legitimate amyloid marker.

Silaffin peptide R5 is key for the biogenesis of silica cell walls of diatoms. Biosilification by... more Silaffin peptide R5 is key for the biogenesis of silica cell walls of diatoms. Biosilification by the R5 peptide has potential in biotechnology, drug development, and materials science due to its ability to precipitate stable, high fidelity silica sheets and particles. A true barrier for the design of novel peptide-based architectures for wider applications has been the limited understanding of the interfacial structure of R5 when precipitating silica nanoparticles. While R5-silica interactions have been studied in detail at flat surfaces, the structure within nanophase particles is still being debated. We herein elucidate the conformation of R5 in its active form within silica particles by combining interface-specific vibrational spectroscopy data with solid-state NMR torsion angles using theoretical spectra. Our calculations show that R5 is structured and undergoes a conformational transition from a strand-type motif in solution to a more curved, contracted structure when interacting with silica precursors.

We propose a novel numerical method able to determine efficiently and effectively the relationshi... more We propose a novel numerical method able to determine efficiently and effectively the relationship of complementarity between portions of protein surfaces. This innovative and general procedure, based on the representation of the molecular iso-electron density surface in terms of 2D Zernike polynomials, allows the rapid and quantitative assessment of the geometrical shape complementarity between interacting proteins, that was unfeasible with previous methods. We first tested the method with a large dataset of known protein complexes obtaining an overall area under the ROC curve of 0.76 in the blind recognition of binding sites and then applied it to investigate the features of the interaction between the Spike protein of SARS-CoV-2 and human cellular receptors. Our results indicate that SARS-CoV-2 uses a dual strategy: its spike protein could also interact with sialic acid receptors of the cells in the upper airways, in addition to the known interaction with Angiotensin-converting e...

The Journal of Physical Chemistry B, 2020

Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sa... more Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sarcolemma resides the multidomain dysferlin protein. Mutations in this protein render it unable to repair the sarcolemma and have been linked to muscular dystrophy. A key step in dysferlin-regulated repair is the binding of the C2A domain to the lipid membrane upon increased intracellular calcium. Mutations mapped to this domain cause loss of binding ability of the C2A domain. There is a crucial need to understand the geometry of dysferlin C2A at a membrane interface as well as cell membrane lipid reorientation when compared to that of a mutant. Here, we describe a comparison between the wild-type dysferlin C2A and a mutation to the conserved aspartic acids in the domain binding loops. To identify both the geometry and the cell membrane lipid reorientation, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the interaction with a model cell membrane composed of phosphotidylserine and phosphotidylcholine. Observed changes in surface pressure demonstrate that calcium-bridged electrostatic interactions govern the initial interaction of the C2A domains docking with a lipid membrane. SFG spectra taken from the amide-I region for the wild type and variant contain features near 1642, 1663, and 1675 cm-1 related to the C2A domain β-sandwich secondary structure, indicating that the domain binds in a specific orientation. Mapping simulated SFG spectra to the experimentally collected spectra indicated that both wild-type and variant domains have nearly the same orientation to the membrane surface. However, examining the ordering of the lipids that make up a model membrane using SFG, we find that the wild type clusters the lipids as seen by the increase in the ratio of the CD3 and CD2 symmetric intensities by 170% for the wild type and by 120% for the variant. This study highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.

Understanding the assembly of proteins at the air water interface (AWI) informs the formation of ... more Understanding the assembly of proteins at the air water interface (AWI) informs the formation of protein films, emulsion properties, and protein aggregation. Determination of protein conformation and orientation at an interface is difficult to resolve with a single experimental or simulation technique alone. To date, the interfacial structure of even one of the most widely studied proteins, lysozyme, at the AWI remains unresolved. In this study, MD simulations, are used to determine if the protein adopts a side-on, head-on, or axial orientation at the AWI with two different forcefields GROMOS-53a6+SPC/E and a99SB-disp+TIP4P-D. Vibrational SFG spectroscopy experiments and spectral SFG calculations validate consistency between the structure determined from MD and experiments. Overall, we show with strong agreement, that lysozyme adopts an axial conformation at pH 7. Further, we provide molecular-level insight as to how pH influences the binding domains of lysozyme resulting in side-on adsorption near the isoelectric point of lysozyme.