Louise Aigrain - Academia.edu (original) (raw)

Papers by Louise Aigrain

Research paper thumbnail of Conséquences de l'échange de domaines évolutivement éloignés sur l'activité et la géométrie de la nadph-cytochrome P450 réductase

Research paper thumbnail of www.mdpi.com/journal/ijms Dynamic Control of Electron Transfers in Diflavin Reductases

Abstract: Diflavin reductases are essential proteins capable of splitting the two-electron flux f... more Abstract: Diflavin reductases are essential proteins capable of splitting the two-electron flux from reduced pyridine nucleotides to a variety of one electron acceptors. The primary sequence of diflavin reductases shows a conserved domain organization harboring two catalytic domains bound to the FAD and FMN flavins sandwiched by one or several non-catalytic domains. The catalytic domains are analogous to existing globular proteins: the FMN domain is analogous to flavodoxins while the FAD domain resembles ferredoxin reductases. The first structural determination of one member of the diflavin reductases family raised some questions about the architecture of the enzyme during catalysis: both FMN and FAD were in perfect position for interflavin transfers but the steric hindrance of the FAD domain rapidly prompted more complex hypotheses on the possible mechanisms for the electron transfer from FMN to external acceptors. Hypotheses of domain reorganization during catalysis in the context...

Research paper thumbnail of And Mutator Derivatives Establish Fidelity Checkpoints

Research paper thumbnail of Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms

Nucleic Acids Research, 2015

DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational ch... more DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intraprotein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s −1 , much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.

Research paper thumbnail of Combining Accurate FRET and Tracking of Single Protein and DNA Molecules in Live Bacteria

Biophysical Journal, 2014

Membrane organization of lipid anchored proteins plays a fundamental role in proper cell signalin... more Membrane organization of lipid anchored proteins plays a fundamental role in proper cell signaling behavior. Several important signaling proteins, such as members of the Src family of kinases, are anchored to the membrane by covalent acyl, alkyl, and glycosylphosphatidylinositol (GPI) moieties. A convenient approach to studying this lipid anchor organization in cells is to image genetically encoded lipidation motifs of various signaling proteins fused to a fluorescent protein. However, previous efforts to reduce this lateral organization to a simple model often ignore the complexity inherent in cell membranes and have contributed to an inconsistent picture of membrane organization. We use two-color fluorescence fluctuation spectroscopy to study the spatial organization of fluorescently tagged lipid anchors in live cell membranes. In order to retain the complexity of this organization, we simultaneously observe several modes of lipid anchor dynamics by employing a multi-dimensional analysis of the time-resolved fluorescence data. Time-correlated single photon counting (TCSPC) data is gathered on a 3-detector system optimized for simultaneous polarization and two-color fluorescence correlation spectroscopy (FCS). The degree of dynamic colocalization of eGFP-and mCherry-fused lipid anchors can be quantified by pulse-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), while single color FCS evaluates the translational mobility of the lipid anchors. The photon counting method also allows for time-resolved fluorescence anisotropy (TRFA) measurements, evaluating the rotational diffusion of the fluorescent protein, as well as fluorescence lifetime measurements to determine energy transfer. Taken together, this simultaneous multi-dimensional analysis of time-resolved fluorescence data offers a powerful approach towards determining the complex organizations of lipid anchors in live cell membranes.

Research paper thumbnail of Optimized Internalization of Fluorescently Labeled Biomolecules into Live Bacteria

Biophysical Journal, 2014

The aim of this work is to investigate the character of the time-course of the blood plasma parti... more The aim of this work is to investigate the character of the time-course of the blood plasma particles sizes and their concentrations in the samples during 30 hours after their preparation. For this purpose we use dynamic light scattering (DLS) that is a powerful technique for studying of the particle sizes in the solutions. We study the fresh heparinized blood plasma samples obtained from the donors (n=10) of different ages. For all samples we obtain particle size distributions (PSDs) of the scattered light by DLS. Each PSD contains from three to five specific peaks (protein complexes). For each peak we observe the mean sizes changing during 30 hours after sample preparation. Then we calculate the volume concentration of particles of each sort using the Rayleigh-Gans-Debye approximation. The time-courses of plasma particles sizes and their concentrations represent the oscillations with decreasing amplitudes and non-regular periods. Such behavior of the system is typical for relaxation transition of the system to another state that is causes by the disbalance of the aggregation and degradation rates. The amplitude and period of these oscillations could be useful parameters for some decease diagnostics.

Research paper thumbnail of Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

Journal of visualized experiments : JoVE, Jan 8, 2015

The ability to study biomolecules in vivo is crucial for understanding their function in a biolog... more The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisiæ, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or s...

Research paper thumbnail of Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion

Nature Communications, 2013

The fidelity of DNA polymerases depends on conformational changes that promote the rejection of i... more The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine singlemolecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.

Research paper thumbnail of The Closed and Compact Domain Organization of the 70-kDa Human Cytochrome P450 Reductase in Its Oxidized State As Revealed by NMR

Journal of Molecular Biology, 2012

The NADPH cytochrome P450 reductase (CPR), a diflavin enzyme, catalyzes the electron transfer (ET... more The NADPH cytochrome P450 reductase (CPR), a diflavin enzyme, catalyzes the electron transfer (ET) from NADPH to the substrate P450. The crystal structures of mammalian and yeast CPRs show a compact organization for the two domains containing FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), with a short interflavin distance consistent with fast ET from the NADPH-reduced FAD to the second flavin FMN. This conformation, referred as "closed", contrasts with the alternative opened or extended domain arrangements recently described for partially reduced or mutant CPR. Internal domain flexibility in this enzyme is indeed necessary to account for the apparently conflicting requirements of having FMN flavin accessible to both the FAD and the substrate P450 at the same interface. However, how interdomain dynamics influence internal and external ETs in CPR is still largely unknown. Here, we used NMR techniques to explore the global, domain-specific and residue-specific structural and dynamic properties of the nucleotide-free human CPR in solution in its oxidized state. Based on the backbone resonance assignment of this 70-kDa protein, we collected residuespecific 15 N relaxation and 1 H-15 N residual dipolar couplings. Surprisingly and in contrast with previous studies, the analysis of these NMR data revealed that the CPR exists in a unique and predominant conformation that highly resembles the closed conformation observed in the crystalline state. Based on our findings and the previous observations of conformational equilibria of the CPR in partially reduced states, we propose that the large-scale conformational transitions of the CPR during the catalytic cycle are tightly controlled to ensure optimal electron delivery.

Research paper thumbnail of Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation

Histochemistry and Cell Biology, 2014

cell viability and demonstrate that, whilst internalization increases with increased voltage, cel... more cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer.

Research paper thumbnail of Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase

EMBO reports, 2009

Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the ... more Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the core of the NADPH cytochrome P450 reductase (CPR). The FMN domain of CPR mediates electron shuttling from the FAD domain to cytochromes P450. Together, both enzymes form the main mixed-function oxidase system that participates in the metabolism of endo-and xenobiotic compounds in mammals. Available CPR structures show a closed conformation, with the two cofactors in tight proximity, which is consistent with FAD-to-FMN, but not FMN-to-P450, electron transfer. Here, we report the 2.5 Å resolution crystal structure of a functionally competent yeasthuman chimeric CPR in an open conformation, compatible with FMN-to-P450 electron transfer. Comparison with closed structures shows a major conformational change separating the FMN and FAD cofactors from 86 Å .

Research paper thumbnail of Internalization of Fluorescent Biomolecules for Long-Lived Single-Molecule Observation in Living Bacteria

Biophysical Journal, 2013

Metalloregulators regulate transcription in response to metal ion concentrations. How they intera... more Metalloregulators regulate transcription in response to metal ion concentrations. How they interact with DNA and change the DNA structure dictates the regulation process. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Here we use single-molecule fluorescence resonance energy transfer (smFRET) measurements to probe the dynamic interactions between CueR, a Cu1þ-responsive MerR-family regulator, and a double-strand DNA in real time one event at a time. Besides seeing its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are the first such examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two novel processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.

Research paper thumbnail of Single-Molecule Fluorescence and FRET Measurements on Internalized Proteins in Living Bacteria

Biophysical Journal, 2013

Calcitriol is the active form of Vitamin D 3. Epidemiological data show that women with Vitamin D... more Calcitriol is the active form of Vitamin D 3. Epidemiological data show that women with Vitamin D deficiency at the time of breast cancer diagnosis are 94% more likely to experience cancer spread and 73% more likely to die over the next 10 years, compared to women with adequate Vitamin D levels. Since vitamin D deficiency is especially common in African American and obese women, this observation may partially explain the relatively poor clinical outcome experienced by these patients [2]. Although current treatments for IBC

Research paper thumbnail of Long-Lived Intracellular Single-Molecule Fluorescence Using Electroporated Molecules

Biophysical Journal, 2013

Studies of biomolecules in vivo are crucial to understand their function in a natural, biological... more Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing electroporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desirable properties of organic fluorophores to perform single-molecule Fö rster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeast Saccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence.

Research paper thumbnail of Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH–cytochrome P450 reductase

Biochemical Journal, 2011

CPR (NADPH–cytochrome P450 reductase) is a multidomain protein containing two flavin-containing d... more CPR (NADPH–cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the inte...

Research paper thumbnail of Beginner’s guide to next-generation sequencing

The Biochemist, 2021

Since the publication of the first draft of the human genome 20 years ago, several novel sequenci... more Since the publication of the first draft of the human genome 20 years ago, several novel sequencing technologies have emerged. Whilst some drive the cost of DNA sequencing down, others address the difficult parts of the genome which remained inaccessible so far. But the next-generation sequencing (NGS) landscape is a fast-changing environment and one can easily get lost between second- and third- generation sequencers, or the pros and cons of short- versus long-read technologies. In this beginner’s guide to NGS, we will review the main NGS technologies available in 2021. We will compare sample preparation protocols and sequencing methods, highlighting the requirements and advantages of each technology.

Research paper thumbnail of Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion

Nature Communications, 2013

The fidelity of DNA polymerases depends on conformational changes that promote the rejection of i... more The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine singlemolecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.

Research paper thumbnail of Conséquences de l'échange de domaines évolutivement éloignés sur l'activité et la géométrie de la nadph-cytochrome P450 réductase

Research paper thumbnail of www.mdpi.com/journal/ijms Dynamic Control of Electron Transfers in Diflavin Reductases

Abstract: Diflavin reductases are essential proteins capable of splitting the two-electron flux f... more Abstract: Diflavin reductases are essential proteins capable of splitting the two-electron flux from reduced pyridine nucleotides to a variety of one electron acceptors. The primary sequence of diflavin reductases shows a conserved domain organization harboring two catalytic domains bound to the FAD and FMN flavins sandwiched by one or several non-catalytic domains. The catalytic domains are analogous to existing globular proteins: the FMN domain is analogous to flavodoxins while the FAD domain resembles ferredoxin reductases. The first structural determination of one member of the diflavin reductases family raised some questions about the architecture of the enzyme during catalysis: both FMN and FAD were in perfect position for interflavin transfers but the steric hindrance of the FAD domain rapidly prompted more complex hypotheses on the possible mechanisms for the electron transfer from FMN to external acceptors. Hypotheses of domain reorganization during catalysis in the context...

Research paper thumbnail of And Mutator Derivatives Establish Fidelity Checkpoints

Research paper thumbnail of Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms

Nucleic Acids Research, 2015

DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational ch... more DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intraprotein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s −1 , much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.

Research paper thumbnail of Combining Accurate FRET and Tracking of Single Protein and DNA Molecules in Live Bacteria

Biophysical Journal, 2014

Membrane organization of lipid anchored proteins plays a fundamental role in proper cell signalin... more Membrane organization of lipid anchored proteins plays a fundamental role in proper cell signaling behavior. Several important signaling proteins, such as members of the Src family of kinases, are anchored to the membrane by covalent acyl, alkyl, and glycosylphosphatidylinositol (GPI) moieties. A convenient approach to studying this lipid anchor organization in cells is to image genetically encoded lipidation motifs of various signaling proteins fused to a fluorescent protein. However, previous efforts to reduce this lateral organization to a simple model often ignore the complexity inherent in cell membranes and have contributed to an inconsistent picture of membrane organization. We use two-color fluorescence fluctuation spectroscopy to study the spatial organization of fluorescently tagged lipid anchors in live cell membranes. In order to retain the complexity of this organization, we simultaneously observe several modes of lipid anchor dynamics by employing a multi-dimensional analysis of the time-resolved fluorescence data. Time-correlated single photon counting (TCSPC) data is gathered on a 3-detector system optimized for simultaneous polarization and two-color fluorescence correlation spectroscopy (FCS). The degree of dynamic colocalization of eGFP-and mCherry-fused lipid anchors can be quantified by pulse-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), while single color FCS evaluates the translational mobility of the lipid anchors. The photon counting method also allows for time-resolved fluorescence anisotropy (TRFA) measurements, evaluating the rotational diffusion of the fluorescent protein, as well as fluorescence lifetime measurements to determine energy transfer. Taken together, this simultaneous multi-dimensional analysis of time-resolved fluorescence data offers a powerful approach towards determining the complex organizations of lipid anchors in live cell membranes.

Research paper thumbnail of Optimized Internalization of Fluorescently Labeled Biomolecules into Live Bacteria

Biophysical Journal, 2014

The aim of this work is to investigate the character of the time-course of the blood plasma parti... more The aim of this work is to investigate the character of the time-course of the blood plasma particles sizes and their concentrations in the samples during 30 hours after their preparation. For this purpose we use dynamic light scattering (DLS) that is a powerful technique for studying of the particle sizes in the solutions. We study the fresh heparinized blood plasma samples obtained from the donors (n=10) of different ages. For all samples we obtain particle size distributions (PSDs) of the scattered light by DLS. Each PSD contains from three to five specific peaks (protein complexes). For each peak we observe the mean sizes changing during 30 hours after sample preparation. Then we calculate the volume concentration of particles of each sort using the Rayleigh-Gans-Debye approximation. The time-courses of plasma particles sizes and their concentrations represent the oscillations with decreasing amplitudes and non-regular periods. Such behavior of the system is typical for relaxation transition of the system to another state that is causes by the disbalance of the aggregation and degradation rates. The amplitude and period of these oscillations could be useful parameters for some decease diagnostics.

Research paper thumbnail of Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

Journal of visualized experiments : JoVE, Jan 8, 2015

The ability to study biomolecules in vivo is crucial for understanding their function in a biolog... more The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisiæ, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or s...

Research paper thumbnail of Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion

Nature Communications, 2013

The fidelity of DNA polymerases depends on conformational changes that promote the rejection of i... more The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine singlemolecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.

Research paper thumbnail of The Closed and Compact Domain Organization of the 70-kDa Human Cytochrome P450 Reductase in Its Oxidized State As Revealed by NMR

Journal of Molecular Biology, 2012

The NADPH cytochrome P450 reductase (CPR), a diflavin enzyme, catalyzes the electron transfer (ET... more The NADPH cytochrome P450 reductase (CPR), a diflavin enzyme, catalyzes the electron transfer (ET) from NADPH to the substrate P450. The crystal structures of mammalian and yeast CPRs show a compact organization for the two domains containing FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), with a short interflavin distance consistent with fast ET from the NADPH-reduced FAD to the second flavin FMN. This conformation, referred as "closed", contrasts with the alternative opened or extended domain arrangements recently described for partially reduced or mutant CPR. Internal domain flexibility in this enzyme is indeed necessary to account for the apparently conflicting requirements of having FMN flavin accessible to both the FAD and the substrate P450 at the same interface. However, how interdomain dynamics influence internal and external ETs in CPR is still largely unknown. Here, we used NMR techniques to explore the global, domain-specific and residue-specific structural and dynamic properties of the nucleotide-free human CPR in solution in its oxidized state. Based on the backbone resonance assignment of this 70-kDa protein, we collected residuespecific 15 N relaxation and 1 H-15 N residual dipolar couplings. Surprisingly and in contrast with previous studies, the analysis of these NMR data revealed that the CPR exists in a unique and predominant conformation that highly resembles the closed conformation observed in the crystalline state. Based on our findings and the previous observations of conformational equilibria of the CPR in partially reduced states, we propose that the large-scale conformational transitions of the CPR during the catalytic cycle are tightly controlled to ensure optimal electron delivery.

Research paper thumbnail of Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation

Histochemistry and Cell Biology, 2014

cell viability and demonstrate that, whilst internalization increases with increased voltage, cel... more cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer.

Research paper thumbnail of Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase

EMBO reports, 2009

Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the ... more Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the core of the NADPH cytochrome P450 reductase (CPR). The FMN domain of CPR mediates electron shuttling from the FAD domain to cytochromes P450. Together, both enzymes form the main mixed-function oxidase system that participates in the metabolism of endo-and xenobiotic compounds in mammals. Available CPR structures show a closed conformation, with the two cofactors in tight proximity, which is consistent with FAD-to-FMN, but not FMN-to-P450, electron transfer. Here, we report the 2.5 Å resolution crystal structure of a functionally competent yeasthuman chimeric CPR in an open conformation, compatible with FMN-to-P450 electron transfer. Comparison with closed structures shows a major conformational change separating the FMN and FAD cofactors from 86 Å .

Research paper thumbnail of Internalization of Fluorescent Biomolecules for Long-Lived Single-Molecule Observation in Living Bacteria

Biophysical Journal, 2013

Metalloregulators regulate transcription in response to metal ion concentrations. How they intera... more Metalloregulators regulate transcription in response to metal ion concentrations. How they interact with DNA and change the DNA structure dictates the regulation process. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Here we use single-molecule fluorescence resonance energy transfer (smFRET) measurements to probe the dynamic interactions between CueR, a Cu1þ-responsive MerR-family regulator, and a double-strand DNA in real time one event at a time. Besides seeing its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are the first such examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two novel processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.

Research paper thumbnail of Single-Molecule Fluorescence and FRET Measurements on Internalized Proteins in Living Bacteria

Biophysical Journal, 2013

Calcitriol is the active form of Vitamin D 3. Epidemiological data show that women with Vitamin D... more Calcitriol is the active form of Vitamin D 3. Epidemiological data show that women with Vitamin D deficiency at the time of breast cancer diagnosis are 94% more likely to experience cancer spread and 73% more likely to die over the next 10 years, compared to women with adequate Vitamin D levels. Since vitamin D deficiency is especially common in African American and obese women, this observation may partially explain the relatively poor clinical outcome experienced by these patients [2]. Although current treatments for IBC

Research paper thumbnail of Long-Lived Intracellular Single-Molecule Fluorescence Using Electroporated Molecules

Biophysical Journal, 2013

Studies of biomolecules in vivo are crucial to understand their function in a natural, biological... more Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing electroporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desirable properties of organic fluorophores to perform single-molecule Fö rster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeast Saccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence.

Research paper thumbnail of Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH–cytochrome P450 reductase

Biochemical Journal, 2011

CPR (NADPH–cytochrome P450 reductase) is a multidomain protein containing two flavin-containing d... more CPR (NADPH–cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the inte...

Research paper thumbnail of Beginner’s guide to next-generation sequencing

The Biochemist, 2021

Since the publication of the first draft of the human genome 20 years ago, several novel sequenci... more Since the publication of the first draft of the human genome 20 years ago, several novel sequencing technologies have emerged. Whilst some drive the cost of DNA sequencing down, others address the difficult parts of the genome which remained inaccessible so far. But the next-generation sequencing (NGS) landscape is a fast-changing environment and one can easily get lost between second- and third- generation sequencers, or the pros and cons of short- versus long-read technologies. In this beginner’s guide to NGS, we will review the main NGS technologies available in 2021. We will compare sample preparation protocols and sequencing methods, highlighting the requirements and advantages of each technology.

Research paper thumbnail of Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion

Nature Communications, 2013

The fidelity of DNA polymerases depends on conformational changes that promote the rejection of i... more The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine singlemolecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.