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Andras Lukacs

University of Pecs, Department of Biophysics, Faculty Member

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I am a physicist by training but I am working in the field of biophysics since graduation. My primary interest is the study of functional dynamics of proteins using ultrafast spectroscopy as well as fluorescence spectroscopy
Phone: +3672535825
Address: Szigeti str 12
Biophysics Department
7624 Pecs, Hungary

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School of the Art Institute of Chicago

Dr.Mahmoud Lotfy

Minufiya University

Sofia University "St. Kliment Ohridski"

Rajapandiyan Panneerselvam

SRM UNIVERSITY

Universidade Federal Rural de Pernambuco

Dipankar Chatterjee

Indian Institute of Science

Virginia Commonwealth University

Massachusetts Institute of Technology (MIT)

shivani R pandya

Parul university

Universiti Pertahanan Nasional Malaysia

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Videos by Andras Lukacs

Presentation of the ultrafast spectroscopy lab at the Department of Biophysics, Medical School, University of Pecs

Ultrafast transient absorption system

5 views

Papers by Andras Lukacs

ATP-dependent conformational dynamics in a photoactivated adenylate cyclase revealed by fluorescence spectroscopy and small-angle X-ray scattering

Communications biology, Feb 2, 2024

Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of... more Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of controlling cellular cyclic adenosine monophosphate (cAMP) levels for optogenetic and other applications. In this work, we use an integrative approach that combines biophysical and structural biology methods to provide insight on the interaction of adenosine triphosphate (ATP) with the dark-adapted state of the photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata (OaPAC). A moderate affinity of the nucleotide for the enzyme was calculated and the thermodynamic parameters of the interaction have been obtained. Stopped-flow fluorescence spectroscopy and small-angle solution scattering have revealed significant conformational changes in the enzyme, presumably in the adenylate cyclase (AC) domain during the allosteric mechanism of ATP binding to OaPAC with small and large-scale movements observed to the best of our knowledge for the first time in the enzyme in solution upon ATP binding. These results are in line with previously reported drastic conformational changes taking place in several class III AC domains upon nucleotide binding.

Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix

Light-activated protein domains provide a convenient, modular, and genetically encodable sensor f... more Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

Functional dynamics of the photoactivated adenylate cyclase OaPAC revealed by fluorescence spectroscopy and small-angle X-ray scattering

Biophysical Journal, Feb 1, 2022

A possible way to relate the effects of SARS-CoV-2 induced changes in transferrin to severe COVID-19 associated diseases

bioRxiv (Cold Spring Harbor Laboratory), Feb 16, 2022

The potentially life-threatening Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) inf... more The potentially life-threatening Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is responsible for the coronavirus pandemic in 2019 (COVID-19). The transferrin as an essential component of iron-metabolism was suggested to be a link between iron transport associated diseases and COVID-19 infection. The effect of SARS-CoV-2 on human whole blood was studied by differential scanning calorimetry. The analysis and deconvolution of the thermal transition curves showed that the T m of transferrin related second peak decreased by 5.16 °C (6.4%) in the presence of SARS-CoV-2 virus. The ratio of the under-curve area of the two main peaks was greatly affected while the total enthalpy of the heat denaturation was nearly unchanged in the presence of the virus. Based on the results it is possible to conclude that SARS-CoV-2 through binding to transferrin can influence it's Fe 3+ uptake by inducing thermodynamic changes. Transferrin may stay in iron-free apo-conformational state, which probably depends on the SARS-CoV-2 concentration. SARS-CoV-2 might induce disturbance in the erythropoiesis due to the free iron overload generated iron toxicity. As a late consequence iron toxicity related hepatocellular carcinoma can even develop. Our work can support the basic role of transferrin in COVID-19 related severe diseases.

Mechanical Characterization and Structural Analysis of Latex-Containing and Latex-Free Intermaxillary Orthodontic Elastics

Polymers, Oct 23, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more

Functional dynamics of proteins revealed by fluorescence and femtosecond transient absorption spectroscopy

Solubility and Thermal Stability of Thermotoga maritima MreB

International Journal of Molecular Sciences, Dec 16, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more

Radical Formation in the Photoactivated Adenylate Cyclase OaPAC Revealed by Ultrafast Spectroscopy

Biophysical Journal, Feb 1, 2020

modes, and find that organizational changes can optimize for different objectives. Cargos with cl... more modes, and find that organizational changes can optimize for different objectives. Cargos with clustered motors are transported efficiently, but are slow to bind to microtubules. Cargos with motors dispersed rigidly on their surface bind microtubules quickly, but are transported inefficiently. Cargos with freelydiffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results point to a functional role for observed changes in motor organization on cargos, and suggest motor diffusivity as a control point for transport, either by modulation of adaptor proteins or changes in lipid composition.

Ultrafast Protein Dynamics Probed by Site Specific Transient IR Spectroscopy

The 22nd International Conference on Ultrafast Phenomena 2020, 2020

Ultrafast spectroscopy can measure real-time protein dynamics, but yields limited structural info... more Ultrafast spectroscopy can measure real-time protein dynamics, but yields limited structural information. Here we use unnatural amino acid substitution to place an IR absorber at specific locations in light activated proteins. Ultrafast pump - transient IR probe spectroscopy then yields unique site-specific data on protein structural dynamics.

The effect of the flexibility of hydrogen bonding network on low-frequency motions of amino acids. Evidence from Terahertz spectroscopy and DFT calculations

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, Feb 1, 2018

Low-frequency modes of l-Asp and l-Asn were studied in the range from 0.1 to 3.0 THz using time-d... more Low-frequency modes of l-Asp and l-Asn were studied in the range from 0.1 to 3.0 THz using time-domain Terahertz spectroscopy and density functional theory calculation. The results show that PBE-D2 shows more success than BLYP-D2 in prediction of THz absorption spectra. To compare their low-frequency modes, we adopted "vibrational character ID strips" proposed by Schmuttenmaer and coworkers [Journal of Physical Chemistry B, 117, 10444(2013)]. We found that the most intense THz absorption peaks of two compounds both involve severe distortion of their hydrogen bonding networks. Due to less rigid hydrogen bonding network in l-Asp, the side chain (carboxyl group) of l-Asp exhibits larger motions than that (carboxamide group) of l-Asn in low-frequency modes.

Photophysics of the Blue Light Using Flavin Domain

Accounts of Chemical Research, Jan 12, 2022

Light activated proteins are at the heart of photobiology and optogenetics, so there is wide inte... more Light activated proteins are at the heart of photobiology and optogenetics, so there is wide interest in understanding the mechanisms coupling optical excitation to protein function. In addition, such light activated proteins provide unique insights into the real-time dynamics of protein function. Using pump-probe spectroscopy, the function of a photoactive protein can be initiated by a sub-100 fs pulse of light, allowing subsequent protein dynamics to be probed from femtoseconds to milliseconds and beyond. Among the most interesting photoactive proteins are the Blue Light Using Flavin (BLUF) domain proteins, which regulate the response to light of a wide range of bacterial and some euglenoid processes. The photosensing mechanism of BLUF domains has long been a subject of debate. In contrast to other photoactive proteins, the electronic and nuclear structure of the chromophore (flavin) is the same in dark-and light-adapted states. Thus, the driving force for photoactivity is unclear. To address this question requires real-time observation of both chromophore excited state processes and their effect on the structure and dynamics of the surrounding protein matrix. In this Account we describe how time resolved infra-red (IR) experiments, coupled with chemical biology, provides important new insights into the signalling mechanism of BLUF domains. IR measurements are sensitive to changes in both chromophore electronic structure and protein hydrogen bonding interactions. These contributions are resolved by isotope labelling chromophore and protein Key References

Conformational Distributions and Proximity Relationships in the Rigor Complex of Actin and Myosin Subfragment-1

Journal of Biological Chemistry, 2000

Cyclic conformational changes in the myosin head are considered essential for muscle contraction.... more

Flexibility of myosin-subfragment-1 in its complex with actin as revealed by fluorescence resonance energy transfer

European journal of biochemistry, Jul 1, 2000

The flexibility of the acto±myosin complex in rigor conditions was characterized by measuring the... more

Short‐Lived Radical Intermediates in the Photochemistry of Glucose Oxidase

ChemPhysChem, Jun 21, 2019

Supporting information for this article is given via a link at the end of the document.

Infrared spectroscopy reveals multi-step multi-timescale photoactivation in the photoconvertible protein archetype dronpa

Nature Chemistry, Jun 11, 2018

Author Contributions SPL, AAG, CRH, AL and JNI measured and collected the data. SPL and CRH analy... more Author Contributions SPL, AAG, CRH, AL and JNI measured and collected the data. SPL and CRH analysed the data. AAG and JNI grew and purified the samples. GAJ performed the DFT calculations. GMG and PD built, developed and managed the ULTRA and LifeTime apparatus used in the measurements. AM designed the dronpa2 protein. PJT and SRM designed the experiment and wrote the paper, with discussion and editorial input from all authors Competing Interest Statement The authors declare no competing financial and non-financial interest. Data Availability Statement The data sets generated during the current study are available from the corresponding author on reasonable request.

Mechanism of Radical Transfer During Photoactivation of the flavoprotein DNA Photolyase

Springer series in chemical physics, 2007

ABSTRACT Ultrafast transient absorption experiments on mutant DNA photolyase imply that radical t... more

Photoselection Polarization Experiments Reveal Ultrafast Electron Hopping Between Distinct Aromatic Residues in the Flavoprotein DNA Photolyase

Springer eBooks, 2009

Flavin-excitation initiated electron transfer along three tryptophan amino acids in DNA photolyas... more

Structural Information about the <i>trans</i>-to-<i>cis</i> Isomerization Mechanism of the Photoswitchable Fluorescent Protein rsEGFP2 Revealed by Multiscale Infrared Transient Absorption

Journal of Physical Chemistry Letters, Jan 27, 2022

The rsEGFP2 is a reversible photoswitchable fluorescent protein (RSFP) used in super-resolved opt... more The rsEGFP2 is a reversible photoswitchable fluorescent protein (RSFP) used in super-resolved optical microscopies, which can be switched between a fluorescent On state and a non-fluorescent Off state. Previous studies have shown that the Off-to-On photoactivation extends over the femtosecond to the millisecond range and involves two picosecond-lifetime excited states and four ground state intermediates, reflecting a trans-to-cis excited state isomerization, a millisecond deprotonation and protein structural reorganizations. Femtosecond to millisecond time resolved multiple probe infrared spectroscopy (TRMPS-IR) is ideal to reveal the structure structural aspects of intermediate species in solution. Here we apply TRMPS-IR to rsEGFP2 and implement a along with the application of Savitzky-Golay derivative analysis, to correct for baseline drifts. The results reveal that a sub-picosecond twisted excited state precursor controls the trans-to-cis isomerization and a previously unreported step assigned to structural relaxation of the β-barrel occurs before the final millisecond deprotonation of the chromophore. TOC

Excited State Vibrations of Isotopically Labeled FMN Free and Bound to a Light–Oxygen–Voltage (LOV) Protein

Journal of Physical Chemistry B, Jul 27, 2020

Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetic... more Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetics. The characterization of their excited state structure and dynamics is thus an important objective. Here we present a detailed study of excited state vibrational spectra of flavin mononucleotide (FMN), in solution and bound to the LOV-2 (Light-Oxygen-Voltage) domain of Avena sativa phototropin. Vibrational frequencies are determined for the optically excited singlet state and the reactive triplet state, through resonant ultrafast femtosecond stimulated Raman spectroscopy (FSRS). To assign the observed spectra, vibrational frequencies of the excited states are calculated using density functional theory, and both measurement and theory are applied to four different isotopologues of FMN. Excited state mode assignments are refined in both states and their sensitivity to deuteration and protein environment are investigated. We show that resonant FSRS provides a useful tool for characterizing photoactive flavoproteins, and is able to highlight chromophore localized modes, and to record hydrogen/deuterium exchange. ‡JNI, CRH and DG contributed equally to this work through protein spectroscopy, FSRS development and calculations respectively.

Presentation of the ultrafast spectroscopy lab at the Department of Biophysics, Medical School, University of Pecs

Ultrafast transient absorption system

5 views

ATP-dependent conformational dynamics in a photoactivated adenylate cyclase revealed by fluorescence spectroscopy and small-angle X-ray scattering

Communications biology, Feb 2, 2024

Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of... more Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of controlling cellular cyclic adenosine monophosphate (cAMP) levels for optogenetic and other applications. In this work, we use an integrative approach that combines biophysical and structural biology methods to provide insight on the interaction of adenosine triphosphate (ATP) with the dark-adapted state of the photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata (OaPAC). A moderate affinity of the nucleotide for the enzyme was calculated and the thermodynamic parameters of the interaction have been obtained. Stopped-flow fluorescence spectroscopy and small-angle solution scattering have revealed significant conformational changes in the enzyme, presumably in the adenylate cyclase (AC) domain during the allosteric mechanism of ATP binding to OaPAC with small and large-scale movements observed to the best of our knowledge for the first time in the enzyme in solution upon ATP binding. These results are in line with previously reported drastic conformational changes taking place in several class III AC domains upon nucleotide binding.

Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix

Light-activated protein domains provide a convenient, modular, and genetically encodable sensor f... more Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

Functional dynamics of the photoactivated adenylate cyclase OaPAC revealed by fluorescence spectroscopy and small-angle X-ray scattering

Biophysical Journal, Feb 1, 2022

A possible way to relate the effects of SARS-CoV-2 induced changes in transferrin to severe COVID-19 associated diseases

bioRxiv (Cold Spring Harbor Laboratory), Feb 16, 2022

The potentially life-threatening Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) inf... more The potentially life-threatening Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is responsible for the coronavirus pandemic in 2019 (COVID-19). The transferrin as an essential component of iron-metabolism was suggested to be a link between iron transport associated diseases and COVID-19 infection. The effect of SARS-CoV-2 on human whole blood was studied by differential scanning calorimetry. The analysis and deconvolution of the thermal transition curves showed that the T m of transferrin related second peak decreased by 5.16 °C (6.4%) in the presence of SARS-CoV-2 virus. The ratio of the under-curve area of the two main peaks was greatly affected while the total enthalpy of the heat denaturation was nearly unchanged in the presence of the virus. Based on the results it is possible to conclude that SARS-CoV-2 through binding to transferrin can influence it's Fe 3+ uptake by inducing thermodynamic changes. Transferrin may stay in iron-free apo-conformational state, which probably depends on the SARS-CoV-2 concentration. SARS-CoV-2 might induce disturbance in the erythropoiesis due to the free iron overload generated iron toxicity. As a late consequence iron toxicity related hepatocellular carcinoma can even develop. Our work can support the basic role of transferrin in COVID-19 related severe diseases.

Mechanical Characterization and Structural Analysis of Latex-Containing and Latex-Free Intermaxillary Orthodontic Elastics

Polymers, Oct 23, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more

Functional dynamics of proteins revealed by fluorescence and femtosecond transient absorption spectroscopy

Solubility and Thermal Stability of Thermotoga maritima MreB

International Journal of Molecular Sciences, Dec 16, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more

Radical Formation in the Photoactivated Adenylate Cyclase OaPAC Revealed by Ultrafast Spectroscopy

Biophysical Journal, Feb 1, 2020

modes, and find that organizational changes can optimize for different objectives. Cargos with cl... more modes, and find that organizational changes can optimize for different objectives. Cargos with clustered motors are transported efficiently, but are slow to bind to microtubules. Cargos with motors dispersed rigidly on their surface bind microtubules quickly, but are transported inefficiently. Cargos with freelydiffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results point to a functional role for observed changes in motor organization on cargos, and suggest motor diffusivity as a control point for transport, either by modulation of adaptor proteins or changes in lipid composition.

Ultrafast Protein Dynamics Probed by Site Specific Transient IR Spectroscopy

The 22nd International Conference on Ultrafast Phenomena 2020, 2020

Ultrafast spectroscopy can measure real-time protein dynamics, but yields limited structural info... more Ultrafast spectroscopy can measure real-time protein dynamics, but yields limited structural information. Here we use unnatural amino acid substitution to place an IR absorber at specific locations in light activated proteins. Ultrafast pump - transient IR probe spectroscopy then yields unique site-specific data on protein structural dynamics.

The effect of the flexibility of hydrogen bonding network on low-frequency motions of amino acids. Evidence from Terahertz spectroscopy and DFT calculations

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, Feb 1, 2018

Low-frequency modes of l-Asp and l-Asn were studied in the range from 0.1 to 3.0 THz using time-d... more Low-frequency modes of l-Asp and l-Asn were studied in the range from 0.1 to 3.0 THz using time-domain Terahertz spectroscopy and density functional theory calculation. The results show that PBE-D2 shows more success than BLYP-D2 in prediction of THz absorption spectra. To compare their low-frequency modes, we adopted "vibrational character ID strips" proposed by Schmuttenmaer and coworkers [Journal of Physical Chemistry B, 117, 10444(2013)]. We found that the most intense THz absorption peaks of two compounds both involve severe distortion of their hydrogen bonding networks. Due to less rigid hydrogen bonding network in l-Asp, the side chain (carboxyl group) of l-Asp exhibits larger motions than that (carboxamide group) of l-Asn in low-frequency modes.

Photophysics of the Blue Light Using Flavin Domain

Accounts of Chemical Research, Jan 12, 2022

Light activated proteins are at the heart of photobiology and optogenetics, so there is wide inte... more Light activated proteins are at the heart of photobiology and optogenetics, so there is wide interest in understanding the mechanisms coupling optical excitation to protein function. In addition, such light activated proteins provide unique insights into the real-time dynamics of protein function. Using pump-probe spectroscopy, the function of a photoactive protein can be initiated by a sub-100 fs pulse of light, allowing subsequent protein dynamics to be probed from femtoseconds to milliseconds and beyond. Among the most interesting photoactive proteins are the Blue Light Using Flavin (BLUF) domain proteins, which regulate the response to light of a wide range of bacterial and some euglenoid processes. The photosensing mechanism of BLUF domains has long been a subject of debate. In contrast to other photoactive proteins, the electronic and nuclear structure of the chromophore (flavin) is the same in dark-and light-adapted states. Thus, the driving force for photoactivity is unclear. To address this question requires real-time observation of both chromophore excited state processes and their effect on the structure and dynamics of the surrounding protein matrix. In this Account we describe how time resolved infra-red (IR) experiments, coupled with chemical biology, provides important new insights into the signalling mechanism of BLUF domains. IR measurements are sensitive to changes in both chromophore electronic structure and protein hydrogen bonding interactions. These contributions are resolved by isotope labelling chromophore and protein Key References

Conformational Distributions and Proximity Relationships in the Rigor Complex of Actin and Myosin Subfragment-1

Journal of Biological Chemistry, 2000

Cyclic conformational changes in the myosin head are considered essential for muscle contraction.... more

Flexibility of myosin-subfragment-1 in its complex with actin as revealed by fluorescence resonance energy transfer

European journal of biochemistry, Jul 1, 2000

The flexibility of the acto±myosin complex in rigor conditions was characterized by measuring the... more

Short‐Lived Radical Intermediates in the Photochemistry of Glucose Oxidase

ChemPhysChem, Jun 21, 2019

Supporting information for this article is given via a link at the end of the document.

Infrared spectroscopy reveals multi-step multi-timescale photoactivation in the photoconvertible protein archetype dronpa

Nature Chemistry, Jun 11, 2018

Author Contributions SPL, AAG, CRH, AL and JNI measured and collected the data. SPL and CRH analy... more Author Contributions SPL, AAG, CRH, AL and JNI measured and collected the data. SPL and CRH analysed the data. AAG and JNI grew and purified the samples. GAJ performed the DFT calculations. GMG and PD built, developed and managed the ULTRA and LifeTime apparatus used in the measurements. AM designed the dronpa2 protein. PJT and SRM designed the experiment and wrote the paper, with discussion and editorial input from all authors Competing Interest Statement The authors declare no competing financial and non-financial interest. Data Availability Statement The data sets generated during the current study are available from the corresponding author on reasonable request.

Mechanism of Radical Transfer During Photoactivation of the flavoprotein DNA Photolyase

Springer series in chemical physics, 2007

ABSTRACT Ultrafast transient absorption experiments on mutant DNA photolyase imply that radical t... more

Photoselection Polarization Experiments Reveal Ultrafast Electron Hopping Between Distinct Aromatic Residues in the Flavoprotein DNA Photolyase

Springer eBooks, 2009

Flavin-excitation initiated electron transfer along three tryptophan amino acids in DNA photolyas... more

Structural Information about the <i>trans</i>-to-<i>cis</i> Isomerization Mechanism of the Photoswitchable Fluorescent Protein rsEGFP2 Revealed by Multiscale Infrared Transient Absorption

Journal of Physical Chemistry Letters, Jan 27, 2022

The rsEGFP2 is a reversible photoswitchable fluorescent protein (RSFP) used in super-resolved opt... more The rsEGFP2 is a reversible photoswitchable fluorescent protein (RSFP) used in super-resolved optical microscopies, which can be switched between a fluorescent On state and a non-fluorescent Off state. Previous studies have shown that the Off-to-On photoactivation extends over the femtosecond to the millisecond range and involves two picosecond-lifetime excited states and four ground state intermediates, reflecting a trans-to-cis excited state isomerization, a millisecond deprotonation and protein structural reorganizations. Femtosecond to millisecond time resolved multiple probe infrared spectroscopy (TRMPS-IR) is ideal to reveal the structure structural aspects of intermediate species in solution. Here we apply TRMPS-IR to rsEGFP2 and implement a along with the application of Savitzky-Golay derivative analysis, to correct for baseline drifts. The results reveal that a sub-picosecond twisted excited state precursor controls the trans-to-cis isomerization and a previously unreported step assigned to structural relaxation of the β-barrel occurs before the final millisecond deprotonation of the chromophore. TOC

Excited State Vibrations of Isotopically Labeled FMN Free and Bound to a Light–Oxygen–Voltage (LOV) Protein

Journal of Physical Chemistry B, Jul 27, 2020

Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetic... more Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetics. The characterization of their excited state structure and dynamics is thus an important objective. Here we present a detailed study of excited state vibrational spectra of flavin mononucleotide (FMN), in solution and bound to the LOV-2 (Light-Oxygen-Voltage) domain of Avena sativa phototropin. Vibrational frequencies are determined for the optically excited singlet state and the reactive triplet state, through resonant ultrafast femtosecond stimulated Raman spectroscopy (FSRS). To assign the observed spectra, vibrational frequencies of the excited states are calculated using density functional theory, and both measurement and theory are applied to four different isotopologues of FMN. Excited state mode assignments are refined in both states and their sensitivity to deuteration and protein environment are investigated. We show that resonant FSRS provides a useful tool for characterizing photoactive flavoproteins, and is able to highlight chromophore localized modes, and to record hydrogen/deuterium exchange. ‡JNI, CRH and DG contributed equally to this work through protein spectroscopy, FSRS development and calculations respectively.

Site-Specific Protein Dynamics Probed by Ultrafast Infrared Spectroscopy of a Noncanonical Amino Acid

Journal of Physical Chemistry B, Oct 9, 2019

Real-time observation of structure change associated with protein function remains a major challe... more Real-time observation of structure change associated with protein function remains a major challenge. Ultrafast pump-probe methods record dynamics in light activated proteins, but the assignment of spectroscopic observables to specific structure changes can be difficult. The BLUF (blue light using flavin) domain proteins are an important class of light sensing flavoprotein. Here we incorporate the unnatural amino acid (UAA) azidophenylalanine (AzPhe) at key positions in the H-bonding environment of the isoalloxazine chromophore of two BLUF domains, PixD and AppABLUF; both proteins retain the red shift on irradiation characteristic of photoactivity. Steady state and ultrafast time resolved infrared (TRIR) difference measurements of the azido mode reveal site-specific information on the nature and dynamics of light driven structure change. AzPhe dynamics are thus shown to be an effective probe of BLUF domain photoactivation, revealing significant differences between the two proteins, and a differential response of the two sites to chromophore excitation.