Functionally relevant electric-field induced perturbations of the prosthetic group of yeast ferrocytochrome c mutants obtained from a vibronic analysis of low-temperature absorption spectra.

We have measured the low temperature (T = 20 K) absorption spectra of the N52A, N52V, N52I, Y67F, and N52AY67F mutants of ferrous Saccharomyces cerevisiae (baker's yeast) cytochrome c. All the bands in the Q0- and Q(v)-band region are split, and the intensity distributions among the split bands are highly asymmetric. The spectra were analyzed by a decomposition into Voigtian profiles. The spectral parameters thus obtained were further analyzed in terms of the vibronic coupling model of Schweitzer-Stenner and Bigman (Schweitzer-Stenner, R.; Bigman, D. J. Phys. Chem. B 2001, 7064-7073) to identify parameters related to electronic and vibronic perturbations of the heme macrocycle. We report th…

The importance of vibronic perturbations in ferrocytochrome c spectra: a reevaluation of spectral properties based on low-temperature optical absorption, resonance Raman, and molecular-dynamics simulations.

We have measured and analyzed the low-temperature (T=10 K) absorption spectrum of reduced horse heart and yeast cytochrome c. Both spectra show split and asymmetric Q(0) and Q(upsilon) bands. The spectra were first decomposed into the individual split vibronic sidebands assignable to B(1g) (nu15) and A(2g) (nu19, nu21, and nu22) Herzberg-Teller active modes due to their strong intensity in resonance Raman spectra acquired with Q(0) and Q(upsilon) excitations. The measured band splittings and asymmetries cannot be rationalized solely in terms of electronic perturbations of the heme macrocycle. On the contrary, they clearly point to the importance of considering not only electronic perturbati…

The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin

International audience; The acknowledged success of the Monod-Wyman-Changeux (MWC) allosteric model stems from its efficacy in accounting for the functional behavior of many complex proteins starting with hemoglobin (the paradigmatic case) and extending to channels and receptors. The kinetic aspects of the allosteric model, however, have been often neglected, with the exception of hemoglobin and a few other proteins where conformational relaxations can be triggered by a short and intense laser pulse, and monitored by time-resolved optical spectroscopy. Only recently the application of time-resolved wide-angle X-ray scattering (TR-WAXS), a direct structurally sensitive technique, unveiled th…

Hysteresis in the temperature dependence of the IR bending vibration of deeply cooled confined water.

Using Fourier Transform Infrared (FTIR) spectroscopy, we investigate the temperature dependence of the bending vibrations of water confined in the pores of a silica hydrogel in the temperature interval of 270-180 K. We also investigate the presence of thermal hysteresis by cooling and reheating temperature scans. The results clearly show the presence, at about 230 K, of a crossover in the temperature dependence of the IR spectra; moreover, the presence of hysteresis is clearly demonstrated. By comparing FTIR data with neutron diffraction data and previous calorimetric data on the same samples, we conclude that the crossover and the hysteretical behavior do not involve a water glass transiti…

The Tempered Polymerization of Human Neuroserpin

Neuroserpin, a member of the serpin protein superfamily, is an inhibitor of proteolytic activity that is involved in pathologies such as ischemia, Alzheimer's disease, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). The latter belongs to a class of conformational diseases, known as serpinopathies, which are related to the aberrant polymerization of serpin mutants. Neuroserpin is known to polymerize, even in its wild type form, under thermal stress. Here, we study the mechanism of neuroserpin polymerization over a wide range of temperatures by different techniques. Our experiments show how the onset of polymerization is dependent on the formation of an intermediate mon…

Dynamics of ligand binding to hemoglobin: quaternary structure dependence

Hemoglobin loaded polymeric nanoparticles: preparation and characterizations.

In the present work polymeric nanoparticles based on Poly (maleic anhydride-alt-butyl vinyl ether) 5% grafted with m-PEG (2000) and 95% grafted with 2-methoxyethanol (VAM41-PEG) were loaded with human hemoglobin (Hb) and characterized from a physicochemical point of view. The assessment of structural and functional features of the loaded Hb was performed and the effect of the introduction of different reducing agents as aimed at minimizing Hb oxidation during the nanoparticles formulation process, was also investigated. Nanoparticles possessing an average diameter of 138 ± 10 nm and physicochemical features suitable for this kind of application were successfully obtained. Although the oxida…

Dynamics of water confined in silica hydrogels studied with elastic and quasi-elastic neutron scattering

Conformational relaxations in hemoglobin studied by time-resolved absorption spectroscopy

Tracking protein motions in solution

Using synchrotrons and XFELs for time-resolved X-ray crystallography and solution scattering experiments on biomolecules

International audience; Time-resolved structural information is key to understand the mechanism of biological processes, such as catalysis and signalling. Recent developments in X-ray sources as well as data collection and analysis methods are making routine time-resolved X-ray crystallography and solution scattering experiments a real possibility for structural biologists. Here we review the information that can be obtained from these techniques and discuss the considerations that must be taken into account when designing a time-resolved experiment.

Different relaxations in myoglobin after photolysis

To clarify the interplay of kinetic hole-burning (KHB), structural relaxation, and ligand migration in myoglobin (Mb), we measured time-resolved absorption spectra in the Soret region after photolysis of carbon monoxide Mb (MbCO) in the temperature interval 120-260 K and in the time window 350 ns to 200 ms. The spectral contributions of both photolyzed (Mb * ) and liganded Mb (MbCO) have been analyzed by taking into account homogeneous bandwidth, coupling to vibrational modes, and static conformational heterogeneity. We succeeded in separating the “time-dependent” spectral changes, and this work provides possibilities to identify the events in the process of ligand rebinding. KHB is domina…

Solvation of a probe molecule by fluid supercooled water in a hydrogel at 200 K

By combining electron paramagnetic resonance (EPR) measurements on a nitroxide probe and differential scanning calorimetry (DSC), we demonstrate existence of liquid supercooled water in a silica hydrogel with high hydration level down to temperatures of at least 198 K. Besides the major fraction of liquid supercooled water, a minor fraction crystallizes at about 236 K during cooling and melts at 246 K during heating. The liquid domains are of sufficient size to solvate the nearly spherical paramagnetic probe molecule TEMPO with a diameter of about 6 angstrom. Analysis of EPR spectra provides the rotational correlation time of the probe that is further used to compare the viscosity of the su…

Observing myoglobin proteinquake with an X-ray free-electron laser

The events following the photodissociation of the bond be- tween myoglobin and its ligand have been extensively studied with a variety of experimental, theoretical and computational methods [1]. The results of these investigations have been rationalized in terms of a model that implies a protein quake- like motion [2], i.e. the propagation of the strain released upon photoexcitation through the protein similar to the prop- agation of acoustic waves during an earthquake. The exper- imental investigations performed so far have been based on spectroscopic measurements or did not have sufficient time- resolution to measure the timescale of such “proteinquake”. We have obtained direct experiment…

Unveiling the timescale of the R-T transition in human hemoglobin.

Time-resolved wide-angle X-ray scattering, a recently developed technique allowing to probe global structural changes of proteins in solution, was used to investigate the kinetics of R-T quaternary transition in human hemoglobin and to systematically compare it to that obtained with time-resolved optical spectroscopy under nearly identical experimental conditions. Our data reveal that the main structural rearrangement associated with the R-T transition takes place approximately 2 mus after the photolysis of hemoglobin at room temperature and neutral pH. This finding suggests that the 20-mus step observed with time-resolved optical spectroscopy corresponds to a small and localized structural…

On the molecular structure of human neuroserpin polymers. Coagulation, fragmentation and latentization control serpin aggregation

The polymerization of serpins is at the root of a large class of diseases; the molecular structure of serpin polymers has been recently debated. In this work, we study the polymerization kinetics of human neuroserpin by Fourier Transform Infra Red spectroscopy and by time-lapse Size Exclusion Chromatography. First, we show that two distinct neuroserpin polymers, formed at 45 and 85 °C, display the same isosbestic points in the Amide I band, and therefore share common secondary structure features. We also find a concentration independent polymerization rate at 45 °C suggesting that the polymerization rate limiting step is the formation of an activated monomeric species. The polymer structure…

Time-resolved X-ray scattering as a tool to probe heme proteins structural dynamics in solution

Time-resolved wide-angle X-ray scattering (TR-WAXS) is a recently developed technique allowing to probe global structural changes of proteins in solution. We have used TR-WAXS to investigate large conformational changes of heme proteins (wild-type and mutant hemoglobin, neuroglobin, etc.) that cannot take place when these macromolecules are in a crystalline environment. Our data revealed detailed information on kinetic and thermodynamic properties of the investigated proteins and demonstrate the potentiality of the TR-WAXS technique.

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering (vol 5, pg 881, 2008)

Immobilization of proteins in silica gel: Biochemical and biophysical properties

The development of silica-based sol-gel techniques compatible with the retention of protein structure and function started more than 20 years ago, mainly for the design of biotechnological devices or biomedical applications. Silica gels are optically transparent, exhibit good mechanical stability, are manufactured with different geometries, and are easily separated from the reaction media. Biomolecules encapsulated in silica gel normally retain their structural and functional properties, are stabilized with respect to chemical and physical insults, and can sometimes exhibit enhanced activity in comparison to the soluble form. This review briefly describes the chemistry of protein encapsulat…

Ferricytochrome c encapsulated in silica hydrogels: correlation between active site dynamics and solvent structure.

Ferricytochrome c encapsulated in silica hydrogels has been prepared by the sol-gel technique following, with some modifications, the procedure originally developed by Ellerby et al. (Science 255 1113 (1992)). A suitable preparation of hydrogels enables having both 'wet' and 'dry' samples. Wet samples have a high water content: as the temperature is lowered below approximately 260 K, water freezes and the samples crack. On the contrary, dry samples have a low water content (hydration h approximately equal 0.35): in these conditions water does not freeze even at cryogenic temperatures and the samples remain transparent and non-cracking. The dynamics of ferricytochrome c and its dependence on…

Quaternary structure specific dynamics and reactivity in human hemoglobin under cryogenic conditions

Functional and dysfunctional conformers of human neuroserpin characterized by optical spectroscopies and Molecular Dynamics

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native neuroserpin and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric species. Based on circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In particular, emission spectra of aromatic res…

Quaternary structure control of geminate ligand rebinding after photolysis of carbonmonoxy hemoglobin

Charge-transfer driven by ultrafast spin-transition in a CoFe Prussian blue analogue

Photoinduced charge-transfer is an important process in nature and technology and is responsible for the emergence of exotic functionalities, such as magnetic order for cyanide-bridged bimetallic coordination networks. Despite its broad interest and intensive developments in chemistry and material sciences, the atomic-scale description of the initial photoinduced process, which couples intermetallic charge-transfer and spin transition, has been debated for decades; it has been beyond reach due to its extreme speed. Here we study this process in a prototype cyanide-bridged CoFe system by femtosecond X-ray and optical absorption spectroscopies, enabling the disentanglement of ultrafast electr…

Inactivation and polymerization of human neuroserpin

Neuroserpin is an inhibitory enzyme, belonging to the family of serpins and involved in several pathologies, such as ischemia, Alzheimer disease, and FENIB (Familial Encephalopathy with Neuroserpin Inclusion Body). Here, we study the mechanism of neuroserpin inactivation and polymerization by different experimental techniques (static and dynamic light scattering, liquid chromatography, Fourier transform infrared spectroscopy, emission spectroscopy). Our results show that at intermediate temperatures (45-55 °C) neuroserpin forms flexible polymers with a size from a few tens to a few hundreds of nanometers. At high temperatures, above 80 °C, our results reveal a different polymeric form, reac…

Two Latent and Two Hyperstable Polymeric Forms of Human Neuroserpin

AbstractHuman neuroserpin (hNS) is a serine protease inhibitor that belongs to the serpin superfamily and is expressed in nervous tissues. The serpin fold is generally characterized by a long exposed loop, termed the reactive center loop, that acts as bait for the target protease. Intramolecular insertion of the reactive center loop into the main serpin β-sheet leads to the serpin latent form. As with other known serpins, hNS pathological mutants have been shown to accumulate as polymers composed of quasi-native protein molecules. Although hNS polymerization has been intensely studied, a general agreement about serpin polymer organization is still lacking. Here we report a biophysical chara…

Near-Infrared Spectra of Water Confined in Silica Hydrogels in the Temperature Interval 365−5 K

We have used a sol−gel technique to obtain optically transparent hydrogels in which water is trapped within a tridimensional disordered silica matrix. A suitable aging of these hydrogels enables to have transparent noncracking samples down to cryogenic temperatures. We report the optical absorption spectra, in the near-infrared region, of water trapped in our silica hydrogels, measured in the temperature range 365−5 K, and we compare them with the same spectra of liquid water, measured in the temperature range 365−263 K. The data show that it is possible to have noncrystallizing water even at 5 K:  indeed, the overtone bands at ∼1.41 μm and at ∼1.155 μmtypical of “weakly bonded” water molec…

Conformational relaxations and kinetic hole burning in hemoglobin

Time-resolved small-angle scattering study of the R to T transition in hemoglobin

The R-T quaternary transition in deoxyhemoglobin studied by NIR spectral relaxation

Hemoglobin dynamics probed by picosecond wide-angle x-ray scattering

Structure and dynamics of water confined in silica hydrogels: X-ray scattering and dielectric spectroscopy studies.

We have used a sol-gel technique to obtain optically transparent hydrogels in which water is confined within a 3D silica matrix. In this work we report X-ray scattering and dielectric spectroscopy measurements on samples having different aging times and compare them with previously obtained results with near-infrared (NIR) absorption spectroscopy. X-ray scattering at room temperature enables to characterize the structure and size of the matrix pores and the non-uniform distribution of water inside the hydrogel. Broad band dielectric spectroscopy in the temperature range 130-280 K enables to study water dynamics. In aged hydrogels two relaxations are clearly evident and show characteristic t…

Femtosecond Structural Dynamics of Proteins

Proteins are the workhorses of living cells, providing essential functions such as structural support, signal transduction, enzymatic catalysis, transport and storage of small ligands. Atomic-resolution structures obtained with conventional X-ray crystallography show proteins essentially as static. In reality, however, proteins move and their motion is crucial for functioning. Although the structure and dynamics of proteins are intimately related, they are not equally well understood. A very large number of protein structures have been determined, but only a few studies have been able to monitor experimentally the dynamics of proteins in real time. In the last two decades, the availability …

Probing protein structural dynamics in a human cell

Spectroscopic markers of the T-R quaternary transition in human hemoglobin

n questo lavoro, usiamo un protocollo sol-gel per intrappolare e confrontare gli stati quaternari R e T di entrambi i deossigenati (deossiHb) ederivati ​​di ossido di carbonio (HbCO) dell'emoglobina umana. La banda di assorbimento ottico del vicino infrarosso III e lo stretching di CO a infrarossibanda sono utilizzati per rilevare l'effetto della struttura quaternaria sulle proprietà spettrali di deoxyHb e HbCO; confronto con mioglobinaconsente una valutazione dei contributi terziari e quaternari ai turni di banda misurati. La RXLa transizione T è indicata per causare un bluspostamento della banda III di ~ 35 cm?1per deoxyHb e uno spostamento rosso della banda di allungamento CO di soli ~ 0…

Probing in cell protein structural changes with time-resolved X-ray scattering

International audience; Investigating protein structural changes inside the cell is a major goal in molecular biology. Here we show that time-resolved wide-angle X-ray scattering is a valuable tool for this purpose. Hemoglobin has been chosen as a model system and its tertiary and quaternary conformational changes following laser flash-photolysis have been tracked in intact red blood cells with nanosecond time resolution.

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.

The necessary chances of a thermodynamically metastable protein: inactivation and polymeritzation of human neuroserpin

Serpins are a wide class of proteins with high structural similarity, characterized by a unique substrate-like inhibitory mechanism that resembles a "molecular mousetrap". The active serpin is characterized by a main 5-stranded β-sheet and an exposed Reactive Centre Loop, which acts as a bait for the target protease. The cleavage of the loop by the protease triggers the insertion of the loop into the β-sheet as a strand and the disruptive translocation of the protease. This peculiar conformational mobility is achievable since serpins fold into a metastable native conformation. This feature gives a selective advantage to the serpin family to develop inhibitory activities, but leaves these pr…

Temperature effects on the performances of the ATHENA X-IFU thermal filters

The X-Ray Integral Field Unit (X-IFU) detector on-board ATHENA is an array of TES micro-calorimeters that will operate at ~50 mK. In the current investigated design, five thermal filters (TF) will be mounted on the cryostat shields to attenuate IR radiative load and avoid energy resolution degradation due to photon shot noise. Each filter consists of a thin polyimide film (~50 nm thick) coated with aluminum (~30 nm thick). Since the TF operate at different temperatures in the range 0.05-300 K, it is relevant to study how temperature affects their mechanical/optical performances (e.g. near edge absorption fine structures of the atomic elements in the filter material). Such results are crucia…

Quaternary structure dependence of kinetic hole burning and conformational substates interconversion in hemoglobin.

Using a sol-gel encapsulation technique, we have prepared samples of CO saturated human adult hemoglobin locked in the R or T quaternary conformation. We report time-resolved spectra of these samples in the Soret region following flash photolysis, in the time interval ranging from 250 ns to 200 ms and in the temperature interval of 100-170 K. A suitable analysis of the measured difference spectra enables us to obtain the spectral contribution of deoxyHb and HbCO molecules as a function of time and/or of the fraction N(t) of deoxyHb molecules. In our experimental time window geminate CO rebinding to hemoglobin in the T quaternary conformation is about 2 orders of magnitude slower than to hem…

Water and proteins confined in silica hydrogels and silica nanoparticles: structural, dynamic and functional studies.

Functional and dysfunctional isoforms of human neuroserpin

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native NS and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric conformer. Using circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In par- ticular, emission spectra of aromatic residues yi…

On the molecular structure of human neuroserpin polymers

The polymerization of serpins is at the root of a large class of diseases; the molecular structure of serpin polymers has been recently debated. In this work, we study the polymerization kinetics of human neuroserpin by Fourier Transform Infra Red spectroscopy and by time-lapse Size Exclusion Chromatography. First, we show that two distinct neuroserpin polymers, formed at 45 and 85°C, display the same isosbestic points in the Amide I' band, and therefore share common secondary structure features. We also find a concentration independent polymerization rate at 45°C suggesting that the polymerization rate-limiting step is the formation of an activated monomeric species. The polymer structures…

Ultrafast myoglobin structural dynamics observed with an X-ray free-electron laser.

Light absorption can trigger biologically relevant protein conformational changes. The light-induced structural rearrangement at the level of a photoexcited chromophore is known to occur in the femtosecond timescale and is expected to propagate through the protein as a quake-like intramolecular motion. Here we report direct experimental evidence of such ‘proteinquake’ observed in myoglobin through femtosecond X-ray solution scattering measurements performed at the Linac Coherent Light Source X-ray free-electron laser. An ultrafast increase of myoglobin radius of gyration occurs within 1 picosecond and is followed by a delayed protein expansion. As the system approaches equilibrium it underg…

Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2] E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release o…

Quaternary structure dependence of hemoglobin ligand rebinding kinetics under cryogenic conditions

Observing heme doming in myoglobin with femtosecond X-ray absorption spectroscopy.

International audience; We report time-resolved X-ray absorption measurements after photolysis of carbonmonoxy myoglobin performed at the LCLS X-ray free electron laser with nearly 100 fs (FWHM) time resolution. Data at the Fe K-edge reveal that the photoinduced structural changes at the heme occur in two steps, with a faster (∼70 fs) relaxation preceding a slower (∼400 fs) one. We tentatively attribute the first relaxation to a structural rearrangement induced by photolysis involving essentially only the heme chromophore and the second relaxation to a residual Fe motion out of the heme plane that is coupled to the displacement of myoglobin F-helix

Conformational substates of ferricytochrome c revealed by combined optical absorption and electronic circular dichroism spectroscopy at cryogenic temperature.

We have investigated the heterogeneity of the Fe(III)–Met80 linkage of horse heart ferricytochrome c by probing the 695 nm charge transfer band with absorption and electronic circular dichroism (ECD) spectroscopy. In order to verify the connection between conformational substates of the Fe(III)–Met80 linkage and the 695 nm band spectral heterogeneity, we have performed experiments as a function of pH (neutral and acidic) and temperature (room and 20 K). At room temperature, the ECD spectrum is blue shifted with respect to the absorption one; the shift is more pronounced at acidic pH and is compatible with the presence of sub-bands. ECD measurements at 20 K highlighted the heterogeneous natu…

Heme deformations in mutant N52V yeast ferrocytochrome c detected by Raman and optical spectroscopy

Tracking protein intra-molecular motions in solution with time-resolved Wide Angle X-ray Scattering

Photocage-initiated time-resolved solution X-ray scattering investigation of protein dimerization

Photocaging in combination with X-ray solution scattering allows for the time-resolved study of protein dynamics in solution. This method is versatile and allows for accurate triggering of protein function.