High pressure NMR studies of hemoproteins. The effect of pressure on the quaternary structure of hemoglobin

Proton NMR spectra for nitrosyl-, aquomet- and deoxy des-Arg(α141)-hemoglobin in H₂O were studied at high pressures up to 1400 atm with attention to the exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds. For aquomethemoglboin, the T state marker signal at 6.4 ppm is insensitive to pressure while the R state marker signal at 6.0 ppm exhibits progressive upfield shift upon pressurization. For nitrosylhemoglobin, the T state signals at 9.6 and 6.5 ppm decrease their intensities upon pressurization while the R state marker signal at 6.0ppm remains unchanged. Pressure-induced spectral changes for some of exchangeable resonances are also encountered for deoxy des-Arg(α141)-hemoglobin while the R and T quaternary structural indicators at 6.0 and 9.4 ppm are insensitive to pressure. These pressure-induced spectral changes for these hemoglobin derivatives are significantly distinguished from those associated with the R-T transition induced by addition of IHP or by variatiuon of pH. It is therefore concluded that pressure induces subtle quaternary structural changes in these hemoglobin derivatives without causing the R-T transition.     

Allosteric transition of aspartokinase I-homoserine dehydrogenase I studied by time-resolved fluorescence

The allosteric transition of threonine-sensitive aspartokinase I-homoserine dehydrogenase I from Escherichia coli has been studied by time-resolved fluorescence spectroscopy. Fluorescence decay can be resolved into 2 distinct classes of tryptophan emitters: a fast component, with a lifetime of about 1.5 ns; and a slow component, with a lifetime of about 4.5 ns. The fluorescence properties of the slow component are modified by the allosteric transition. In the T-form of the enzyme stabilized by threonine, the lifetime of the slow component is longer, with a red-shifted spectrum; its accessibility to quenching by acrylamide becomes slightly higher without any decrease of fluorescence anisotropy. These results indicate a change in polarity of the slow component environment. The quaternary structure change associated with the allosteric transition probably involves global movements of structural domains without leading to any local mobility on the nanosecond time-scale. We suggest that the slow component corresponds to the unique tryptophan of the buried kinase domain.     

Tyr39 of Ran Preserves the Ran·GTP Gradient by Inhibiting GTP Hydrolysis

Ran is a member of the superfamily of small GTPases, which cycle between a GTP-bound “on” and a GDP-bound “off” state. Ran regulates nuclear transport. In order to maintain a gradient of excess Ran·GTP within the nucleoplasm and excess Ran·GDP within the cytoplasm, the hydrolysis of Ran·GTP in the nucleoplasm should be prevented, whereas in the cytoplasm, hydrolysis is catalyzed by Ran·GAP (GTPase-activating protein). In this article, we investigate the GTPase reaction of Ran in complex with its binding protein Ran-binding protein 1 by time-resolved Fourier transform infrared spectroscopy: We show that the slowdown of the intrinsic hydrolysis of RanGTP is accomplished by tyrosine 39, which is probably misplacing the attacking water. We monitored the interaction of Ran with RanGAP, which reveals two reactions steps. By isotopic labeling of Ran and RanGAP, we were able to assign the first step to a small conformational change within the catalytic site. The following bond breakage is the rate-limiting step of hydrolysis. An intermediate of protein-bound phosphate as found for Ras or Rap systems is kinetically unresolved. This demonstrates that despite the structural similarity among the G-domain of the GTPases, different reaction mechanisms are utilized.     

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.     

Oligomerization processes limit photoactivation and recovery of the orange carotenoid protein

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection by quenching of the excess of light-harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy-quenching activity. Here, we probed photoinduced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation a numbed state forms, which remains in a non-photoexcitable structural state for at least ≈0.5 μs after absorption of a first photon.     

Tri-mode optical biopsy probe with fluorescence endomicroscopy,Raman spectroscopy,and time-resolved fluorescence spectroscopy

We present an endoscopic probe that combines three distinct optical fibre technologies including: A high-resolution imaging fibre for optical endomicroscopy, a multimode fibre for time-resolved fluorescence spectroscopy, and a hollow-core fibre with multimode signal collection cores for Raman spectroscopy. The three fibers are all enclosed within a 1.2 mm diameter clinical grade catheter with a 1.4 mm end cap. To demonstrate the probe's flexibility we provide data acquired with it in loops of radii down to 2 cm. We then use the probe in an anatomically accurate model of adult human airways, showing that it can be navigated to any part of the distal lung using a commercial bronchoscope. Finally, we present data acquired from fresh ex vivo human lung tissue. Our experiments show that this minimally invasive probe can deliver real-time optical biopsies from within the distal lung - simultaneously acquiring co-located high-resolution endomicroscopy and biochemical spectra.     

Time-resolved infrared spectroscopy of pH-induced aggregation of the Alzheimer Abeta(1-28) peptide

Aggregation of the Alzheimer's disease-related Aβ_1-28 peptide was induced by a rapid, sub-millisecond pH jump and monitored by time-resolved infrared spectroscopy on the millisecond to second time-scale. The release of protons was induced by the photolysis of a caged compound, 1-(2-nitrophenyl)ethyl sulfate (NPE-sulfate). The pH jump generated in our experimental setup is used to model the Aβ peptide structural conversions that may occur in the acidic endosomal/lysosomal cell compartment system. The aggregation of the Aβ_1-28 peptide induced by the pH jump from 8.5 to < 6 yields an antiparallel β-sheet structure. The kinetics of the structural transition is biphasic, showing an initial rapid phase with a transition from random coil to an oligomeric β-sheet form with a time constant of 3.6 s. This phase is followed by a second slower transition, which yields larger aggregates during 48.0 s.     

Binding of a spin-labeled phenylalanine analog to sickle hemoglobin: EPR and NMR studies

We have synthesized a spin-label analog of phenylalanine as a competitive inhibitor probe of the sickle hemoglobin aggregation process. Sickle hemoglobin gelation measurements indicate that the spin-label phenylalanine analog is a potent inhibitor of deoxy sickle hemoglobin aggregation. We have also used spin label EPR and high-resolution proton NMR to study the interaction of the phenylalanine analog with hemoglobin, and find that the kinetic off-rate is comparable to, or slower than the hemoglobin rotational rate (i.e., greater than or equal to 10(8) s-1), and that at least one, and perhaps two significant localized interaction region(s) exist within a few angstroms of the beta chain N- and C-termini. Correlation with other known structural information suggests that the observed interaction sites may be relevant to the mechanism for inhibition of sickle hemoglobin aggregation.     

Time evolution of the quaternary structure of Escherichia coli aspartate transcarbamoylase upon reaction with the natural substrates and a slow, tight-binding inhibitor

Here, we present a study of the conformational changes of the quaternary structure of Escherichia coli aspartate transcarbamoylase, as monitored by time-resolved small-angle X-ray scattering, upon combining with substrates, substrate analogs, and nucleotide effectors at temperatures between 5 and 22 °C, obviating the need for ethylene glycol. Time-resolved small-angle X-ray scattering time courses tracking the T → R structural change after mixing with substrates or substrate analogs appeared to be a single phase under some conditions and biphasic under other conditions, which we ascribe to multiple ligation states producing a time course composed of multiple rates. Increasing the concentration of substrates up to a certain point increased the T → R transition rate, with no further increase in rate beyond that point. Most strikingly, after addition of N-phosphonacetyl-l-aspartate to the enzyme, the transition rate was more than 1 order of magnitude slower than with the natural substrates. These results on the homotropic mechanism are consistent with a concerted transition between structural and functional states of either low affinity, low activity or high affinity, high activity for aspartate. Addition of ATP along with the substrates increased the rate of the transition from the T to the R state and also decreased the duration of the R-state steady-state phase. Addition of CTP or the combination of CTP/UTP to the substrates significantly decreased the rate of the T → R transition and caused a shift in the enzyme population towards the T state even at saturating substrate concentrations. These results on the heterotropic mechanism suggest a destabilization of the T state by ATP and a destabilization of the R state by CTP and CTP/UTP, consistent with the T and R state crystallographic structures of aspartate transcarbamoylase in the presence of the heterotropic effectors.     

Synchrotron-excited time-resolved fluorescence spectroscopy of adenosine,protonated adenosine and 6N, 6N-dimethyladenosine in aqueous solution at room temperature

The fluorescence behavior of adenosine in neutral solution has been studied by time-resolved spectroscopy using synchrotron excitation and timecorrelated single photon counting, and by decay time measurements. Three emissions have been identified and correlated with three excitation spectra. The assignment of these transitions has been made by comparison with similar measurements on ⁶N, ⁶N-dimethyladenosine (6 DMA), and on adenosine in acid solution (ADO H⁺). It is proposed that two of the transitions of adenosine which correlate with 6DMA originate from coplanar and orthogonal rotational conformers of the amino group. The other transition, correlating with ADO H⁺ may originate either from the ³H-imino tautomer, or from a differently solvated rotational conformer.A partial presentation of this work has been made at the Second Congress of the European Society for Photobiology Padova, Italy, 6–10 September 1987     

Delayed fluorescence observed in the nanosecond time region at 77 K originates directly from the photosystem II reaction center

The excited-state dynamics of delayed fluorescence in photosystem (PS) II at 77 K were studied by time-resolved fluorescence spectroscopy and decay analysis on three samples with different antenna sizes: PS II particles and the PS II reaction center from spinach, and the PS II core complexes from Synechocystis sp. PCC 6803. Delayed fluorescence in the nanosecond time region originated from the 683-nm component in all three samples, even though a slight variation in lifetimes was detected from 15 to 25 ns. The relative amplitude of the delayed fluorescence was higher when the antenna size was smaller. Energy transfer from the 683-nm pigment responsible for delayed fluorescence to antenna pigment(s) at a lower energy level was not observed in any of the samples examined. This indicated that the excited state generated by charge recombination was not shared with antenna pigments under the low-temperature condition, and that delayed fluorescence originates directly from the PS II reaction center, either from chlorophyll a_D1 or P680. Supplemental data on delayed fluorescence from spinach PS I complexes are included.     

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.     

Effect of hydrostatic pressure on ligand binding to hemoglobin.

Increase in hydrostatic pressure to 1000 atm increased the affinity of human and menhaden (Brevoortia tyrannus) hemoglobins for oxygen. With necessary assumptions about the form of the equilibrium curve, and after correction for changes in pH and volume due to pressure, the increase in affinity is about 2-fold for both hemoglobins. At pH 6.5, Hill's n for menhaden hemoglobin is near 1, and it is believed to remain in the T state, whereas human hemoglobin undergoes a T to R transition. This suggests that the R-T equilibrium is not disturbed by pressure. In direct experiments the binding of a fluorescent effector (8 hydroxy-1,3,6-pyrene (trisulfonic acid) to deoxyhemoglobin was not changed by pressure. The binding of n-butylisocyanide to hemoglobin and to myoglobin is also greater at high pressures, similarly suggesting that the R-T transition is not involved in the pressure effect.     

A third quaternary structure of human hemoglobin A at 1.7-A resolution.

Previous crystallographic studies have shown that human hemoglobin A can adopt two stable quaternary structures, one for deoxyhemoglobin (the T-state) and one for liganded hemoglobin (the R-state). In this paper we report our finding of a second quaternary structure (the R2-state) for liganded hemoglobin A. The magnitudes of the spatial differences between the R- and R2-states are as large as those between the R- and T-states. Of particular interest are the structural changes that occur as a result of R-T and R-R2 transitions at the so-called "switch" region of the critical alpha 1 beta 2 interface. In the R-state, His-97 beta 2 is positioned between Thr-38 alpha 1 and Thr-41 alpha 1, whereas in transition to the T-state His 97 beta 2 must "jump" a turn in the alpha 1 C helix to form nonpolar contacts with Thr-41 alpha 1 and Pro-44 alpha 1. This facet of the R-T transition presents a major steric barrier to the quaternary structure change. In the R2-state, His-97 beta 2 simply rotates away from threonines 38 alpha 1 and 41 alpha 1, breaking contact with these residues and allowing water access to the center of the alpha 1 beta 2 interface. With the switch region in an open position in the R2-state, His-97 beta 2 should be able to move by Thr-41 alpha 1 and make the transition to the T-state with a steric barrier that is less than that for the R-T transition. Thus the R2-state may function as a stable intermediate along a R-R2-T pathway. The T-, R-, and R2-states must coexist in solution. That is, the fact that these states can be crystallized implies that they are all energetically accessible structures. What remains to be determined are the T-to-R, T-to-R2, and R-to-R2 equilibrium constants for hemoglobin under various solution conditions and ligation states. Although this may prove to be difficult, we discuss previously published results which indicate that low concentrations of inorganic anions or low pH may favor the R2-state and at least one alpha 1 beta 2 interface mutation stabilizes a quaternary structure that is very similar to the R2-state.     

Changes of cerebral blood oxygenation and optical pathlength during activation and deactivation in the prefrontal cortex measured by time-resolved near infrared spectroscopy

To determine the alterations in optical characteristics and cerebral blood oxygenation (CBO) during activation and deactivation, we evaluated the changes in mean optical pathlength (MOP) and CBO induced by a verbal fluency task (VFT) and driving simulation in the right and left prefrontal cortex (PFC), employing a newly developed time-resolved near infrared spectroscopy, which allows quantitative measurements of the evoked-CBO changes by determining the MOP with a sampling time of 1 s. The results demonstrated differences in MOP in the foreheads with the subjects and wavelength; however, there was no significant difference between the right and left foreheads (p > 0.05). Also, both the VFT and driving simulation task did not affect the MOP significantly as compared to that before the tasks (p > 0.05). In the bilateral PFCs, the VFT caused increases of oxyhemoglobin and total hemoglobin associated with a decrease of deoxyhemoglobin, while the driving simulation task caused decreases of oxyhemoglobin and total hemoglobin associated with an increase of deoxyhemoglobin; there were no significant differences in evoked-CBO changes between the right and left PFC. The present results will be useful for quantitative measurement of hemodynamic changes during activation and deactivation in the adults by near infrared spectroscopy.     

Visualization of excitation energy transfer processes in plants and algae

Development of the time-resolved fluorescence spectroscopy in the pico-second time range and its application to the energy transfer processes in many photosynthetic organisms is reviewed here. This method enabled visualization of energy transfer processes by three-dimensional expression of fluorescence spectra and discrimination of kinetic components and spectral components. The second generation of the ultrafast fluorescence spectroscopy is the femto-second (fs) fluorescence up-conversion, and this has enabled analyses of the transfer processes from carotenoids to chlorophylls with a resolution of less than 100 fs. For future progress, a further development of the spectroscopy is indispensable as well as structural data at atomic resolution. This revised version was published online in June 2006 with corrections to the Cover Date.     

Opportunities and challenges for time-resolved studies of protein structural dynamics at X-ray free-electron lasers

X-ray free-electron lasers (XFELs) are revolutionary X-ray sources. Their time structure, providing X-ray pulses of a few tens of femtoseconds in duration; and their extreme peak brilliance, delivering approximately 10¹² X-ray photons per pulse and facilitating sub-micrometre focusing, distinguish XFEL sources from synchrotron radiation. In this opinion piece, I argue that these properties of XFEL radiation will facilitate new discoveries in life science. I reason that time-resolved serial femtosecond crystallography and time-resolved wide angle X-ray scattering are promising areas of scientific investigation that will be advanced by XFEL capabilities, allowing new scientific questions to be addressed that are not accessible using established methods at storage ring facilities. These questions include visualizing ultrafast protein structural dynamics on the femtosecond to picosecond time-scale, as well as time-resolved diffraction studies of non-cyclic reactions. I argue that these emerging opportunities will stimulate a renaissance of interest in time-resolved structural biochemistry.     

Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport

The evolution of oxygen transport hemoglobins occurred on at least two independent occasions. The earliest event led to myoglobin and red blood cell hemoglobin in animals. In plants, oxygen transport "leghemoglobins" evolved much more recently. In both events, pentacoordinate heme sites capable of inert oxygen transfer evolved from hexacoordinate hemoglobins that have unrelated functions. High sequence homology between hexacoordinate and pentacoordinate hemoglobins in plants has poised them for potential structural analysis leading to a molecular understanding of this important evolutionary event. However, the lack of a plant hexacoordinate hemoglobin structure in the exogenously ligand-bound form has prevented such comparison. Here we report the crystal structure of the cyanide-bound hexacoordinate hemoglobin from barley. This presents the first opportunity to examine conformational changes in plant hexacoordinate hemoglobins upon exogenous ligand binding, and reveals structural mechanisms for stabilizing the high-energy pentacoordinate heme conformation critical to the evolution of reversible oxygen binding hemoglobins.     

Time-resolved OH → EH transition of the aberrant ba3 oxidase from Thermus thermophilus

The kinetics of single-electron injection into the oxidized nonrelaxed state (O_H → E_H transition) of the aberrant ba₃ cytochrome oxidase from Thermus thermophilus, noted for its lowered efficiency of proton pumping, was investigated by time-resolved optical spectroscopy. Two main phases of intraprotein electron transfer were resolved. The first component (τ ∼ 17 μs) reflects oxidation of Cu_A and reduction of the heme groups (low-spin heme b and high-spin heme a₃ in a ratio close to 50:50). The subsequent component (τ ∼ 420 μs) includes reoxidation of both hemes by Cu_B. This is in significant contrast to the O_H → E_H transition of the aa₃-type cytochrome oxidase from Paracoccus denitrificans, where the fastest phase is exclusively due to transient reduction of the low-spin heme a, without electron equilibration with the binuclear center. On the other hand, the one-electron reduction of the relaxed O state in ba₃ oxidase was similar to that in aa₃ oxidase and only included rapid electron transfer from Cu_A to the low-spin heme b. This indicates a functional difference between the relaxed O and the pulsed O_H forms also in the ba₃ oxidase from T. thermophilus.     

Real-time optical studies of respiratory Complex I turnover

Reduction of Complex I (NADH:ubiquinone oxidoreductase I) from Escherichia coli by NADH was investigated optically by means of an ultrafast stopped-flow approach. A locally designed microfluidic stopped-flow apparatus with a low volume (0.2 μl) but a long optical path (10 mm) cuvette allowed measurements in the time range from 270 μs to seconds. The data acquisition system collected spectra in the visible range every 50 μs. Analysis of the obtained time-resolved spectral changes upon the reaction of Complex I with NADH revealed three kinetic components with characteristic times of < 270 μs, 0.45–0.9 ms and 3–6 ms, reflecting reduction of different FeS clusters and FMN. The rate of the major (τ = 0.45–0.9 ms) component was slower than predicted by electron transfer theory for the reduction of all FeS clusters in the intraprotein redox chain. This delay of the reaction was explained by retention of NAD⁺ in the catalytic site. The fast optical changes in the time range of 0.27–1.5 ms were not altered significantly in the presence of 10-fold excess of NAD⁺ over NADH. The data obtained on the NuoF E95Q variant of Complex I shows that the single amino acid replacement in the catalytic site caused a strong decrease of NADH binding and/or the hydride transfer from bound NADH to FMN.