Finite-element modeling of time-dependent sodium exchange across the hollow fiber of a hemodialyzer by coupling with a blood pool model.

INTRODUCTION: Hollow fiber models describe the exchange of solutes between blood and dialysate across the membrane of a single fiber of the hemodialysis filter (hemodialyzer). This work aims to develop a new approach to simulate the solute exchange in a hollow fiber in a dynamic and realistic way. Sodium was chosen as our solute of interest due to its importance in hemodialysis as an osmotic regulator. METHODS: A 2-dimensional (2D) hollow fiber model based on the finite element method (FEM) is coupled to a simple blood pool model to dynamically update the concentration of the solute entering the dialyzer. The resulting coupled model maintains the geometrical detail of the 2D fiber representation and gains a dynamic, blood-side inlet solute concentration. In vitro dialysis sessions were carried out for model validation, by implementing a combination of blood volume loss and/or sodium concentration steps. Plasmatic sodium concentration was recorded by blood gas sampling. Dialysate inlet and outlet conductivities were continuously recorded. RESULTS: Simulated plasmatic sodium concentration was compared with data from the blood gas samples. A mean error of 1.76 ± 1.03 mM was found for the complete dataset, along with a 3.87 mM maximum error. The simulated outlet dialysate sodium concentration was compared with the recorded outlet dialysate conductivity: a very high correlation was found on the whole dataset (R2 = 0.992). CONCLUSIONS: Coupling our FEM hollow fiber model to a simple blood pool model proved to be an effective approach for dynamical analysis of the properties of the hemodialyzer.

Phthalocyanine tetrasulfonates bind to multiple sites on natively-folded prion protein.

The phthalocyanine tetrasulfonates (PcTS), a class of cyclic tetrapyrroles, bind to the mammalian prion protein, PrP. Remarkably, they can act as anti-scrapie agents to prevent the formation and spread of infectious, misfolded PrP. While the effects of phthalocyanines on the diseased state have been investigated, the interaction between PcTS and PrP has not yet been extensively characterized. Here we use multiple, complementary assays (surface plasmon resonance, isothermal titration calorimetry, fluorescence correlation spectroscopy, and tryptophan fluorescence quenching) to characterize the binding of PcTS to natively-folded hamster PrP(90-232), in order to determine binding constants, ligand stoichiometry, influence of buffer ionic strength, and the effects of chelated metal ions. We found that binding strength depends strongly on chelated metal ions, with Al(3+)-PcTS binding the weakest and free-base PcTS the strongest of the three types tested (Al(3+), Zn(2+), and free-base). Buffer ionic strength also affected the binding, with K(d) increasing along with salt concentration. The binding isotherms indicated the presence of at least two different binding sites with micromolar affinities and a total stoichiometry of ~4-5 PcTS molecules per PrP molecule.

Structural plasticity and functional implications of internal cavities in distal mutants of type 1 non-symbiotic hemoglobin AHb1 from Arabidopsis thaliana.

The increasing number of nonsymbiotic plant hemoglobins discovered in genomic studies in the past decade raises intriguing questions about their physiological role. Among them, the nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana deserves particular attention, as it combines an extremely high oxygen affinity with an internal hexacoordination of the distal histidine HisE7 to the heme iron in the absence of exogenous ligands. In order to gain insight into the structure-function relationships of the protein, the ligand binding properties of mutants of two conserved residues of the distal cavity, HisE7 --> Leu and PheB10 --> Leu, were investigated by experimental and computational studies and compared to results determined for the wild type (wt) protein. The Fe(2+)-deoxy HisE7 --> Leu mutant exists, as expected, in the pentacoordinated form, while a mixture of penta- and hexacoordinated forms is found for the PheB10 --> Leu mutant, with an equilibrium shifted toward the pentacoordinated form with respect to the wt protein. Spectroscopic studies of the complexes of CO and CN(-) with AHb1 and its mutants show a subtle interplay of steric and electrostatic effects by distal residues on the ligand binding to the heme. Moreover, stopped-flow and flash photolysis experiments reveal substantial kinetic differences triggered by those mutations, which are particularly manifested in the enhanced geminate rebinding and bimolecular association rate. These findings are discussed in light of the drastic alterations found by molecular dynamics simulations in the nature and distribution of internal cavities in the protein matrix of the mutants, revealing an extremely large sensitivity of the protein structure to changes in distal HisE7 and PheB10 residues. Overall, data are consistent with the putative NO-dioxygenase activity attributed to AHb1.

Cis-trans photoisomerization of fluorescent-protein chromophores.

Photochromic variants of fluorescent proteins are opening the way to a number of opportunities for high-sensitivity regioselective studies in the cellular environment and may even lead to applications in information and communication technology. Yet, the detailed photophysical processes at the basis of photoswitching have not been fully clarified. In this paper, we used synthetic FP chromophores to clarify the photophysical processes associated with the photochromic behavior. In particular, we investigated the spectral modification of synthetic chromophore analogues of wild-type green fluorescent protein (GFP), Y66F GFP (BFPF), and Y66W GFP (CFP) upon irradiation in solutions of different polarities. We found that the cis-trans photoisomerization mechanism can be induced in all the chromophores. The structural assignments were carried out both by NMR measurements and DFT calculations. Remarkably, we determined for the first time the spectra of neutral trans isomers in different solvents. Finally, we calculated the photoconversion quantum yields by absorption measurements under continuous illumination at different times and by a nanosecond laser-flash photolysis method. Our results indicate that cis-trans photoisomerization is a general mechanism of FP chromophores whose efficiency is modulated by the detailed mutant-specific protein environment.

Characterization of ligand migration mechanisms inside hemoglobins from the analysis of geminate rebinding kinetics.

The presence of internal hydrophobic cavities and packing defects has been demonstrated for several small globular proteins, including hemoglobins. The reduced thermodynamic stability appears to be compensated for by the capability of controlling ligand diffusion through the protein matrix to the active site, possibly by stocking more than one reactant molecule in selected sites. Photolysis of carbon monoxide complexes of hemoglobins encapsulated in silica gels leads to multiphasic geminate rebinding kinetics at room temperature, reflecting rebinding also from different temporary docking sites inside the protein matrix. A careful analysis of the ligand rebinding kinetics allows the determination of the microscopic rates for the underlying reactions, including those governing the migration to and from the docking sites. This chapter describes the experimental approach used to characterize the ligand rebinding kinetics for heme proteins in silica gels after nanosecond laser flash photolysis and the computational methods necessary to retrieve the kinetic parameters.

Ligand migration in nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana.

AHb1 is a hexacoordinated type 1 nonsymbiotic hemoglobin recently discovered in Arabidopsis thaliana. To gain insight into the ligand migration inside the protein, we studied the CO rebinding kinetics of AHb1 encapsulated in silica gels, in the presence of glycerol. The CO rebinding kinetics after nanosecond laser flash photolysis exhibits complex ligand migration patterns, consistent with the existence of discrete docking sites in which ligands can temporarily be stored before rebinding to the heme at different times. This finding may be of relevance to the physiological NO dioxygenase activity of this protein, which requires sequential binding of two substrates, NO and O2, to the heme.

Different roles of protein dynamics and ligand migration in non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana.

The ligand rebinding kinetics after photolysis of the CO complexes of Arabidopsis thaliana hemoglobins AHb1 and AHb2 in solution show very different amplitudes in the geminate phase, reflecting different migration pathways of the photodissociated ligand in the system of internal cavities accessible from the heme. The dependence of the geminate phase on CO concentration, temperature, encapsulation in silica gels and presence of glycerol confirms a remarkable difference in the internal structure of the two proteins and a dramatically different role of protein dynamics in regulating the reactivity with CO. This finding strongly supports the idea that they have distinct physiological functions.

Green fluorescent protein ground states: the influence of a second protonation site near the chromophore.

The photophysical properties of most green fluorescent protein mutants (GFPs) are strongly affected by pH. This effect must be carefully taken into account when using GFPs as fluorescent probes or indicators. Usually, the pH-dependence of GFPs is rationalized on the basis of the ionization equilibrium of the chromophore phenol group. Yet many different mutants show spectral behavior that cannot be explained by ionization of this group alone. In this study, we propose a general model of protonation comprising two ionization sites (2S model). Steady-state optical measurements at different pH and temperature and pH-jump relaxation experiments were combined to highlight the thermodynamic and kinetic properties of paradigmatically different GFP variants. Our experiments support the 2S model. For the case of mutants in which E222 is the second protonation site, thermodynamic coupling between this residue's and the chromophore's ionization reactions was demonstrated. In agreement with the 2S model predictions, X-ray analysis of one of these mutants showed the presence of two chromophore populations at high pH.

The reactivity with CO of AHb1 and AHb2 from Arabidopsis thaliana is controlled by the distal HisE7 and internal hydrophobic cavities.

The nonsymbiotic hemoglobins, AHb1 and AHb2, have recently been isolated from Arabidopsis thaliana. Using steady-state and time-resolved spectroscopic methods, we show that Fe2+ AHb1 contains a mixture of penta- and hexacoordinated heme, while Fe2+ AHb2 is fully hexacoordinated. In the CO complexes, polar interactions and H-bonds with the ligand are stronger for AHb1 than for AHb2. The ligand binding kinetics are substantially different, reflecting the distribution between the penta- and hexacoordinated species, and indicate that protein dynamics and ligand migration pathways are very specific for each of the two proteins. In particular, a very small, non-exponential geminate rebinding observed in AHb1 suggests that the distal heme cavity is connected with the exterior by a relatively open channel. The large, temperature-dependent geminate rebinding observed for AHb2 implies a major role of protein dynamics in the ligand migration from the distal cavity to the solvent. The structures of AHb1 and AHb2, modeled on the basis of the homologous rice hemoglobin, exhibit a different cavity system that is fully compatible with the observed ligand binding kinetics. Overall, these kinetic and structural data are consistent with the putative NO-dioxygenase activity previously attributed to AHb1, whereas the role of AHb2 remains elusive.

Time-resolved methods in Biophysics. 2. Monitoring haem proteins at work with nanosecond laser flash photolysis.

Haem proteins have long been the most studied proteins in biophysics, and have become paradigms for the characterization of fundamental biomolecular processes as ligand binding and regulatory conformational transitions. The presence of the haem prosthetic group, the absorbance spectrum of which has a ligation sensitive region conveniently located in the UV-visible range, has offered a powerful and sensitive tool for the investigation of molecular functions. The central Fe atom is capable of reversibly binding diatomic ligands, including O(2), CO, and NO. The Fe-ligand bond is photolabile, and a reactive unligated state can be transiently generated with a pulsed laser. The photodissociated ligands quickly rebind to the haem and the process can be monitored by transient absorbance methods. The ligand rebinding kinetics reflects protein dynamics and ligand migration within the protein inner cavities. The characterization of these processes was done in the past mainly by low temperature experiments. The use of silica gels to trap proteins allows the characterization of internal ligand dynamics at room temperature. In order to show the potential of the laser flash photolysis techniques, combined with modern numerical analysis methods, we report experiments conducted on two non-symbiotic haemoglobins from Arabidopsis thaliana. The comparison between time courses recorded on haemoglobins in solution and encapsulated in silica gels allows for the highlighting of different interplays between protein dynamics and ligand migration.

Kinetics of acid-induced spectral changes in the GFPmut2 chromophore.

We have used a nanosecond pH-jump technique, coupled with simultaneous transient absorption and fluorescence emission detection, to characterize the dynamics of the acid-induced spectral changes in the GFPmut2 chromophore. Disappearance of the absorbance at 488 nm and the green fluorescence emission occurs with a thermally activated, double exponential relaxation. To understand the source of the two transients we have introduced mutations in amino acid residues that interact with the chromophore (H148G, T203V, and E222Q). Results indicate that the faster transient is associated with proton binding from the solution, while the second process, smaller in amplitude, is attributed to structural rearrangement of the amino acids surrounding the chromophore. The protonation rate shows a 3-fold increase for the H148G mutant, demonstrating that His148 plays a key role in protecting the chromophore from the solvent. The deprotonation rate for T203V is an order of magnitude smaller, showing that the hydrogen bond with the hydroxyl of Thr203 is important in stabilizing the deprotonated form of the chromophore. A kinetic model suggests that, in addition to protecting the chromophore from the solvent, His148 may act as the primary acceptor for the protons on the way to the chromophore.