Calculation of the electronic structure and spectra of model cytochrome P450 compound I

The electronic structure and spectra of the oxyferryl (Fe=O) compound I P450 heme species, the transient putative active intermediate of cytochrome P450s, have been calculated employing a full protoporphyrin IX heme model representation. The principal aim of this work was to compare the computed spectra of this species with the observed transient spectra attributed to it. Computations were made using both nonlocal density functional theory (DFT) and semiempirical INDO/CI methods to characterize the electronic structure of the compound I P450 species. Both methods resulted in a similar antiferromagnetic doublet as the ground state with a ferromagnetic quartet excited state partner, slightly higher in energy. The INDO/ROHF/CI semiempirical method was used to calculate the spectrum of the protoporphyrin IX P450 compound I heme species in its lowest energy antiferromagnetic doublet state at the DFT optimized geometry. As a reference, the spectrum of the ferric resting form of the protoporphyrin IX P450 heme species was also calculated. The computed shifts in the Soret and Q bands of compound I relative to the resting state were both in good agreement with the corresponding experimentally observed shifts in the transient spectra of cytochrome P450cam (Biochem. Biophys. Res. Commun. 201 (1994) 1464) and chloroperoxidase (Biochem. Biophys. Res. Commun. 94 (1980) 1123) both ascribed to their common compound I heme site. This consistency provides additional, independent support for the assignment of compound I as the origin of the reported observed transient spectra.     

A fluorometric approach to local electric field measurements in a voltage-gated ion channel

Site-specific electrostatic measurements have been limited to soluble proteins purified for in vitro spectroscopic characterization or proteins of known structure; however, comparable measurements have not been made for functional membrane bound proteins. Here, using an electrochromic fluorophore, we describe a method to monitor localized electric field changes in a voltage-gated potassium channel. By coupling the novel probe Di-1-ANEPIA to cysteines in Shaker and tracking field-induced optical changes, in vivo electrostatic measurements were recorded with submillisecond resolution. This technique reports dynamic changes in the electric field during the gating process and elucidates the electric field profile within Shaker. The extension of this method to other membrane bound proteins, including transporters, will yield insight into the role of electrical forces on protein function.     

Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce activation of platelets and vascular cells, while PAR2 is preferentially cleaved by trypsin. In pathological situations, other proteolytic enzymes may be generated in the circulation and could modify the responses of PARs by cleaving their extracellular domains. To assess the ability of such proteases to activate or inactivate PARs, we designed a strategy for locating cleavage sites on the exofacial NH(2)-terminal fragments of the receptors. The first extracellular segments of PAR1 (PAR1E) and PAR2 (PAR2E) expressed as recombinant proteins in Escherichia coli were incubated with a series of proteases likely to be encountered in the circulation during thrombosis or inflammation. Kinetic and dose-response studies were performed, and the cleavage products were analyzed by MALDI-TOF mass spectrometry. Thrombin cleaved PAR1E at the Arg41-Ser42 activation site at concentrations known to induce cellular activation, supporting a native conformation of the recombinant polypeptide. Plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3 cleaved at multiple sites and would be expected to disable PAR1 by cleaving COOH-terminal to the activation site. Cleavage specificities were further confirmed using activation site defective PAR1E S42P mutant polypeptides. Surface plasmon resonance studies on immobilized PAR1E or PAR1E S42P were consistent with cleavage results obtained in solution and allowed us to determine affinities of PAR1E-thrombin binding. FACS analyses of intact platelets confirmed the cleavage of PAR1 downstream of the Arg41-Ser42 site. Mass spectrometry studies of PAR2E predicted activation of PAR2 by trypsin through cleavage at the Arg36-Ser37 site, no effect of thrombin, and inactivation of the receptor by plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3. The inhibitory effect of elastase was confirmed on native PAR1 and PAR2 on the basis of Ca(2+) signaling studies in endothelial cells. It was concluded that none of the main proteases generated during fibrinolysis or inflammation appears to be able to signal through PAR1 or PAR2. This strategy provides results which can be extended to the native receptor to predict its activation or inactivation, and it could likewise be used to study other PARs or protease-dependent processes.     

Initiation of Sodium Spikelets in Basal Dendrites of Neocortical Pyramidal Neurons

Cortical information processing relies critically on the processing of electrical signals in pyramidal neurons. Electrical transients mainly arise when excitatory synaptic inputs impinge upon distal dendritic regions. To study the dendritic aspect of synaptic integration one must record electrical signals in distal dendrites. Since thin dendritic branches, such as oblique and basal dendrites, do not support routine glass electrode measurements, we turned our effort towards voltage-sensitive dye recordings. Using the optical imaging approach we found and reported previously that basal dendrites of neocortical pyramidal neurons show an elaborate repertoire of electrical signals, including backpropagating action potentials and glutamate-evoked plateau potentials. Here we report a novel form of electrical signal, qualitatively and quantitatively different from backpropagating action potentials and dendritic plateau potentials. Strong glutamatergic stimulation of an individual basal dendrite is capable of triggering a fast spike, which precedes the dendritic plateau potential. The amplitude of the fast initial spikelet was actually smaller that the amplitude of the backpropagating action potential in the same dendritic segment. Therefore, the fast initial spike was dubbed “spikelet”. Both the basal spikelet and plateau potential propagate decrementally towards the cell body, where they are reflected in the somatic whole-cell recordings. The low incidence of basal spikelets in the somatic intracellular recordings and the impact of basal spikelets on soma-axon action potential initiation are discussed.     

Modeling and analysis of calcium signaling events leading to long-term depression in cerebellar Purkinje cells

Modeling and simulation of the calcium signaling events that precede long-term depression of synaptic activity in cerebellar Purkinje cells are performed using the Virtual Cell biological modeling framework. It is found that the unusually high density and low sensitivity of inositol-1,4,5-trisphosphate receptors (IP3R) are critical to the ability of the cell to generate and localize a calcium spike in a single dendritic spine. The results also demonstrate the model's capability to simulate the supralinear calcium spike observed experimentally during coincident activation of the parallel and climbing fibers. The sensitivity of the calcium spikes to certain biological and geometrical effects is investigated as well as the mechanisms that underlie the cell's ability to generate the supralinear spike. The sensitivity of calcium release rates from the IP3R to calcium concentrations, as well as IP3 concentrations, allows the calcium spike to form. The diffusion barrier caused by the small radius of the spine neck is shown to be important, as a threshold radius is observed above which a spike cannot be formed. Additionally, the calcium buffer capacity and diffusion rates from the spine are found to be important parameters in shaping the calcium spike.     

Pom33, a novel transmembrane nucleoporin required for proper nuclear pore complex distribution

The biogenesis of nuclear pore complexes (NPCs) represents a paradigm for the assembly of high-complexity macromolecular structures. So far, only three integral pore membrane proteins are known to function redundantly in NPC anchoring within the nuclear envelope. Here, we describe the identification and functional characterization of Pom33, a novel transmembrane protein dynamically associated with budding yeast NPCs. Pom33 becomes critical for yeast viability in the absence of a functional Nup84 complex or Ndc1 interaction network, which are two core NPC subcomplexes, and associates with the reticulon Rtn1. Moreover, POM33 loss of function impairs NPC distribution, a readout for a subset of genes required for pore biogenesis, including members of the Nup84 complex and RTN1. Consistently, we show that Pom33 is required for normal NPC density in the daughter nucleus and for proper NPC biogenesis and/or stability in the absence of Nup170. We hypothesize that, by modifying or stabilizing the nuclear envelope–NPC interface, Pom33 may contribute to proper distribution and/or efficient assembly of nuclear pores.     

Characterization and application of a new optical probe for membrane lipid domains

In this article, we characterize the fluorescence of an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. In large unilamellar lipid vesicles (LUVs), its emission spectrum shifts up to 30 nm to the blue with increasing cholesterol concentration. Independently, it displays a comparable blue shift in liquid-ordered relative to liquid-disordered phases. The cumulative effect is a 60-nm difference in emission spectra for cholesterol containing LUVs in the liquid-ordered state versus cholesterol-free LUVs in the liquid-disordered phase. Given these optical properties, we use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and nonlinear optical microscopy. The dye shows green and red fluorescence in liquid-ordered and -disordered domains, respectively. We propose that this reflects the relative rigidity of the molecular packing around the dye molecules in the two phases. We also observe a sevenfold stronger second harmonic generation signal in the liquid-disordered domains, consistent with a higher concentration of the dye resulting from preferential partitioning into the disordered phase. The efficacy of the dye for reporting lipid domains in cell membranes is demonstrated in polarized migrating neutrophils.     

Use of SANS and biophysical techniques to reveal subtle conformational differences between native apo-calmodulin and its unfolded states

Apo-calmodulin, a small, mainly α, soluble protein is a calcium-dependent protein activator. It is made of two N- and C-terminal domains having a sequence homology of 70%, an identical folding but different stabilities, and is thus an interesting system for unfolding studies. The use of small angle neutron scattering (SANS) and other biophysical techniques has permitted to reveal conformational difference between native and thermal denatured states of apo-calmodulin. The results show that secondary and tertiary structures of apo-calmodulin evolve in a synchronous way, indicating the absence in the unfolding pathway of molten-globule state sufficiently stable to affect transition curves. From SANS experiments, at 85 °C, apo-calmodulin adopts a polymer chain conformation with some residual local structures. After cooling down, apo-calmodulin recovers a compact state, with a secondary structure close to the native one but with a higher radius of gyration and a different tyrosine environment. In fact on a timescale of few minutes, heat denaturation of apo-calmodulin is partially reversible, but on a time scale of hours (for SANS experiments), the long exposure to heat may lead to a non-reversibility due to some chemical perturbation of the protein. In fact, from Mass Spectrometry measurements, we got evidence of dehydration and deamidation of heated apo-calmodulin.     

Computational analysis of calcium signaling and membrane electrophysiology in cerebellar Purkinje neurons associated with ataxia

ABSTRACT: BACKGROUND: Mutations in the smooth endoplasmic reticulum (sER) calcium channel Inositol Trisphosphate Receptor type 1 (IP3R1) in humans with the motor function coordination disorders Spinocerebellar Ataxia Types 15 and 16 (SCA15/16) and in a corresponding mouse model, the IP3R1delta18/delta18 mice, lead to reduced IP3R1 levels. We posit that increasing IP3R1 sensitivity to IP3 in ataxias with reduced IP3R1 could restore normal calcium response. On the other hand, in mouse models of the human polyglutamine (polyQ) ataxias, SCA2, and SCA3, the primary finding appears to be hyperactive IP3R1-mediated calcium release. It has been suggested that the polyQ SCA1 mice may also show hyperactive IP3R1. Yet, SCA1 mice show downregulated gene expression of IP3R1, Homer, metabotropic glutamate receptor (mGluR), smooth endoplasmic reticulum Ca-ATP-ase (SERCA), calbindin, parvalbumin, and other calcium signaling proteins. RESULTS: We create a computational model of pathological alterations in calcium signaling in cerebellar Purkinje neurons to investigate several forms of spinocerebellar ataxia associated with changes in the abundance, sensitivity, or activity of the calcium channel IP3R1. We find that increasing IP3R1 sensitivity to IP3 in computational models of SCA15/16 can restore normal calcium response if IP3R1 abundance is not too low. The studied range in IP3R1 levels reflects variability found in human and mouse ataxic models. Further, the required fold increases in sensitivity are within experimental ranges from experiments that use IP3R1 phosphorylation status to adjust its sensitivity to IP3. Results from our simulations of polyglutamine SCAs suggest that downregulation of some calcium signaling proteins may be partially compensatory. However, the downregulation of calcium buffer proteins observed in the SCA1 mice may contribute to pathology. Finally, our model suggests that the calcium-activated voltage-gated potassium channels may provide an important link between calcium metabolism and membrane potential in Purkinje cell function. CONCLUSION: Thus, we have established an initial platform for computational evaluation and prediction of ataxia pathophysiology. Specifically, the model has been used to investigate SCA15/16, SCA1, SCA2, and SCA3. Results suggest that experimental studies treating mouse models of any of these ataxias with appropriately chosen peptides resembling the C-terminal of IP3R1 could adjust receptor sensitivity, and thereby modulate calcium release and normalize IP3 response. In addition, the model supports the hypothesis of IP3R1 supersensitivity in SCA1.