Photoacoustic Calorimetry Studies of O2-Sensing FixL and (R200, I209) Variants from Sinorhizobium meliloti Reveal Conformational Changes Coupled to Ligand Photodissociation from the Heme-PAS Domain.

FixL is an oxygen-sensing heme-PAS protein that regulates nitrogen fixation in the root nodules of plants. In this paper, we present the first photothermal studies of the full-length wild-type FixL protein from Sinorhizobium meliloti and the first thermodynamic profile of a full-length heme-PAS protein. Photoacoustic calorimetry studies reveal a quadriphasic relaxation for SmFixL*WT and the five variant proteins (SmFixL*R200H, SmFixL*R200Q, SmFixL*R200E, SmFixL*R200A, and SmFixL*I209M) with four intermediates from <20 ns to ∼1.5 µs associated with the photodissociation of CO from the heme. The altered thermodynamic profiles of the full-length SmFixL* variant proteins confirm that the conserved heme domain residues R200 and I209 are important for signal transduction. In contrast, the truncated heme domain, SmFixLH128-264, shows only a single, fast monophasic relaxation at <50 ns associated with the fast disruption of a salt bridge and release of CO to the solvent, suggesting that the full-length protein is necessary to observe the conformational changes that propagate the signal from the heme domain to the kinase domain.

Role of Conserved Residues and F322 in the Extracellular Vestibule of the Rat P2X7 Receptor in Its Expression, Function and Dye Uptake Ability.

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.

Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.

The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33-239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33-239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33-239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33-239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33-239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33-239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33-239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle.

Ligand-induced structural changes in the Escherichia coli ferric citrate transporter reveal modes for regulating protein-protein interactions.

Outer-membrane TonB-dependent transporters, such as the Escherichia coli ferric citrate transporter FecA, interact with the inner-membrane protein TonB through an energy-coupling segment termed the Ton box. In FecA, which regulates its own transcription, the Ton box is preceded by an N-terminal extension that interacts with the inner-membrane protein FecR. Here, site-directed spin labeling was used to examine the structural basis for transcriptional signaling and Ton box regulation in FecA. EPR spectroscopy indicates that regions of the N-terminal domain are in conformational exchange, consistent with its role as a protein binding element; however, the local fold and dynamics of the domain are not altered by substrate or TonB. Distance restraints derived from pulse EPR were used to generate models for the position of the extension in the apo, substrate-, and TonB-bound states. In the apo state, this domain is positioned at the periplasmic surface of FecA, where it interacts with the Ton box and blocks access of the Ton box to the periplasm. Substrate addition rotates the transcriptional domain and exposes the Ton box, leading to a disorder transition in the Ton box that may facilitate interactions with TonB. When a soluble fragment of TonB is bound to FecA, the transcriptional domain is displaced to one edge of the barrel, consistent with a proposed ß-strand exchange mechanism. However, neither substrate nor TonB displaces the N-terminus further into the periplasm. This result suggests that the intact TonB system mediates both signaling and transport by unfolding portions of the transporter.

Photothermal studies of CO photodissociation from peroxidases from horseradish and soybean.

In this work, the results of photoacoustic calorimetry (PAC) studies involving CO photodissociation from horseradish peroxidase (HRP) and soybean peroxidase (SBP) are discussed. Both proteins contain Fe-protoporphyrin IX active sites and relatively open distal heme pockets (i.e., direct solvent access). In addition, it has been shown previously that SBP binds a Tris molecule in the distal pocket near the heme group potentially regulating ligand binding to the heme iron. Results of PAC studies indicate a fast (< approximately 50 ns) relaxation for both HRP and SBP subsequent to CO photolysis in both phosphate and Tris buffers and with varying concentrations of Tris. However, the molar volume/enthalpy changes associated with CO release are distinct between the two proteins. In the case of HRP, CO photolysis results in an enthalpy change of approximately 2 kcal mol(-1) and volume change of approximately -12 mL mol(-1) attributed to solvation/structural changes regardless of buffer conditions. In contrast, SBP exhibits buffer and ionic strength dependent enthalpy changes ranging from approximately -23 kcal mol(-1) in 50 mM phosphate buffer to approximately 6 kcal mol(-1) in Tris buffer with volume changes similar to those observed in HRP. The results are consistent with a model in which photodissociation of CO from ferrous HRP or SBP leads to CO migration from the distal heme pocket to the bulk solvent with a corresponding input of a water molecule all occurring in < approximately 50 ns. The differences in enthalpies are attributed to variations in hydrogen bond formation between the incoming water molecule(s) and the protein matrix in both HRP and SBP.

Photophysical studies of the trans to cis isomerization of the push-pull molecule: 1-(pyridin-4-yl)-2-(N-methylpyrrol-2-yl)ethene (mepepy).

Organic molecules possessing intramolecular charge-transfer properties (D-pi-A type molecules) are of key interest particularly in the development of new optoelectronic materials as well as photoinduced magnetism. One such class of D-pi-A molecules that is of particular interest contains photoswitchable intramolecular charge-transfer states via a photoisomerizable pi-system linking the donor and acceptor groups. Here we report the photophysical and electronic properties of the trans to cis isomerization of 1-(pyridin-4-yl)-2-(N-methylpyrrol-2-yl)ethene ligand (mepepy) in aqueous solution using photoacoustic calorimetry (PAC) and theoretical methods. Density functional theory (DFT) calculations demonstrate a global energy difference between cis and trans isomers of mepepy to be 8 kcal mol(-1), while a slightly lower energy is observed between the local minima for the trans and cis isomers (7 kcal mol(-1)). Interestingly, the trans isomer appears to exhibit two ground-state minima separated by an energy barrier of approximately 9 kcal mol(-1). Results from the PAC studies indicate that the trans to cis isomerization results in a negligible volume change (0.9 +/- 0.4 mL mol(-1)) and an enthalpy change of 18 +/- 3 kcal mol(-1). The fact that the acoustic waves associated with the trans to cis transition of mepepy overlap in frequency with those of a calorimetric reference implies that the conformational transition occurs faster than the approximately 50 ns response time of the acoustic detector. Comparison of the experimental results with theoretical studies provide evidence for a mechanism in which the trans to cis isomerization of mepepy results in the loss of a hydrogen bond between a water molecule and the pyridine ring of mepepy.

Evidence for fast conformational change upon ligand dissociation in the HemAT class of bacterial oxygen sensors.

Here we report the results of transient absorption and photoacoustic calorimetry studies of CO photodissociation from the heme domain of the bacterial oxygen sensor HemAT-Bs. The results indicate that CO photolysis is accompanied by an overall DeltaH of -19 kcal mol(-1) and DeltaV of +4 ml mol(-1) as well as a red-shifted kinetic difference spectrum all occurring in <50 ns. Analysis of the DeltaH/DeltaV reveals that a conformational change takes place with a DeltaH(conf) of -40 kcal mol(-1) and DeltaV(conf) of -22 ml mol(-1). These thermodynamic changes are consistent with an increase in the solvent accessible surface area of the protein upon ligand dissociation, as observed in the X-ray structure of the ferric CN-bound and CN free forms of HemAT-Bs.