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1.
Biochemistry ; 63(1): 116-127, 2024 Jan 02.
Article in English | MEDLINE | ID: mdl-38127721

ABSTRACT

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.


Subject(s)
Hemeproteins , Sinorhizobium meliloti , Protein Kinases/metabolism , Histidine Kinase/genetics , Histidine Kinase/metabolism , Sinorhizobium meliloti/chemistry , Heme/chemistry , Ligands , Hemeproteins/metabolism , Oxygen/metabolism , Calorimetry , Bacterial Proteins/chemistry
2.
Int J Mol Sci ; 21(22)2020 Nov 10.
Article in English | MEDLINE | ID: mdl-33182845

ABSTRACT

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.


Subject(s)
Receptors, Purinergic P2X7/genetics , Receptors, Purinergic P2X7/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Conserved Sequence , HEK293 Cells , Humans , Ion Channel Gating , Kinetics , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Domains , Protein Interaction Domains and Motifs , Rats , Receptors, Purinergic P2X7/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Static Electricity
3.
Biochemistry ; 52(15): 2638-48, 2013 Apr 16.
Article in English | MEDLINE | ID: mdl-23517233

ABSTRACT

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.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Colicins/chemistry , Colicins/metabolism , Electron Spin Resonance Spectroscopy/methods , Escherichia coli Proteins/genetics , Fluorescence Polarization , Membrane Proteins/genetics , Membrane Transport Proteins/chemistry , Mutation , Protein Binding , Protein Conformation , Protein Multimerization , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism
4.
J Mol Biol ; 423(5): 818-30, 2012 Nov 09.
Article in English | MEDLINE | ID: mdl-22982293

ABSTRACT

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.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Receptors, Cell Surface/chemistry , Electron Spin Resonance Spectroscopy , Escherichia coli Proteins/metabolism , Ligands , Models, Molecular , Protein Binding , Receptors, Cell Surface/metabolism , Signal Transduction , Transcription, Genetic
5.
Biochim Biophys Acta ; 1794(11): 1558-65, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19595798

ABSTRACT

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.


Subject(s)
Carbon Monoxide/metabolism , Horseradish Peroxidase/metabolism , Peroxidases/metabolism , Calorimetry , Models, Molecular , Myoglobin/metabolism , Photochemical Processes , Glycine max/enzymology , Thermodynamics
6.
J Phys Chem A ; 112(36): 8310-5, 2008 Sep 11.
Article in English | MEDLINE | ID: mdl-18700732

ABSTRACT

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.

7.
FEBS Lett ; 581(23): 4512-8, 2007 Sep 18.
Article in English | MEDLINE | ID: mdl-17765225

ABSTRACT

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.


Subject(s)
Bacterial Proteins/chemistry , Hemeproteins/chemistry , Oxygen/chemistry , Bacterial Proteins/metabolism , Calorimetry , Carbon Monoxide/chemistry , Carbon Monoxide/metabolism , Heme/chemistry , Heme/metabolism , Hemeproteins/metabolism , Ligands , Models, Molecular , Oxygen/metabolism , Protein Binding , Protein Conformation , Thermodynamics
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