Human cytochrome C natural variants: Studying the membrane binding properties of G41S and Y48H by fluorescence energy transfer and molecular dynamics.

Cytochrome C (cyt C), the protein involved in oxidative phosphorylation, plays several other crucial roles necessary for both cell life and death. Studying natural variants of cyt C offers the possibility to better characterize the structure-to-function relationship that modulates the different activities of this protein. Naturally mutations in human cyt C (G41S and Y48H) occur in the protein central Ω-loop and cause thrombocytopenia 4.

Conformational Dynamics of Lipoxygenases and Their Interaction with Biological Membranes.

Lipoxygenases (LOXs) are a family of enzymes that includes different fatty acid oxygenases with a common tridimensional structure. The main functions of LOXs are the production of signaling compounds and the structural modifications of biological membranes. These features of LOXs, their widespread presence in all living organisms, and their involvement in human diseases have attracted the attention of the scientific community over the last decades, leading to several studies mainly focused on understanding their catalytic mechanism and designing effective inhibitors.

Head or tail? A molecular dynamics approach to the complex structure of TNF-associated factor TRAF2.

Tumor necrosis factor receptor-associated factor proteins (TRAFs) are trimeric proteins that play a fundamental role in signaling, acting as intermediaries between the tumor necrosis factor (TNF) receptors and the proteins that transmit the downstream signal. The monomeric subunits of all the TRAF family members share a common tridimensional structure: a C-terminal globular domain and a long coiled-coil tail characterizing the N-terminal section. In this study, the dependence of the TRAF2 dynamics on the length of its tail was analyzed .

The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor.

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation.

Polymorphism on human aromatase affects protein dynamics and substrate binding: spectroscopic evidence.

Human aromatase is a member of the cytochrome P450 superfamily, involved in steroid hormones biosynthesis. In particular, it converts androgen into estrogens being therefore responsible for the correct sex steroids balance. Due to its capacity in producing estrogens it has also been considered as a promising target for breast cancer therapy.

The Puzzling Problem of Cardiolipin Membrane-Cytochrome c Interactions: A Combined Infrared and Fluorescence Study.

The interaction of cytochrome c (cyt c) with natural and synthetic membranes is known to be a complex phenomenon, involving both protein and lipid conformational changes. In this paper, we combined infrared and fluorescence spectroscopy to study the structural transformation occurring to the lipid network of cardiolipin-containing large unilamellar vesicles (LUVs). The data, collected at increasing protein/lipid ratio, demonstrate the existence of a multi-phase process, which is characterized by: (i) the interaction of cyt c with the lipid polar heads; (ii) the lipid anchorage of the protein on the membrane surface; and (iii) a long-distance order/disorder transition of the cardiolipin acyl chains.

Protein Assembly: Defining the Strength of Protein-Protein Interactions Coupling the Theory with Experiments.

In this paper we report a procedure to analyze protein homodimer interfaces.We approached the problem by means of a topological methodology. In particular, we analyzed the subunits interface of about 50 homodimers and we have defined a few parameters that allow to organize these proteins in six different classes.

Disclosing Allostery Through Protein Contact Networks.

Proteins are located in the twilight zone between chemistry and biology, where a peculiar kind of complexity starts. Proteins are the smallest 'devices' showing a sensible adaptation to their environment by the production of appropriate behavior when facing a specific stimulus. This fact qualifies (from the 'effector' side) proteins as nanomachines working as catalysts, motors, or switches.

Allostery: The Rebound of Proteins.

The discovery of hemoglobin allosteric properties is briefly summarized and contextualized in the frame of the main biochemical revelations that characterized the first half of the XX century. In particular, the historical background of DNA, RNA, and protein structure research is recalled and the new role that protein-protein interaction may have on allosteric regulation is discussed.

A role of Gln596 in fine-tuning mammalian ALOX15 specificity, protein stability and allosteric properties.

His596 of human ALOX12 has been suggested to interact with the COO-group of arachidonic acid during ALOX catalysis. In mammalian ALOX15 orthologs Gln596 occupies this position and this amino acid exchange might contribute to the functional differences between the two ALOX-isoforms. To explore the role of Gln596 for ALOX15 functionality we mutated this amino acid to different residues in rabbit and human ALOX15 and investigated the impact of these mutations on structural, catalytic and allosteric enzyme properties.

Non-symmetrical structural behavior of a symmetric protein: the case of homo-trimeric TRAF2 (tumor necrosis factor-receptor associated factor 2).

The oligomeric state of TRAF2 (tumor necrosis factor-receptor associated factor 2), a TNF (tumor necrosis factor) receptor-associated factor, is crucial for membrane binding and probably plays a fundamental role in regulating the protein function in vivo. In this study we have combined molecular dynamics with the protein contact network approach to characterize the interaction of the three identical subunits of TRAF2. The average structure obtained after a 225 ns simulation reveals that two clusters of different size are formed, one of which includes almost completely two subunits, while the third monomer appears to be more independent.

Studying the TRAF2 binding to model membranes: The role of subunits dissociation.

The ability of a C-terminal truncated form of TRAF2 to bind synthetic vesicles has been quantitatively studied by steady-state fluorescence energy transfer from the protein to large unilamellar vesicles (LUVs) prepared with different lipid mixtures. The dissociation constants, the free energy of binding, and the average number of phospholipids interacting with truncated TRAF2 have been evaluated from the corresponding binding curves. The results indicate that the protein strongly interacts with the lipid bilayer, preferentially in the monomeric state.

New insight into the interaction of TRAF2 C-terminal domain with lipid raft microdomains.

In this study we provide the first evidence of the interaction of a truncated-TRAF2 with lipid raft microdomains. We have analyzed this interaction by measuring the diffusion coefficient of the protein in large and giant unilamellar vesicles (LUVs and GUVs, respectively) obtained both from synthetic lipid mixtures and from natural extracts. Steady-state fluorescence measurements performed with synthetic vesicles indicate that this truncated form of TRAF2 displays a tighter binding to raft-like LUVs with respect to the control (POPC-containing LUVs), and that this process depends on the protein oligomeric state.

Exploring the stability of dimers through protein structure topology.

Protein homodimers pose some intriguing questions about the relation between structure and stability. We approached the problem by means of a topological methodology based on protein contact networks. We correlated local interface descriptors with structure and energy global properties of the systems under analysis.

TNFR-Associated Factor-2 (TRAF2): Not Only a Trimer.

TNF receptor-associated factors (TRAFs) are characterized by an oligomeric structure that plays a fundamental role in the binding process with membrane receptors. In this work, we studied the trimer-to-monomer (T ↔ 3M) equilibrium transition of the TRAF2 C-terminal domain using both chemical (dilution/guanidinium hydrochloride) and mechanical stress (high pressure) to induce the dissociation of the native protein into subunits. The experimental results and computer simulations indicate that stable monomers exist and that their population accounts for 15% of the total TRAF2 molecules already at a physiological intracellular concentration (≈1 μM), being instead the predominant species in the nanomolar concentration range.

Dynamics and flexibility of human aromatase probed by FTIR and time resolved fluorescence spectroscopy.

Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site.

Modules identification in protein structures: the topological and geometrical solutions.

The identification of modules in protein structures has major relevance in structural biology, with consequences in protein stability and functional classification, adding new perspectives in drug design. In this work, we present the comparison between a topological (spectral clustering) and a geometrical (k-means) approach to module identification, in the frame of a multiscale analysis of the protein architecture principles. The global consistency of an adjacency matrix based technique (spectral clustering) and a method based on full rank geometrical information (k-means) give a proof-of-concept of the relevance of protein contact networks in structure determination.

Probing conformational changes in lipoxygenases upon membrane binding: fine-tuning by the active site inhibitor ETYA.

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme-membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms.

Correlation spectroscopy and molecular dynamics simulations to study the structural features of proteins.

In this work, we used a combination of fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulation methodologies to acquire structural information on pH-induced unfolding of the maltotriose-binding protein from Thermus thermophilus (MalE2). FCS has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.

Role of Arg403 for thermostability and catalytic activity of rabbit 12/15-lipoxygenase.

12/15-Lipoxygenases (12/15-LOX) have been implicated in inflammatory and hyperproliferative diseases but the numerous aspects of structural biology of these enzymes are far from clear. Early mutagenesis data and structural modeling of enzyme-substrate complexes suggested that Arg403, which is localized at the entrance of the putative substrate binding pocket, might interact with the fatty acid carboxylic group. On the other hand, side-chain of Arg403 is a part of an ionic network with the residues of α2-helix, which undergoes pronounced conformation changes upon inhibitor binding.

Rat and human fatty acid amide hydrolases: overt similarities and hidden differences.

Fatty acid amide hydrolase (FAAH) is a membrane protein that plays a relevant role in the metabolism of fatty acid amides and esters. It degrades important neurotransmitters such as oleamide and anandamide, and it has been involved in a number of human pathological conditions, representing therefore a valuable target for biochemical and pharmacological research. In this study, we have investigated in vitro the structure-function relationship of rat and human FAAHs.

Conversion of cytochrome c into a peroxidase: inhibitory mechanisms and implication for neurodegenerative diseases.

A further function of cytochrome c (cyt c), beyond respiration, is realized outside mitochondria in the apoptotic program. In the early events of apoptosis, the interaction of cyt c with a mitochondrion-specific phospholipid, cardiolipin (CL), brings about a conformational transition of the protein and acquirement of peroxidase activity. The hallmark of cyt c with peroxidase activity is its partial unfolding accompanied by loosening of the Fe sixth axial bond and an enhanced access of the heme catalytic site to small molecules like H2O2.

Ligand-induced formation of transient dimers of mammalian 12/15-lipoxygenase: a key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium.

Tight association of N-terminal and catalytic subunits of rabbit 12/15-lipoxygenase is important for protein stability and catalytic activity.

12/15-Lipoxygenases (12/15-LOXs) have been implicated in inflammatory and hyperproliferative diseases but the structural biology of these enzymes is not well developed. Most LOXs constitute single polypeptide chain proteins that fold into a two-domain structure. In the crystal structure the two domains are tightly associated, but small angle X-ray scattering data and dynamic fluorescence studies suggested a high degree of structural flexibility involving movement of the N-terminal domain relative to catalytic subunit.

Characterization of monomeric substates of ascorbate oxidase.

Ascorbate oxidase (AAO) is a large, multidomain, dimeric protein whose folding/unfolding pathway is characterized by a complex, multistep process. Here we used fluorescence correlation spectroscopy to demonstrate the formation of partially folded monomers by pH-induced full dissociation into subunits. Hence, the structural features of monomeric AAO could be studied by fluorescence and CD spectroscopy.