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Septins comprise a family of proteins involved in a variety of cellular processes and related to several human pathologies. They are constituted by three structural domains: the N- and C-terminal domains, highly variable in length and composition, and the central domain, involved in the guanine nucleotide (GTP) binding. Thirteen different human septins are known to form heterogeneous complexes or homofilaments, which are stabilized by specific interactions between the different interfaces present in the domains. In this work, we have investigated by in-solution small-angle x-ray scattering the structural and thermodynamic properties of a human septin 3 construct, SEPT3-GC, which contains both of both interfaces (G and NC) responsible for septin-septin interactions. In order to shed light on the role of these interactions, small-angle x-ray scattering measurements were performed in a wide range of temperatures, from 2 up to 56°C, both with and without a nonhydrolysable form of GTP (GTPγS). The acquired data show a temperature-dependent coexistence of monomers, dimers, and higher-order aggregates that were analyzed using a global fitting approach, taking into account the crystallographic structure of the recently reported SEPT3 dimer, PDB:3SOP. As a result, the enthalpy, entropy, and heat capacity variations that control the dimer-monomer dissociation equilibrium in solution were derived and GTPγS was detected to increase the enthalpic stability of the dimeric species. Moreover, a temperature increase was observed to induce dissociation of SEPT3-GC dimers into monomers just preceding their reassembling into amyloid aggregates, as revealed by the Thioflavin-T fluorescence assays.

The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U1 unfolded state, whereas the successive addition of Cl− ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A1 and A2 states, respectively). A second unfolded state (U2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle x-ray scattering. The results show that in the acidic-unfolded U1 form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U1 appear structurally different: in the A1 state cyt-c is dimeric and less compact, whereas in the A2 form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.

Small-angle neutron and x-ray scattering experiments have been performed on type 2 tissular transglutaminase to characterize the conformational changes that bring about Ca(2+) activation and guanosine triphosphate (GTP) inhibition. The native and a proteolyzed form of the enzyme, in the presence and in the absence of the two effectors, were considered. To describe the shape of transglutaminase in the different conformations, a Monte Carlo method for calculating small-angle neutron scattering profiles was developed by taking into account the computer-designed structure of the native transglutaminase, the results of the Guinier analysis, and the essential role played by the solvent-exposed peptide loop for the conformational changes of the protein after activation. Although the range of the neutron scattering data is rather limited, by using the Monte Carlo analysis, and because the structure of the native protein is available, the distribution of the protein conformations after ligand interaction was obtained. Calcium activation promotes a rotation of the C-terminal with respect to the N-terminal domain around the solvent-exposed peptide loop that connects the two regions. The psi angle between the longest axes of the two pairs of domains is found to be above 50 degrees, larger than the psi value of 35 degrees calculated for the native transglutaminase. On the other hand, the addition of GTP makes possible conformations characterized by psi angles lower than 34 degrees. These results are in good agreement with the proposed enzyme activity regulation: in the presence of GTP, the catalytic site is shielded by the more compact protein structure, while the conformational changes induced by Ca(2+) make the active site accessible to the substrate.

Deoxyguanosine-5′-monophosphate in water self-associates into stable structures, which include liquid-crystalline hexagonal and cholesteric phases. The structural unit is a four-stranded helix, composed of stacked Hoogsteen-bonded guanosine quartets. By using the osmotic stress method, we recently measured the force between helices in KCl solutions up to 2M. In addition to the long-range electrostatic force, a short-range hydration repulsive contribution was recognized. The hydration repulsion is exponential, and shows a decay length independent from the ionic strength of the solution. Here, we report that more concentrated KCl solutions cause condensation of the guanosine helix in a hexagonal phase with constant equilibrium separation of ∼7A between helix surfaces. Long-range attraction, which induces the self-assembly, and short-range repulsion, which prevents the contact between the helices, are implied. By using osmotic stress, the force needed to push helices closer from the spontaneously assumed position has been measured. The attractive force was then estimated as a difference between the net force and the repulsive contribution, revealing an exponential decay length about two times larger than that of the short-range repulsion. The agreement with the helix interaction theory introduced recently by Kornyshev and Leikin (Kornyshev, A. A., and S. Leikin, 1997. Theory of interaction between helical molecules. J . Phys . Chem . 107:3656–3674) suggests that the repulsive and attractive forces originate from helix-specific interactions.

Septins are proteins from the GTP-binding family and participate in cell division cycle performing functions such as secretion and cytoskeletal division. They can also be found in neurodegenerative conditions as Alzheimer's and Parkinson's diseases, forming highly organized fiber-like aggregates known as amyloids. In this work, we used small angle x-ray scattering (SAXS) to investigate the formation and time evolution of septins aggregates under the influence of temperature and concentration.The SAXS measurements were performed with the GTPase domain of human Septin 2 (SEPT2G) at 0.5 and 1 mg/mL and temperatures from 4 to 45°C. At 4°C, our results demonstrate that SEPT2G is self-aggregated as a dimer at 0.5mg/mL, whereas dimers coexist with cylinder-like aggregates (36 nm-long and 12 nm-cross section) at 1mg/mL. At this temperature, the protein does not evolve over one hour of observation.As the temperature was increased to 15°C we verified that, initially coexisting with the protein dimer and cylinders, a small amount of larger aggregates is also present in solution. However, the number of very large aggregates increases with time concomitantly with the decrease of cylinder amount in the solution. Analyzing the samples at 37°C it's not possible to observe cylinders anymore and the amount of dimers decreases from 50% to 20% in less than 1 hour. For 45°C this effect is even more accentuated: the percentage of dimers is only 6% in solution.In conclusion, our results showed the coexistence of dimers of SEPT2G with small fibers and larger aggregates in solution that evolve not only with concentration and temperature but also with time.This work is supported by FAPESP and CNPq.The authors thank the LNLS SAXS beam line staff.

Lipid-protein interactions are regarded as one of the key factors in several biophysical relevant processes. In the present work, we studied the influence of Cytochrome-c (Cyto-c) on unilamellar vesicles composed by mixing an anionic lipid (the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, POPG, or the cardiolipin, CL) with the zwitterionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), by means of small-angle X-ray scattering. Cyto-c is a component of the electron transport chain, where it has a pivotal role in transferring electrons in the inner mitochondrial membrane (IMM), which is composed of a significant amount of anionic lipids. CL is a structurally unique anionic phospholipid, containing two phosphatidylglycerol groups and four acyl chains and it is found predominantly in the IMM. The interaction of Cyto-c and CL-containing mixed vesicles has been extensively studied, and it is appointed as the major responsible for the role of Cyto-c in the cell respiration and also in the cell programmed death (apoptosis), as recently evidenced. Present results show that, in the absence of anionic lipids, Cyto-c is not able to change the structural features of POPC unilamellar vesicles. In the mixed systems, however, Cyto-c induces the formation of a multilamellar vesicles, with a bilayer-bilayer repetition distance of 11 nm, circa 4 nm larger than the staking of POPC multilamellar vesicles. This information indicates that Cyto-c, a globular protein with 3.0 nm of diameter, is probably located among the bilayers. Furthermore, such effect takes place as soon as Cyto-c is mixed in the system, as confirmed by absorption spectroscopy. The time-evolution of such process was also investigated and, interestingly, it is faster in POPG-containing vesicles than in the CL ones. Moreover, the number of staked bilayers is larger in the POPG systems.

Phase behaviour and structural properties of monoacylglycerides in water have been investigated for a long time, due to their extended polymorphism. In particular, monoolein (MO) in water shows several mesophases, characterized by a high disordered conformation of the hydrocarbon chains. At such conditions, an embedded protein can influence the physical properties of the lipid matrix, depending on the protein size and polarity. We take advantage of the structural properties of monoolein and cytochrome-c to extensively study the temperature effects on the cubic transition from Pn3m to Im3m by means of small-angle X-ray scattering technique (SAXS) and electronic absorption spectroscopy (EAS). To do so, we made samples composed of monoolein (50 mg/ml) in the presence of 1, 10 and 50 mg/ml of cytochrome-c. Ours preliminary SAXS results indicate that cyto-c is able to change the monoolein water channels, from cubic Pn3m to Im3m. Moreover, such kinetic behaviour is too slow, taking place within some days. EAS measurements indicate that the incorporation of cyto-c within the Monoolein water channels begins after two or three days (after the sample preparation). Besides, after one week of sample preparation the amount of cyto-c within the Monoolein water channels is equal to 60% and 34% for 1 and 10 mg/ml of cyto-c, respectively. Interestingly, increasing the temperature, the unity cell parameter decreases, indicating that water is going out from the unity cell and the symmetry of the liquid crystal phase can changes to another cubic or to hexagonal, depending on the temperature and the sample composition. We believe that these results could bring more insights on the protein-liquid crystal interaction.

The effect of GuHCl and of NaCl on the structural properties of the hemocyanin (Hc) from Carcinus aestuarii has been studied by small angle x-ray scattering (SAXS) using synchrotron radiation. SAXS data collected as a function of perturbant concentration have been used to analyze conformational states of hexameric holo and apoHc as well as the holo and apoforms of the monomeric subunit CaeSS2. In the case of the holoprotein in GuHCl, two concentration domains were identified: at lower concentration, the perturbant induces aggregation of Hc molecules, whereas at higher concentration the aggregates dissociate with concomitant denaturation of the protein. In contrast, with apoHc the denaturation occurs at rather low GuHCl, pointing to an important effect of the active site bound copper for the stabilization of Hc tertiary structure. The effects of NaCl are similar to those of GuHCl as far as CaeSS2 is concerned, namely oligomerization precedes denaturation, whereas in the case of the hexameric form no aggregation occurs. To improve data analysis, on the basis of the current models for Hc monomers and oligomers, the fraction of each aggregation state and/or unfolded protein has been determined by fitting experimental SAXS curves with form factors calculated from Monte Carlo methods. In addition, a global analysis has been carried out on the basis of a thermodynamic model involving an equilibrium between a monomer in a nativelike and denatured form as well as a class of equilibria among the monomer and other aggregates.

The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of approximately 35-45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0-9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(1), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some re-folding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects.