Molecular and kinetic properties of copper nitrite reductase from Sinorhizobium meliloti 2011 upon substituting the interfacial histidine ligand coordinated to the type 2 copper active site for glycine.

A copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide in the denitrifier Sinorhizobium meliloti 2011 (SmNirK), a microorganism used as bioinoculant in alfalfa seeds. Wild type SmNirK is a homotrimer that contains two copper centers per monomer, one of type 1 (T1) and other of type 2 (T2). T2 is at the interface of two monomers in a distorted square pyramidal coordination bonded to a water molecule and three histidine side chains, H171 and H136 from one monomer and H342 from the other. We report the molecular, catalytic, and spectroscopic properties of the SmNirK variant H342G, in which the interfacial H342 T2 ligand is substituted for glycine. The molecular properties of H342G are similar to those of wild type SmNirK. Fluorescence-based thermal shift assays and FTIR studies showed that the structural effect of the mutation is only marginal. However, the kinetic reaction with the physiological electron donor was significantly affected, which showed a âˆ¼ 100-fold lower turnover number compared to the wild type enzyme. UV-Vis, EPR and FTIR studies complemented with computational calculations indicated that the drop in enzyme activity are mainly due to the void generated in the protein substrate channel by the point mutation. The main structural changes involve the filling of the void with water molecules, the direct coordination to T2 copper ion of the second sphere aspartic acid ligand, a key residue in catalysis and nitrite sensing in NirK, and to the loss of the 3 N-O coordination of T2.

Interaction of the antibiotic peptide nisin with anionic membranes in different phase-states: a vibrational study.

Interactions between the antibiotic peptide nisin and multilamellar vesicles of phosphoglycerol lipids in different phase-states were studied using vibrational spectroscopy. The infrared amide I' band of nisin, both in solution and in the membrane-bound state, was analyzed in the temperature range comprised between 20 and 60 °C in order to study its conformational behavior. Nisin presented mainly unordered and ß-turns conformations. Their relative populations varied according to the environment and as the temperature increased: ß turns were more favored in the membrane-bound state than in solution, but at higher temperatures the disordered conformation was dominant in both states. Spectral changes of specific infrared bands belonging to the hydrocarbon and polar moieties of lipids were also analyzed to evaluate the perturbation of the lipid membrane order. Nisin interactions with the membrane polar region induced a high restriction to water incorporation, promoting a small increase in the temperature of the lipid phase transition. Raman spectra of nisin/phosphoglycerol systems at ambient temperature were also analyzed. They revealed that the peptide incorporation to a membrane in the fluid phase caused drastic structural modifications in the hydrophobic region of the bilayer. Although nisin may be able to disrupt the hydrophobic portion of the bilayer in the gel phase, the most of the peptide molecule remained at the membrane surface interacting with the polar headgroups. This work provides evidence of a differential effect of nisin on anionic membranes, depending on the phase-state of the lipid.

Thermal unfolding of calreticulin. Structural and thermodynamic characterization of the transition.

Calreticulin (CRT) is a calcium-binding protein that participates in several cellular processes including the control of protein folding and homeostasis of Ca2+. Its folding, stability and functions are strongly controlled by the presence of Ca2+. The oligomerization state of CRT is also relevant for its functions. We studied the thermal transitions of monomers and oligomers of CRT by differential scanning calorimetry (DSC), circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) in the presence and absence of Ca2+. We found three and two components for the calorimetric transition in the presence and absence of Ca2+ respectively. The presence of several components was also supported by CD and FTIR spectra acquired as a function of the temperature. The difference between the heat capacity of the native and the unfolded state strongly suggests that interactions between protein domains also contribute to the heat uptake in a calorimetry experiment. We found that once unfolded at high temperature the process is reversible and the native state can be recovered upon cooling only in the absence of Ca2+. We also propose a new simple method to obtain pure CRT oligomers.

Relevance of the protein macrodipole in the membrane-binding process. Interactions of fatty-acid binding proteins with cationic lipid membranes.

The fatty acid-binding proteins L-BABP and Rep1-NCXSQ bind to anionic lipid membranes by electrostatic interactions. According to Molecular Dynamics (MD) simulations, the interaction of the protein macrodipole with the membrane electric field is a driving force for protein binding and orientation in the interface. To further explore this hypothesis, we studied the interactions of these proteins with cationic lipid membranes. As in the case of anionic lipid membranes, we found that both proteins, carrying a negative as well as a positive net charge, were bound to the positively charged membrane. Their major axis, those connecting the bottom of the ß-barrel with the α-helix portal domain, were rotated about 180 degrees as compared with their orientations in the anionic lipid membranes. Fourier transform infrared (FTIR) spectroscopy of the proteins showed that the positively charged membranes were also able to induce conformational changes with a reduction of the ß-strand proportion and an increase in α-helix secondary structure. Fatty acid-binding proteins (FABPs) are involved in several cell processes, such as maintaining lipid homeostasis in cells. They transport hydrophobic molecules in aqueous medium and deliver them into lipid membranes. Therefore, the interfacial orientation and conformation, both shown herein to be electrostatically determined, have a strong correlation with the specific mechanism by which each particular FABP exerts its biological function.

Conformational changes, from ß-strand to α-helix, of the fatty acid-binding protein ReP1-NCXSQ in anionic lipid membranes: dependence with the vesicle curvature.

We studied the conformational changes of the fatty acid-binding protein ReP1-NCXSQ in the interface of anionic lipid membranes. ReP1-NCXSQ is an acidic protein that regulates the activity of the Na+/Ca2+ exchanger in squid axon. The structure is a flattened barrel composed of two orthogonal ß-sheets delimiting an inner cavity and a domain of two α-helix segments arranged as a hairpin. FTIR and CD spectroscopy showed that the interactions with several anionic lipids in the form of small unilamellar vesicles (SUVs) induced an increase in the proportion of helix secondary structure. Lower amount or no increase in α-helix was observed upon the interaction with anionic lipids in the form of large unilamellar vesicles (LUVs). The exception was 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) that was equally efficien to to induce the conformational change both in SUVs and in LUVs. In solution, the infrared spectra of ReP1-NCXSQ at temperatures above the unfolding displayed a band at 1617 cm-1 characteristic of aggregated strands. This band was not observed when the protein interacted with DMPG, indicating inhibition of aggregation in the interface. Similarly to the observed in L-BABP, another member of the fatty acid binding proteins, a conformational change in ReP1-NCXSQ was coupled to the gel to liquid-crystalline lipid phase transition.

Molecular Insight into the Line Tension of Bilayer Membranes Containing Hybrid Polyunsaturated Lipids.

Line tension (γ) is a key parameter for the structure and dynamics of membrane domains. It was proposed that hybrid lipids, with mixed saturated and unsaturated acyl chains, participate in the relaxation of γ through different mechanisms. In this work, we used molecular dynamics simulations of the coarse-grained MARTINI model to measure γ in liquid-ordered-liquid-disordered (Lo-Ld) membranes, with increasingly larger relative proportion of the hybrid polyunsaturated lipid PAPC (4:0-5:4PC) to DAPC (di5:4PC) (i.e., XH). We also calculated an elastic contribution to γ by the Lo-Ld thickness mismatch, tilt moduli, and bending moduli, as predicted by theory. We found that an increase in XH decreased the overall γ value and the elastic contribution to line tension. The effect on the elastic line tension is driven by a reduced hydrophobic mismatch. Changes in the elastic constants of the phases due to an increase in XH produced a slightly larger elastic γ term. In addition to this elastic energy, other major contributions to γ are found in these model membranes. Increasing XH decreases both elastic and nonelastic contributions to γ. Finally, PAPC also behaves as a linactant, relaxing γ through an interfacial effect, as predicted by theoretical results. This study gives insight into the actual contribution of distinct energy terms to γ in bilayers containing polyunsaturated hybrid lipids.

Interactions of the fatty acid-binding protein ReP1-NCXSQ with lipid membranes. Influence of the membrane electric field on binding and orientation.

The regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ) is a 15kDa soluble, intracellular protein that regulates the activity of the Na(+)/Ca(2+) exchanger in the squid axon. It is a member of the cellular retinoic acid-binding proteins family and the fatty acid-binding proteins superfamily. It is composed of ten beta strands defining an inner cavity and a domain of two short alpha helix segments. In this work, we studied the binding and orientation of ReP1-NCXSQ in anionic and zwitterionic lipid membranes using molecular dynamics (MD) simulations. Binding to lipid membranes was also measured by filtration binding assay. ReP1-NCXSQ acquired an orientation in the anionic membranes with the positive end of the macrodipole pointing to the lipid membrane. Potential of mean force calculations, in agreement with experimental measurements, showed that the binding to the anionic interfaces in low ionic strength was stronger than the binding to anionic interfaces in high ionic strength or to zwitterionic membranes. The results of MD showed that the electrostatic binding can be mediated not only by defined patches or domains of basic residues but also by a global asymmetric distribution of charges. A combination of dipole-electric field interaction and local interactions determined the orientation of ReP1-NCXSQ in the interface.

Calreticulin-dimerization induced by post-translational arginylation is critical for stress granules scaffolding.

Protein arginylation mediated by arginyl-tRNA protein transferase is a post-translational modification that occurs widely in biology, it has been shown to regulate protein and properties and functions. Post-translational arginylation is critical for embryogenesis, cardiovascular development and angiogenesis but the molecular effects of proteins arginylated in vivo are largely unknown. In the present study, we demonstrate that arginylation reduces CRT (calreticulin) thermostability and induces a greater degree of dimerization and oligomerization. R-CRT (arginylated calreticulin) forms disulfide-bridged dimers that are increased in low Ca(2+) conditions at physiological temperatures, a similar condition to the cellular environment that it required for arginylation of CRT. Moreover, R-CRT self-oligomerizes through non-covalent interactions that are enhanced at temperatures above 40 °C, condition that mimics the heat shock treatment where R-CRT is the only isoespecies of CRT that associates in cells to SGs (stress granules). We show that in cells lacking CRT the scaffolding of larger SGs is impaired; the transfection with CRT (hence R-CRT expression) restores SGs assembly whereas the transfection with CRT mutated in Cys146 does not. Thus, R-CRT disulfide-bridged dimers (through Cys146) are essential for the scaffolding of larger SGs under heat shock, although these dimers are not required for R-CRT association to SGs. The alteration in SGs assembly is critical for the normal cellular recover of cells after heat induced stress. We conclude that R-CRT is emerging as a novel protein that has an impact on the regulation of SGs scaffolding and cell survival.

Conformational changes of ß2-human glycoprotein I and lipid order in lipid-protein complexes.

We studied the conformation of ß2-human glycoprotein (ß2GPI) in solution and bound to the anionic lipids palmitoyl oleoyl phosphatidylglycerol (POPG), dimiristoyl phosphatidylglycerol (DMPG) and dipalmitoyl phosphatidylglycerol (DPPG) as a function of the temperature. We used the infrared amide I' band to study the protein conformation, and the position of the antisymmetric stretching band of the methylene groups in the lipid hydrocarbon chains to study the lipid order. Lipid-protein complexes were studied in media of low and high ionic strengths. In solution, ß2GPI displayed a conformational pre-transition in the range 47-50°C, characterized by a shift in the band of ß secondary structure, previous to the main unfolding at 64°C. When the protein was bound to the anionic lipid membranes at 25°C, a similar shift as in the pre-transition in solution was observed, together with an increase in the band corresponding to α-helix secondary structure. Lipid-protein complexes formed large aggregates within the temperature range 10≅60°C. At temperatures above the protein unfolding, the complexes were disrupted to yield vesicles with bound protein. This finding indicated that the native fold was required for the formation of the lipid-protein aggregates. Cycles of heating and cooling showed hysteresis in the formation of aggregates.

Kinetics of lipid-membrane binding and conformational change of L-BABP.

We designed an experimental approach to differentiate the kinetics of protein binding to a lipid membrane from the kinetics of the associated conformational change in the protein. We measured the fluorescence intensity of the single Trp6 in chicken liver bile acid-binding protein (L-BABP) as a function of time after mixing the protein with lipid membranes. We mixed the protein with pure lipid membranes, with lipid membranes in the presence of a soluble quencher, and with lipid membranes containing a fluorescence quencher attached to the lipid polar head group. We fitted simultaneously the experimental curves to a three-state kinetic model. We conclude that in a first step, the binding of L-BABP to the interfacial region of the anionic lipid polar head groups occurred simultaneously with a conformational change to the partly unfolded state. In a second slower step, Trp6 buried within the polar head group region, releasing contacts with the aqueous phase.

Fine carbohydrate recognition of Euphorbia milii lectin.

Glycans are key structures involved in biological processes such as cell attachment, migration, and invasion. Information coded on cell-surface glycans is frequently deciphered by proteins, as lectins, that recognize specific carbohydrate topology. Here, we describe the fine carbohydrate specificity of Euphorbia milii lectin (EML). Competitive assays using various sugars showed that GalNAc was the strongest inhibitor, and that the hydroxyl axial position of C4 and acetamido on C2 of GalNAc are critical points of EML recognition. A hydrophobic locus adjacent to GalNAc is also an important region for EML binding. Direct binding assays of EML revealed a stereochemical requirement for a structure adjacent to terminal GalNAc, showing that GalNAc residue is a necessary but not sufficient condition for EML interaction. The capacity of EML to bind epithelial tumor cells makes it a potentially useful tool for study of some over-expressed GalNAc glycoconjugates.

Chicken liver bile acid-binding protein is in a compact partly folded state at acidic pH. Its relevance to the interaction with lipid membranes.

Chicken liver bile acid-binding protein (formerly known as chicken liver basic fatty acid-binding protein) binds to anionic lipid membranes acquiring a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B., and Montich, G. (2003) Biochim. Biophys. Acta 1611, 98-106]. To understand the mechanisms of its interactions with membranes, we have investigated the presence of partly folded states in solution. Using fluorescence spectroscopy of the single Trp residue, circular dichroism in the far- and near-UV, Fourier transform infrared spectroscopy, and size-exclusion chromatography, we found that L-BABP was partly unfolded at pH 2.5 and low ionic strength, retaining some of its secondary structure. Addition of 0.1 M NaCl at pH 2.5 or decreasing the pH to 1.5 produced a more compact partly folded state, with a partial increase of secondary structure and none of tertiary structure. Fluorescence emission spectra of this state indicate that the Trp residue is within an environment of low polarity, similar to the native state. This environment is not produced by the insertion of the Trp into soluble aggregates as revealed by size-exclusion chromatography, fluorescence anisotropy, and infrared spectroscopy. The presence of partly folded states under acidic conditions in solution suggests the possibility that membrane binding of L-BABP occurs via this state.

Conformational flexibility of avidin: the influence of biotin binding.

Ligand binding to proteins is a key process in cell biochemistry. The interaction usually induces modifications in the unfolding thermodynamic parameters of the macromolecule due to the coupling of unfolding and binding equilibria. In addition, these modifications can be attended by changes in protein structure and/or conformational flexibility induced by ligand binding. In this work, we have explored the effect of biotin binding on conformation and dynamic properties of avidin by using infrared spectroscopy including kinetics of hydrogen/deuterium exchange. Our results, along with previously thermodynamic published data, indicate a clear correlation between thermostability and protein compactness. In addition, our results also help to interpret the thermodynamic binding parameters of the exceptionally stable biotin:AVD complex.

Molecular dynamics simulation study of the interaction of trehalose with lipid membranes.

The interactions of the cryoprotective agent trehalose with a lipid membrane made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine at 323 K were studied by means of molecular dynamics simulations. It was observed that trehalose binds to the phospholipid headgroups with its main axis parallel to the membrane normal. Trehalose establishes hydrogen bonds with the carbonyl and phosphate groups and replaces water molecules from the lipid headgroup. Notably, the number of hydrogen bonds (HBs) that the membrane made with its environment was conserved after trehalose binding. The HBs between lipid and trehalose have a longer lifetime than those established between lipid and water. The binding of the sugar does not produce changes either in the lipid area or in the lipid order parameter. The effect of trehalose on the dipole potential is in agreement with experimental results. The contribution of the different components to the membrane dipole potential was analyzed. It was observed that the binding of trehalose produces changes in the different components and the sugar itself contributes to the surface potential due to the polarization of its hydroxyl in the interface.

Protein stability induced by ligand binding correlates with changes in protein flexibility.

The interaction between ligands and proteins usually induces changes in protein thermal stability with modifications in the midpoint denaturation temperature, enthalpy of unfolding, and heat capacity. These modifications are due to the coupling of unfolding with binding equilibrium. Furthermore, they can be attained by changes in protein structure and conformational flexibility induced by ligand interaction. To study these effects we have used bovine serum albumin (BSA) interacting with three different anilinonaphthalene sulfonate derivatives (ANS). These ligands have different effects on protein stability, conformation, and dynamics. Protein stability was studied by differential scanning calorimetry and fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism and infrared spectroscopy including kinetics of hydrogen/deuterium exchange. The order of calorimetric midpoint of denaturation was: 1,8-ANS-BSA > 2,6-ANS-BSA > free BSA >> (nondetected) bis-ANS-BSA. Both 1,8-ANS and 2,6-ANS did not substantially modify the secondary structure of BSA, whereas bis-ANS induced a distorted alpha-helix conformation with an increase of disordered structure. Protein flexibility followed the order: 1,8-ANS-BSA < 2,6-ANS-BSA < free BSA << bis-ANS-BSA, indicating a clear correlation between stability and conformational flexibility. The structure induced by an excess of bis-ANS to BSA is compatible with a molten globule-like state. Within the context of the binding landscape model, we have distinguished five conformers (identified by subscript): BSA(1,8-ANS), BSA(2,6-ANS), BSA(free), BSA(bis-ANS), and BSA(unfolded) among the large number of possible states of the conformational dynamic ensemble. The relative population of each distinguishable conformer depends on the type and concentration of ligand and the temperature of the system.

Interactions of chicken liver basic fatty acid-binding protein with lipid membranes.

The interactions of chicken liver basic fatty acid-binding protein (Lb-FABP) with large unilamellar vesicles (LUVs) of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidylglycerol (POPG) were studied by binding assays, Fourier transform infrared (FT-IR) spectroscopy, monolayers at air-water interface, and low-angle X-ray diffraction. Lb-FABP binds to POPG LUVs at low ionic strength but not at 0.1 M NaCl. The infrared (IR) spectra of the POPG membrane-bound protein showed a decrease of the band corresponding to beta-structures as compared to the protein in solution. In addition, a cooperative decrease of the beta-edge band above 70 degrees C in solution was also evident, while the transition was less cooperative and took place at lower temperature for the POPG membrane-bound protein. Low- and wide-angle X-ray diffraction experiments with lipid multilayers indicate that binding of the protein produces a rearrangement of the membrane structure, increasing the interlamellar spacing and decreasing the compactness of the lipids.

Vanadate inhibits the ATPase activity and DNA binding capability of bacterial MutS. A structural model for the vanadate-MutS interaction at the Walker A motif.

MutS, a member of the ABC ATPases superfamily, is a mismatch DNA-binding protein constituent of the DNA post-replicative mismatch repair system (MMRS). In this work, it is shown that the ATPase activity of Pseudomonas aeruginosa and Escherichia coli MutS is inhibited by ortho- and decavanadate. Structural comparison of the region involved in the ATP binding of E.coli MutS with the corresponding region of other ABC ATPases inhibited by vanadate, including the myosin- orthovanadate-Mg complex, showed that they are highly similar. From these results it is proposed that the orthovanadate inhibition of MutS ATPase can take place by a similar mechanism to that described for other ATPases. Docking of decavanadate on the ATP-binding region of MutS showed that the energetically more favorable interaction of this compound would take place with the complex MutS- ADP-Mg, suggesting that the inhibitory effect could be produced by a steric impediment of the protein ATP/ADP exchange. Besides the effect observed on the ATPase activity, vanadate also affects the DNA-binding capability of the protein, and partially inhibits the oligomerization of MutS and the temperature-induced inactivation of the protein. From the results obtained, and considering that vanadate is an intracellular trace component, this compound could be considered as a new modulator of the MMRS.