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The role of magnetosome associated proteins on the in vitro synthesis of magnetite nanoparticles has gained interest, both to obtain a better understanding of the magnetosome biomineralization process and to be able to produce novel magnetosome-like biomimetic nanoparticles. Up to now, only one recombinant protein has been used at the time to in vitro form biomimetic magnetite precipitates, being that a scenario far enough from what probably occurs in the magnetosome. In the present study, both Mms6 and MamC from Magnetococcus marinus MC-1 have been used to in vitro form biomimetic magnetites. Our results show that MamC and Mms6 have different, but complementary, effects on in vitro magnetite nucleation and growth. MamC seems to control the kinetics of magnetite nucleation while Mms6 seems to preferably control the kinetics for crystal growth. Our results from the present study also indicate that it is possible to combine both proteins to tune the properties of the resulting biomimetic magnetites. In particular, by changing the relative ratio of these proteins, better faceted and/or larger magnetite crystals with, consequently, different magnetic moment per particle could be obtained. This study provides with tools to obtain new biomimetic nanoparticles with a potential utility for biotechnological applications.

Understanding the early molecular mechanisms governing amyloid aggregation is crucial to learn how to prevent it. Here, we used a site-directed mutagenesis approach to explore the molecular mechanism of nucleation of amyloid structure in the N47A Spc-SH3 domain. The changes in the native state stability produced by a series of mutations on each structural element of the domain were uncorrelated with the changes in the aggregation rates, although the overall aggregation mechanism was not altered. Analysis of the thioflavin T initial rates based on a simple kinetic model allowed us to extract thermodynamic magnitudes of the precursor states of nucleation and map the regions of the protein participating in the structure of the amyloidogenic precursors. This structure differs from that of the folding transition state of the SH3 domains, strongly suggesting that the regions of the conformational landscape leading to amyloid formation are divergent from those leading to the native fold.

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination.

The CD2AP (CD2-associated protein) and CIN85 (Cbl-interacting protein of 85 kDa) adaptor proteins each employ three Src homology 3 (SH3) domains to cluster protein partners and ensure efficient signal transduction and down-regulation of tyrosine kinase receptors. Using NMR, isothermal titration calorimetry and small-angle X-ray scattering methods, we have characterized several binding modes of the N-terminal SH3 domain (SH3A) of CD2AP and CIN85 with two natural atypical proline-rich regions in CD2 (cluster of differentiation 2) and Cbl-b (Casitas B-lineage lymphoma), and compared these data with previous studies and published crystal structures. Our experiments show that the CD2AP-SH3A domain forms a type II dimer with CD2 and both type I and type II dimeric complexes with Cbl-b. Like CD2AP, the CIN85-SH3A domain forms a type II complex with CD2, but a trimeric complex with Cbl-b, whereby the type I and II interactions take place at the same time. Together, these results explain how multiple interactions among similar SH3 domains and ligands produce a high degree of diversity in tyrosine kinase, cell adhesion or T-cell signaling pathways., (© 2013 FEBS.)

Understanding the earliest molecular events during nucleation of the amyloid aggregation cascade is of fundamental significance to prevent amyloid related disorders. We report here an experimental kinetic analysis of the amyloid aggregation of the N47A mutant of the α-spectrin SH3 domain (N47A Spc-SH3) under mild acid conditions, where it is governed by rapid formation of amyloid nuclei. The initial rates of formation of amyloid structures, monitored by thioflavine T fluorescence at different protein concentrations, agree quantitatively with high-order kinetics, suggesting an oligomerization pre-equilibrium preceding the rate-limiting step of amyloid nucleation. The curves of native state depletion also follow high-order irreversible kinetics. The analysis is consistent with the existence of low-populated and heterogeneous oligomeric precursors of fibrillation that form by association of partially unfolded protein monomers. An increase in NaCl concentration accelerates fibrillation but reduces the apparent order of the nucleation kinetics; and a double mutant (K43A, N47A) Spc-SH3 domain, largely unfolded under native conditions and prone to oligomerize, fibrillates with apparent first order kinetics. On the light of these observations, we propose a simple kinetic model for the nucleation event, in which the monomer conformational unfolding and the oligomerization of an amyloidogenic intermediate are rapidly pre-equilibrated. A conformational change of the polypeptide chains within any of the oligomers, irrespective of their size, is the rate-limiting step leading to the amyloid nuclei. This model is able to explain quantitatively the initial rates of aggregation and the observed variations in the apparent order of the kinetics and, more importantly, provides crucial thermodynamic magnitudes of the processes preceding the nucleation. This kinetic approach is simple to use and may be of general applicability to characterize the amyloidogenic intermediates and oligomeric precursors of other disease-related proteins.

CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. It contains three N-terminal SH3 domains that are able to interact among others with CD2, ALIX, c-Cbl and Ubiquitin. To understand the role of the individual SH3 domains of this adaptor protein we have performed a complete structural, thermodynamic and dynamic characterization of the separate domains using NMR and DSC. The energetic contributions to the stability and the backbone dynamics have been related to the structural features of each domain using the structure-based FoldX algorithm. We have found that the N-terminal SH3 domain of both adaptor proteins CD2AP and CIN85 are the most stable SH3 domains that have been studied until now. This high stability is driven by a more extensive network of intra-molecular interactions. We believe that this increased stabilization of N-terminal SH3 domains in adaptor proteins is crucial to maintain the necessary conformation to establish the proper interactions critical for the recruitment of their natural targets.

To understand and tackle amyloid-related diseases, it is crucial to investigate the factors that modulate amyloid formation of proteins. Our previous studies proved that the N47A mutant of the α-spectrin SH3 (Spc-SH3) domain forms amyloid fibrils quickly under mildly acidic conditions. Here, we analyze how experimental conditions influence the kinetics of assembly and the final morphology of the fibrils. Early formation of curly fibrils occurs after a considerable conformational change of the protein and the concomitant formation of small oligomers. These processes are strongly accelerated by an increase in salt concentration and temperature, and to a lesser extent by a reduction in pH. The rate-limiting step in these events has a high activation enthalpy, which is significantly reduced by an increase in NaCl concentration. At low-to-moderate NaCl concentrations, the curly fibrils convert to straight and twisted amyloid fibrils after long incubation times, but only in the presence of soluble species in the mixture, which suggests that the curly fibrils and the twisted amyloid fibrils are diverging assembly pathways. The results suggest that the influence of environmental variables on protein solvation is crucial in determining the nucleation kinetics, the pathway of assembly, and the final fibril morphology., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 muM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (K(d) = 132 +/- 13 muM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein-protein complexes.

We investigated the relationship between thermodynamic stability and amyloid aggregation propensity for a set of single mutants of the alpha-spectrin SH3 domain (Spc-SH3). Whilst mutations destabilizing the domain at position 56 did not enhance fibrillation, the N47A mutation increased the rate of amyloid fibril formation by 10-fold. Even under conditions of identical thermodynamic stability, the aggregation rate was much higher for the N47A mutant than for the WT domain. We conclude that the N47A mutation does not change the apparent mechanism of fibrillation or the morphology of the amyloid fibrils, and that its amyloidogenic property is due to its effect upon the rate of the conformational events leading to nucleation and not to its overall destabilizing effect.

Evidence is accumulating that normally folded proteins retain a significant tendency to form amyloid fibrils through a direct assembly of monomers in their native-like conformation. However, the factors promoting such processes are not yet well understood. The acylphosphatase from Sulfolobus solfataricus (Sso AcP) aggregates under conditions in which a native-like state is initially populated and forms, as a first step, aggregates in which the monomers maintain their native-like topology. An unstructured N-terminal segment and an edge beta-strand were previously shown to play a major role in the process. Using kinetic experiments on a set of Sso AcP variants we shall show that the major event of the first step is the establishment of an inter-molecular interaction between the unstructured segment of one Sso AcP molecule and the globular unit of another molecule. This interaction is determined by the primary sequence of the unstructured segment and not by its physico-chemical properties. Moreover, we shall show that the conversion of these initial aggregates into amyloid-like protofibrils is an intra-molecular process in which the Sso AcP molecules undergo conformational modifications. The obtained results allow the formulation of a model for the assembly of Sso AcP into amyloid-like aggregates at a molecular level.

CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. CD2AP interacts, as an adaptor protein, with different natural targets, such as CD2, nefrin, c-Cbl and podocin. These proteins are believed to interact to one of the three SH3 domains that are positioned in the N-terminal region of CD2AP. To understand the network of interactions between the natural targets and the three SH3 domains (SH3-A, B and C), we have started to determine the structures of the individual SH3 domains. Here we present the high-resolution structure of the SH3-C domain derived from NMR data. Full backbone and side-chain assignments were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from high-resolution 600 and 800 MHz NOESY spectra, together with phi and psi torsion angle restraints based on the analysis of 1HN, 15N, 1Halpha, 13Calpha, 13CO and 13Cbeta chemical shifts. Structures were calculated using CYANA and refined in water using RECOORD. The three-dimensional structure of CD2AP SH3-C contains all the features that are typically found in other SH3 domains, including the general binding site for the recognition of polyproline sequences.

In this work we compare the dynamics and conformational stability of Pseudomonas mendocina lipase enzyme and its F180P/S205G mutant that shows higher activity and stability for use in washing powders. Our NMR analyses indicate virtually identical structures but reveal remarkable differences in local dynamics, with striking correspondence between experimental data (i.e., (15)N relaxation and H/D exchange rates) and data from Molecular Dynamics simulations. While overall the cores of both proteins are very rigid on the pico- to nanosecond timescale and are largely protected from H/D exchange, the two point mutations stabilize helices alpha1, alpha4, and alpha5 and locally destabilize the H-bond network of the beta-sheet (beta7-beta9). In particular, it emerges that helix alpha5, undergoing some fast destabilizing motions (on the pico- to nanosecond timescale) in wild-type lipase, is substantially rigidified by the mutation of Phe180 for a proline at its N terminus. This observation could be explained by the release of some penalizing strain, as proline does not require any "N-capping" hydrogen bond acceptor in the i+3 position. The combined experimental and simulated data thus indicate that reduced molecular flexibility of the F180P/S205G mutant lipase underlies its increased stability, and thus reveals a correlation between microscopic dynamics and macroscopic thermodynamic properties. This could contribute to the observed altered enzyme activity, as may be inferred from recent studies linking enzyme kinetics to their local molecular dynamics.

The small size and lack of disulphide bonds or cofactors in the Histidine-containing phosphocarrier protein (HPr) makes it an attractive system with which to study structure, interaction to its enzymatic partners, and its stability and folding. Here we give an overview on the immense work that has been performed on this protein and we will show that HPr has been widely used as a model protein to study important aspects in modern Structural Biology.

We have used site-directed mutagenesis in combination with a battery of biophysical techniques to probe the stability and folding behavior of a small globular protein, the histidine-containing phosphocarrier protein (HPr). Specifically, the four phenylalanine residues (2, 22, 29, and 48) of the wild-type protein were individually replaced by single tryptophans, thus introducing site-specific probes for monitoring the behavior of the protein. The folding of the tryptophan mutants was investigated by NMR, DSC, CD, intrinsic fluorescence, fluorescence anisotropy, and fluorescence quenching. The heat-induced denaturation of all four mutants, and the GdnHCl-induced unfolding curves of F2W, F29W, and F48W, can be fitted adequately to a two-state model, in agreement with the observations for the wild-type protein. The GdnHCl unfolding transitions of F22W, however, showed the accumulation of an intermediate state at low concentrations of denaturant. Kinetic refolding studies of F2W, F29W, and F48W showed a major single phase, independent of the probe used (CD, fluorescence, and fluorescence anisotropy) and similar to that of the wild-type protein. In contrast, F22W showed two phases in the fluorescence experiments corresponding to the two phases previously observed in ANS binding studies of the wild-type protein [Van Nuland et al. (1998) Biochemistry 37, 622-637]. Residue 22 was found from NMR studies to be part of the binding interface on HPr for ANS. These observations indicate that the second slow phase reflects a local, rather than a global, rearrangement from a well-structured highly nativelike intermediate state to the fully folded native state that has less hydrophobic surface exposed to the solvent. The detection of the second slow phase by the use of selective labeling of different regions of the protein with fluorophores illustrates the need for an integrated approach in order to understand the intricate details of the folding reactions of even the simplest proteins.

The thermal denaturation of streptokinase from Streptococcus equisimilis (SK) together with that of a set of fragments encompassing each of its three domains has been investigated using differential scanning calorimetry (DSC). Analysis of the effects of pH, sample concentration and heating rates on the DSC thermograms has allowed us to find conditions where thermal unfolding occurs unequivocally under equilibrium. Under these conditions, pH 7.0 and a sample concentration of less than approximately 1.5 mg x mL(-1), or pH 8.0, the heat capacity curves of intact SK can be quantitatively described by three independent two-state transitions, each of which compares well with the two-state transition observed for the corresponding isolated SK domain. The results indicate that each structural domain of SK behaves as a single cooperative unfolding unit under equilibrium conditions. At pH 7.0 and high sample concentration, or at pH 6.0 at any concentration investigated, the thermal unfolding of domain A was accompanied by the time-dependent formation of aggregates of SK. This produces a severe deformation of the DSC curves, which become concentration dependent and kinetically controlled, and thus precludes their proper analysis by standard deconvolution methods. A simple model involving time-dependent, high-order aggregation may account for the observed effects. Limited-proteolysis experiments suggest that in the aggregates the N-terminal segment 1-63 and the whole of SK domain C are at least partially structured, while domain B is highly unstructured. Unfolding of domain A, under conditions where the N-terminal segment 1-63 has a high propensity for beta sheet structure and a partially formed hydrophobic core, gives rise to rapid aggregation. It is likely that this region is able to act as a nucleus for the aggregation of the full-length protein.

The stability and folding thermodynamics of two SH3-domains, belonging to Fyn and Abl proteins, have been studied by scanning calorimetry and urea-induced unfolding. They undergo an essentially two-state unfolding with parameters similar to those of the previously studied alpha-spectrin SH3 domain. The correlations between the thermodynamic parameters (heat capacity increment, delta Cp,U, the proportionality factor, m, and the Gibbs energy, delta Gw298) of unfolding and some integral structural parameters, such as polar and non-polar areas exposed upon domain denaturation, have been analyzed. The experimental data on delta Cp,U and the m-factor of the linear extrapolation model (LEM) obey the simple empirical correlations deduced elsewhere. The Gibbs energies calculated from the DSC data were compared with those found by fitting urea-unfolding curves to the LEM and the denaturant-binding model (DBM). The delta Gw298 values found with DBM correlate better with the DSC data, while those obtained with LEM are systematically smaller. The systematic difference between the parameters calculated with LEM and DBM are explained by an inherent imperfection of the LEM.

Proteolytic studies have enabled two of the three putative domains of the fibrinolytic protein streptokinase to be isolated and characterized (Conejero-Lara F et al., 1996, Protein Sci 5:2583-2591). The N-terminal domain, however, could not be isolated in these experiments because of its susceptibility to proteolytic cleavage. To complete the biophysical characterization of the domain structure of streptokinase we have overexpressed, purified, and characterized the N-terminal region of the protein, residues 1-146. The results show this is cooperatively folded with secondary structure content and overall stability closely similar to those of the equivalent region in the intact protein.

The contrasting roles of streptokinase (SK) domains in binding human Glu1-plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2-B-C, A2-B, B-C, and B) binds Plg with similar affinity, at a level approximately 100- to 1,000-fold lower than intact SK. Experiments using 10 mM 6-aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B-C and A2-B-C) does not alter significantly their affinities for Plg. In addition, Plg-binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SK and the two-chain complex A1 x A2-B-C) show a significantly higher affinity for Plg, which could not be completely inhibited by saturating amounts of 6-AHA. These results show that SK domain B interacts with Plg in a lysine-dependent manner and that although domains A and C do not appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native-like Plg activator complex.

Differential scanning calorimetry and Fourier-transform infrared spectroscopy have been used to characterize the thermal stability of bacteriorhodopsin (BR) cleaved within different loops connecting the helical rods. The results are compared to those of the native protein. We show that the denaturation temperature and enthalpy of BR cleaved at peptide bond 71-72 or 155-156 are lower than those of the intact protein, and that these values become even lower for the BR cleaved at both peptide bonds. The effect of cleavage on the denaturation temperature and enthalpy values seems to be additive as has been previously suggested [Khan, T. W., Sturtevant, J. M., & Engelman, D. M. (1992) Biochemistry 31, 8829]. The thermal denaturation of all the samples was irreversible and scan-rate dependent. When cleaved at the 71-72 bond BR follows quantitatively the predictions of the two-state kinetic model at pH 9.5, with an activation energy of 374 kJ/mol, similar to that of native BR. Calorimetry experiments with different populations of intact and cleaved BR provide direct evidence for some intermolecular cooperativity upon denaturation. The denatured samples maintain a large proportion of alpha helices and beta structure, a fact which seems to be related to their low denaturation enthalpy as compared to that of water-soluble, globular proteins.

Streptococcus equisimilis streptokinase (SK) is a single-chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al., 1996, Protein Sci 5:693-704) that SK is composed of three structural domains (A, B, and C) with a C-terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two-chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46 degrees C and its stability is largely independent of the presence of the other domains. The high-temperature transition in intact SK (at approximately 63 degrees C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63-Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (A1 and A2) adopt unstructured conformations when separated. A1 binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native-like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.

The 205-316 C-terminal fragment of thermolysin has been studied by differential scanning calorimetry at pH values 2.5, 3.0, 3.5, 4.0 and 5.0 and at a constant ionic strength of 130 mM. The thermal unfolding of the fragment occurs at thermodynamic equilibrium under our experimental conditions. The effect of sample concentration at the different pH values on the calorimetric traces is consistent with a monomer-dimer equilibrium of the folded fragment, which undergoes thermal unfolding into individual fragments. Equilibrium sedimentation experiments at 10 degrees C and different pH values confirm the presence of the association equilibrium and provide the value of the dimerization constants. The global analysis of the calorimetric, heat capacity curves has been carried out by a multidimensional fitting to the model N2<-->2N<-->2U. The analysis leads to a complete thermodynamic characterization of both the association and unfolding processes of the fragment. The resulting thermodynamic functions suggest a partially unfolded structure for both the monomeric and dimeric fragment, as well as a conformational change linked to the association process. Our results are discussed in terms of the structural information currently available and compared with the energetics of unfolding of the shorter 255-316 dimeric C-terminal fragment of thermolysin (Conejero-Lara, F., De Filippis, V., Fontana, A. and Mateo, P.L. (1994) FEBS Lett. 344, 154-156). The presence of the additional 50 residues increases the relative population of the 205-316 monomeric fragment versus that of the 255-316 fragment.

The thermal denaturation of bovine beta-lactoglobulin B was investigated by high-sensitivity differential scanning microcalorimetry between pH 1.5 and 3.0 in 20 mM phosphate buffer. The process was found to be a reversible, two-state transition. Progressive addition of guanidine hydrochloride at pH 3.0 leads to the appearance of a low-temperature calorimetric endotherm, corresponding to the cold renaturation of the protein. Circular dichroism experiments have confirmed the low and high temperature denaturation processes, and have shown some structural differences between both denatured states of beta-lactoglobulin B.