Anabel Lostao

Controlling the Number of Proteins with Dip‐Pen Nanolithography

This work was supported by projects MAT2009-13977-C03, Nanomateria-DGA, and PI091/08. D.M. thanks the Ministerio de Ciencia y Tecnologia for a RyC contract. A. L. thanks ARAID for financial support. R. de M. is indebted to MICINN for receiving a predoctoral FPU fellowship. Funding from the European Network of Excellence MAGMANet is also acknowledged.

How Fmn Binds to Anabaena Apoflavodoxin: A Hydrophobic Encounter at an Open Binding Site

Molecular recognition begins when two molecules approach and establish interactions of certain strength. The mechanisms of molecular recognition reactions between biological molecules are not well known, and few systems have been analyzed in detail. We investigate here the reaction between an apoprotein and its physiological cofactor (apoflavodoxin and flavin mononucleotide) that binds reversibly to form a non-covalent complex (flavodoxin) involved in electron transfer reactions. We have analyzed the fast binding reactions between the FMN cofactor (and shorter analogs) and wild type (and nine mutant apoflavodoxins where residues interacting with FMN in the final complex have been replaced). The x-ray structures of two such mutants are reported that show the mutations are well tolerated by the protein. From the calculated microscopic binding rate constants we have performed a Φ analysis of the transition state of complex formation that indicates that the binding starts by interaction of the isoalloxazine-fused rings in FMN with residues of its hydrophobic binding site. In contrast, the phosphate in FMN, known to contribute most to the affinity of the final holoflavodoxin complex, is not bound in the transition state complex. Both the effects of ionic strength and of phosphate concentration on the wild type complex rate constant agree with this scenario. As suggested previously by nmr data, it seems that the isoalloxazine-binding site may be substantially open in solution. Interestingly, although FMN is a charged molecule, electrostatic interactions seem not to play a role in directing the binding, unlike what has been reported for other biological complexes. The binding can thus be best described as a hydrophobic encounter at an open binding site.

Size-dependent properties of magnetoferritin.

We report a detailed experimental study of maghemite nanoparticles, with sizes ranging from 1.6 to 6 nm, synthesized inside a biological mould of apoferritin. The structural characterization of the inorganic cores, using TEM and x-ray diffraction, reveals a low degree of crystalline order, possibly arising from the nucleation and growth of multiple domains inside each molecule. We have also investigated the molecular structure by means of atomic force microscopy in liquid. We find that the synthesis of nanoparticles inside apoferritin leads to a small, but measurable, decrease in the external diameter of the protein, probably associated with conformational changes. The magnetic response of the maghemite cores has been studied by a combination of techniques, including ac susceptibility, dc magnetization and Mössbauer spectroscopy. From the equilibrium magnetic response, we have determined the distribution of magnetic moments per molecule. The results show highly reduced magnetic moments. This effect cannot be ascribed solely to the canting of spins located at the particle surface but, instead, it suggests that magnetoferritin cores have a highly disordered magnetic structure in which the contributions of different domains compensate each other. Finally, we have also determined, for each sample, the distribution of the activation energies required for the magnetization reversal and, from this, the size-dependent magnetic anisotropy constant K. We find that K is enormously enhanced with respect to the maghemite bulk value and that it increases with decreasing size. The Mössbauer spectra suggest that low-symmetry atomic sites, probably located at the particle surface and at the interfaces between different crystalline domains, are the likely source of the enhanced magnetic anisotropy.

Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O -glycosylation

Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.

Salt-induced stabilization of apoflavodoxin at neutral pH is mediated through cation-specific effects

Electrostatic contributions to the conformational stability of apoflavodoxin were studied by measurement of the proton and salt‐linked stability of this highly acidic protein with urea and temperature denaturation. Structure‐based calculations of electrostatic Gibbs free energy were performed in parallel over a range of pH values and salt concentrations with an empirical continuum method. The stability of apoflavodoxin was higher near the isoelectric point (pH 4) than at neutral pH. This behavior was captured quantitatively by the structure‐based calculations. In addition, the calculations showed that increasing salt concentration in the range of 0 to 500 mM stabilized the protein, which was confirmed experimentally. The effects of salts on stability were strongly dependent on cationic species: K+, Na+, Ca2+, and Mg2+ exerted similar effects, much different from the effect measured in the presence of the bulky choline cation. Thus cations bind weakly to the negatively charged surface of apoflavodoxin. The similar magnitude of the effects exerted by different cations indicates that their hydration shells are not disrupted significantly by interactions with the protein. Site‐directed mutagenesis of selected residues and the analysis of truncation variants indicate that cation binding is not site‐specific and that the cation‐binding regions are located in the central region of the protein sequence. Three‐state analysis of the thermal denaturation indicates that the equilibrium intermediate populated during thermal unfolding is competent to bind cations. The unusual increase in the stability of apoflavodoxin at neutral pH affected by salts is likely to be a common property among highly acidic proteins.

Apoflavodoxin: Structure, stability, and FMN binding

Flavodoxins are one domain alpha/beta electron transfer proteins that participate in photosynthetic reactions. All flavodoxins carry a molecule of flavin mononucleotide (FMN), non-covalently bound, that confers redox properties to the protein. There are two structurally distinct flavodoxins, short ones and long flavodoxins; the latter contain an extra loop with unknown function. We have undertaken the study of the stability and folding of the apoflavodoxin from Anabaena (a long flavodoxin) and the analysis of the interaction between the apoflavodoxin and FMN. Our studies indicate that apoflavodoxin folds in a few seconds to a form that is competent in FMN binding. The stability of this apoflavodoxin is low and its urea denaturation can be described by a two-state mechanism. The role of the different parts of the apoflavodoxin in the stability and structure of the whole protein is being investigated using mutagenesis and specific cleavage to generate apoflavodoxin fragments. The X-ray structure of apoflavodoxin is very similar to that of its complex with FMN, the main difference being the conformation of the two aromatic residues that sandwich FMN in the complex. In apoflavodoxin these groups interact with each other so closing the FMN binding site. Despite this fact, apoflavodoxin binds FMN tightly and rapidly, and the resulting holoflavodoxin displays a high conformational stability. We have found that one role of the aromatic residues that interact with FMN is to help to retain bound the reduced form of the cofactor whose complex with apoflavodoxin is otherwise too weak.

A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2.

Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions.

Unbinding Molecular Recognition Force Maps of Localized Single Receptor Molecules by Atomic Force Microscopy

Atomic force microscopy is a technique capable to study biological recognition processes at the single-molecule level. In this work we operate the AFM in a force-scan based mode, the jumping mode, where simultaneous topographic and tip-sample adhesion maps are acquired. This approach obtains the unbinding force between a well-defined receptor molecule and a ligand attached to the AFM tip. The method is applied to the avidin-biotin system. In contrast with previous data, we obtain laterally resolved adhesion maps of avidin-biotin unbinding forces highly correlated with single avidin molecules in the corresponding topographic map. The scanning rate 250 pixel s(-1) (2 min for a 128 x 128 image) is limited by the hydrodynamic drag force. We are able to build a rupture-force distribution histogram that corresponds to a single defined molecule. Furthermore, we find that due to the motility of the polymer used as spacer to anchor the ligand to the tip, its direction at rupture does not generally coincide with the normal to the tip-sample, this introduces an appreciable error in the measured force.

Nanoscale Positioning of Inorganic Nanoparticles using Biological Ferritin Arrays Fabricated by Dip-Pen Nanolithography

In this manuscript we demonstrate the spatially controlled immobilization of ferritin proteins by directly writing them on a wide range of substrates of technological interest. Optical and fluorescence microscopy, AFM and TOF-SIMS studies confirm the successful deposition of the protein on those surfaces. Control on nanostructure shape and size, by miniaturizing the dot-like features down to a 100 nm, demonstrates the particular capabilities of the DPN approach. Ultimately, this study gives the opportunity to design nanoparticle-based arrays regarding the growing interest in the use of nanoparticles as structural and functional elements for fabricating nanodevices. Herein, we demonstrate how the protein shell of ferritins can be removed by a simple heat-treatment process while maintaining the encapsulated inorganic nanoparticle intact on the same location of the nanoarray. As a result, this study establishes how direct-write DPN approach could give the opportunity to design not only protein-based nanoarrays but also nanoparticle-based nanoarrays with high-resolution and control.

Electron-Nuclear Double Resonance and Hyperfine Sublevel Correlation Spectroscopic Studies of Flavodoxin Mutants from Anabaena sp. PCC 7119

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.