High-burnup Zr-based nuclear fuel claddings exhibit accelerated irradiation growth, corrosion and hydrogen pick-up, all correlated with the emergence of c-component dislocation loops. We made use of sub-nm-resolution atom probe tomography to characterize the nanoscale chemistry of c-loops in fuel cladding from boiling water reactor operation. We found segregation of Fe, Ni and Sn to dislocation lines and depletion of Sn and O inside the loops, resulting in nearly pure Zr islands. We also observed nucleation of suboxide inside one c-loop, pointing to a possible mechanism of accelerated in-reactor corrosion. Such Zr-islands might also promote hydride precipitation and associated degradation.

This work indicates that the matrix content of the alloying elements iron, chromium, and nickel in as-produced commercial Zircaloy-2-type materials is lower than what has been indicated by many previous studies. Atom probe tomography in voltage pulse mode was used to study the matrix content of solutes in Zircaloy-2 of type LK3/L and a similar model alloy, called Alloy 2, of the same heat treatment. Both alloys were analyzed in the as-produced state and after reactor exposure. In the as-produced materials, the concentrations of iron, chromium, and nickel were all below the detection limits of around 10 wt. ppm. After reactor exposure, these alloying elements were observed to reside in clusters at loops, and the matrix content (including clusters) of iron had increased to about 1,200 wt. ppm in the fueled region of the rod and to about half that value in the plenum region. The chromium content in the fueled region was approximately 100 wt. ppm, and the nickel content was approximately 200 wt. ppm. In the plenum region, the content of these elements was lower. However, due to an uneven distribution of clusters, there was a wide scatter in the measured concentrations in the irradiated materials. Additionally, the matrix concentrations of solute elements in (nonirradiated) Zircaloy-2 were investigated for a series of samples subjected to α annealing at 770°C followed by cooling at different rates. From these measurements, the solubilities at 770°C were estimated to be around 65 wt. ppm for chromium, at least 37 wt. ppm for iron, and below 9 wt. ppm for nickel. Slow cooling resulted in virtually no iron, chromium, or nickel in the matrix. The concentration of aluminum in the matrix was observed to be between 10 and 20 wt. ppm for all α-annealed samples and for the as-produced materials of commercial heat treatment.

At high fluence and prolonged reactor operation Zr-based nuclear fuel claddings exhibit accelerated (often termed ‘breakaway’) irradiation growth, corrosion, and hydrogen pick-up. These degradation processes are correlated with the emergence of c-component dislocation loops on basal planes caused by damage from fast neutrons. We made use of the unique capabilities of atom probe tomography – namely chemical characterization with sub-nm resolution in 3D – to analyze the nano-chemistry of c-loops formed in Zircaloy-2 type cladding tubes during boiling water reactor operation. We found segregation of Fe, Ni and Sn to dislocation lines and – more surprisingly – depletion of Sn and O in the strain field of the stacking fault inside the loops, resulting in nearly pure Zr islands. We furthermore found nucleation of ZrO (‘suboxide’) inside one c-loop, pointing to a possible mechanism of accelerated in-reactor corrosion. Such pure Zr-islands might to also promote hydride precipitation and associated degradation.

PSD95-Disc large-Zonula occludens (PDZ) domains are among the most abundant modular domains in the human proteome. They typically bind short carboxy-terminal sequence motifs of their ligand proteins, which may be transmembrane proteins such as ion channels and GPCRs, as well as soluble proteins. The identity of the endogenous ligands of many PDZ domains remains unclear despite more than two decades of PDZ research. Combinatorial peptide phage display and bioinformatics predictions have contributed to shed light on PDZ-mediated interactions. However, the efficiency of these methods for the identification of interactions of potential biological relevance is hampered by different biases. Proteomic peptide-phage display (ProP-PD) was developed to overcome these limitations. Here we describe a ProP-PD protocol for the identification of C-terminal PDZ domain ligands. The method efficiently identifies peptide ligands within a proteome of interest, and pinpoint targets of potential biological relevance.

A recently published study of high temperature nitridation of iron chromium aluminum alloys (FeCrAl) at 900°C in N2–H2 has redundantly shown the formation of locally confined corrosion pockets reaching several microns into the alloy. These nitrided pockets form underneath chromia islands laterally surrounded by the otherwise protective alumina scale. Chromia renders a nitrogen‐permeable defect under the given conditions and the presence of aluminum in the alloy. In light of these findings on FeCrAl, a focused ion beam–scanning electron microscope tomography study has been undertaken on an equally nitrided FeNiCrAl sample to characterize its nitridation corrosion features chemically and morphologically. The alloy is strengthened by a high number of chromium carbide precipitates, which are also preferential chromia formation sites. Besides the confirmation of the complete encapsulation of the corrosion pocket from the alloy by a closed and dense aluminum nitride rim, very large voids have been found in the said pockets. Furthermore, metallic particles comprising nickel and iron are deposited on top of the outer oxide scale above such void regions.

Whereas microstructure evolution in adiabatic shear bands have been thoroughly studied, reports on the stability of hardening precipitates during shear localisation are scarce. We report an atomic scale investigation of solute distribution in adiabatic shear bands in a precipitation strengthened Ni-Fe-based superalloy, showing that the hardening particles are completely dissolved. Temperature estimations indicate that peak temperatures in the shear band above the solvus limits of the precipitates are not unrealistic, and thus diffusion-assisted transformations during the severe plastic deformation cannot be ruled out.

In article number 1900316, Martin Andersson and co-workers develop a new method for protein structure analysis. Atom probe tomography (APT) is used to simultaneously asses the 3D structure and chemical composition of proteins with high resolution. The key concept of the method is to embed proteins in an amorphous silicon oxide glass, from which ultra-sharp needles are prepared and analyzed with APT.

Surface modifications in the nanoscale regime have shown promising potential in the combat against bacterial adhesion and colonization of surfaces. However, detailed knowledge of how the bacteria-substrate interactions occur is still limited. Herein we have used a gradient in nanostructure density on a surface, realized by immobilizing 40 nm sized silicon dioxide nanoparticles with increasing distance on a glass surface, to systematically study the initial attachment of Staphylococcus epidermidis with or without the presence of human fibrinogen. By using a parallel plate laminar flow chamber, we found a near-linear positive correlation between the adhesion of S. epidermidis with increasing nanoparticle density on unmodified (hydrophilic) nanogradients as well as on gradients where polyethylene glycol was immobilized on the surface in-between nanoparticles. However, if the nanostructured gradient was precoated with human fibrinogen the opposite relationship was observed, although the adsorbed amount of fibrinogen was found to be higher on nanostructured than on smooth surfaces. Our results highlight that even minute changes of the nanotopography on a surface can have profound impact on initial attachment of S. epidermidis to biomaterial surfaces and that the presence of nanostructures strongly hampered the cell's ability to bind to adsorbed fibrinogen, possibly due to changes in the orientation or secondary structure of the fibrinogen molecule upon adsorption.

Determination of the 3D structure of proteins and other biomolecules is a major goal in structural biology, to provide insights to their biological function. Such structures are historically unveiled experimentally by X-ray crystallography or NMR spectroscopy, and in recent years using cryo-electron microscopy. Here, a method for structural analysis of individual proteins on the sub-nanometer scale using atom probe tomography is described. This technique offers a combination of high-resolution analysis of biomolecules in 3D, and the chemical sensitivity of mass spectrometry. As a model protein, the well-characterized antibody IgG is used. IgG is encapsulated in an amorphous solid silica matrix via a sol-gel process to provide the requisite support for atom probe analysis. The silica synthesis is tuned to resemble physiological conditions. The 3D reconstructions show good agreement with the protein databank IgG crystal structure. This suggests that the silica-embedding strategy can open the field of atom probe tomography to the analysis of biological molecules. In addition to high-resolution structural information, the technique may potentially provide chemical information on the atomic scale using isotopic labeling. It is envisaged that this method may constitute a useful complement to existing tools in structural biology, particularly for the examination of proteins with low propensity for crystallization.

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.

Although the evolution of irradiation-induced dislocation loops has been well correlated with irradiation-induced growth phenomena, the effect of alloying elements on this evolution remains elusive, especially at low fluences. To develop a more mechanistic understanding of the role iron has on loop formation, we used state-of-the-art techniques to study a proton-irradiated Zr-0.1Fe alloy and proton- and neutron-irradiated Zircaloy-2. The two alloys were irradiated with 2-MeV protons up to 7 dpa at 350 degrees C and Zircaloy-2 up to 14.7 x 10(25) n. m(-2), approximately 24 dpa, in a boiling water reactor at approximately 300 degrees C. Baseline transmission electron microscopy showed that the Zr3Fe secondary-phase particles in the binary system were larger and fewer in number than the Zr(Fe,Cr)(2) and Zr2(Fe,Ni) particles in Zircaloy-2. An analysis of the irradiated binary alloy revealed only limited dissolution of Ze(3)Fe, suggesting little dispersion of iron into the matrix, while at the same time a higher < a >-loop density was observed compared with Zircaloy-2 at equivalent proton dose levels. We also found that the redistribution of iron during irradiation led to the formation of iron nanoclusters. A delay in the onset of < c >-loop nucleation in proton-irradiated Zircaloy-2 compared with the binary alloy was observed. The effect of iron redistributed from secondary-phase particles because of dissolution on the density and morphology of < a > and < c > loops is described. The implication this may have on irradiation induced growth of zirconium fuel cladding is also discussed.

A key function of reversible protein phosphorylation is to regulate protein-protein interactions, many of which involve short linear motifs (3-12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale.

Protein-protein interactions involving intrinsically disordered proteins are important for cellular function and common in all organisms. However, it is not clear how such interactions emerge and evolve on a molecular level. We performed phylogenetic reconstruction, resurrection and biophysical characterization of two interacting disordered protein domains, CID and NCBD. CID appeared after the divergence of protostomes and deuterostomes 450–600 million years ago, while NCBD was present in the protostome/deuterostome ancestor. The most ancient CID/NCBD formed a relatively weak complex (Kd∼5 µM). At the time of the first vertebrate-specific whole genome duplication, the affinity had increased (Kd∼200 nM) and was maintained in further speciation. Experiments together with molecular modeling using NMR chemical shifts suggest that new interactions involving intrinsically disordered proteins may evolve via a low-affinity complex which is optimized by modulating direct interactions as well as dynamics, while tolerating several potentially disruptive mutations.

We report phosphomimetic proteomic peptide-phage display, a powerful large-scale method for finding ligands of short linear motif binding domains that simultaneously pinpoint functional Ser/Thr phosphosites in three steps. First, we computationally designed an oligonucleotide library encoding all human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. Second, we incorporated these oligonucleotides into a phage library. Third, we screened the six PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for binding and identified known and novel ligands from the human proteome, and whether these interactions may be regulated by ligand phosphorylation. We demonstrate that the Scribble PDZ domains preferentially bind to ligands with phosphomimetic mutations at two distinct positions, and show that the equilibrium dissociation constant for Scribble PDZ1 with the C-terminal peptide of RPS6KA2 is enhanced over four-fold by phosphorylation. We elucidate the molecular determinants of phosphopeptide binding through NMR structure determination and mutational analysis. Finally, we discuss the role of Ser/Thr phosphorylation as a switching mechanism of PDZ domain interactions.

The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.

Osseointegration of dental implants occurs on a hierarchy of length scales down to the atomic level. A deeper understanding of the complex processes that take place at the surface of an implant on the smallest scale is of interest for the development of improved biomaterials. To date, transmission electron microscopy (TEM) has been utilized for examination of the bone-implant interface, providing details on the nanometer level. In this study we show that TEM imaging can be complemented with atom probe tomography (APT) to reveal the chemical composition of a Ti-based dental implant in a human jaw on the atomic level of resolution. As the atom probe technique has equal sensitivity for all elements, it allows for 3 dimensional characterizations of osseointegrated interfaces with unprecedented resolution. The APT reconstructions reveal a Ca-enriched zone in the immediate vicinity of the implant surface. A surface oxide of some 5nm thickness was measured on the titanium implant, with a sub-stoichiometric composition with respect to TiO2. Minor incorporation of Ca into the thin oxide film was also evident. We conclude that the APT technique is capable of revealing chemical information from the bone-implant interface in 3D with unprecedented resolution, thus providing important insights into the mechanisms behind osseointegration. STATEMENT OF SIGNIFICANCE:Osseointegration of dental implants occurs on a hierarchy of length scales down to the atomic level. A deeper understanding of the complex processes that take place at the surface of an implant on the smallest scale is of interest for the development of improved biomaterials. To date, transmission electron microscopy (TEM) has been utilized for examination of the bone-implant interface, providing details on the nanometer level. In this study we show that TEM imaging can be complemented with atom probe tomography (APT) to reveal the chemical composition of a Ti-based dental implant in a human jaw on the atomic level of resolution. Correlative microscopy ensures the accuracy of APT reconstructions and helps provide both chemical and structural information of the bone-implant interface on the smallest of length scales.

The E6 protein from human papillomavirus (HPV) plays an important role during productive infection and is a potential drug target. We have previously designed a high affinity bivalent protein binder for the E6 protein, a fusion between a helix from the E6 associated protein and PDZØ9, an engineered variant (L391F/K392M) of the second PDZ domain from synapse associated protein 97 (SAP97 PDZ2). How the substitutions improve the affinity of SAP97 PDZ2 for HPV E6 is not clear and it is not known to what extent they affect the specificity for cellular targets. Here, we explore the specificity of wild type SAP97 PDZ2 and PDZØ9 through proteomic peptide phage display. In addition, we employ a double mutant cycle of SAP97 PDZ2 in which the binding kinetics for nine identified potential cellular peptide ligands are measured and compared with those for the C-terminal E6 peptide. The results demonstrate that PDZØ9 has an increased affinity for all peptides, but at the cost of specificity. Furthermore, there is a peptide dependent coupling free energy between the side chains at positions 391 and 392. This corroborates our previous allosteric model for PDZ domains, involving sampling of intramolecular energetic pathways.

Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance.