Vinculin is a ubiquitously expressed focal adhesion protein that plays an important role in cell-matrix and cell-to-cell junctions. Metavinculin, a muscle-specific splice variant of vinculin, contains a 68-amino acid disordered insert region in its actin binding tail domain (MVt). Mutations in this insert are linked to cardiomyopathies. This study investigates the solution structures and structural ensembles of wild-type (WT) and two mutant MVts, ΔLeu954 and R975W, which have been associated with cardiomyopathies, using small-angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations. SAXS analyses revealed subtle differences in the estimated maximum dimensions and corroborated the elongated shape of the MVts. Quantitative comparisons of SAXS profiles indicated similarity between the WT and ΔLeu954, whereas R975W exhibited differences in the small-angle region. MD simulations demonstrated reduced conformational flexibility and greater packing of the insert in WT compared to mutants. Notably, a salt-bridge observed between R975 and D907 in a WT simulation provides a structural basis for the destabilization caused by the R975W mutation. These findings provide insights into the structure and dynamics of WT and mutant MVt, reflecting the promise of SAXS combined with MD simulations to elucidate the structural properties of proteins with structural disorder.

Combining size exclusion chromatography-small angle X-ray scattering (SEC-SAXS) and molecular dynamics (MD) analysis is a promising approach to investigate protein behavior in solution, particularly for understanding conformational changes due to substrate binding in cytochrome P450s (CYPs). This study investigates conformational changes in CYP119, a thermophilic CYP from Sulfolobus acidocaldarius that exhibits structural flexibility similar to mammalian CYPs. Although the crystal structure of ligand-free (open state) and ligand-bound (closed state) forms of CYP119 is known, the overall structure of the enzyme in solution has not been explored until now. It was found that theoretical scattering profiles from the crystal structures of CYP119 did not align with the SAXS data, but conformers from MD simulations, particularly starting from the open state (46 % of all frames), agreed well. Interestingly, a small percentage of closed-state conformers also fit the data (9 %), suggesting ligand-free CYP119 samples ligand-bound conformations. Ab initio SAXS models for N-His tagged CYP119 revealed a tail-like unfolded structure impacting protein flexibility, which was confirmed by in silico modeling. SEC-SAXS analysis of N-His CYP119 indicated pentameric structures in addition to monomers in solution, affecting the stability and activity of the enzyme. This study adds insights into the conformational dynamics of CYP119 in solution.

This study combines molecular dynamics (MD) simulations with solution X-ray scattering measurements to investigate the range of conformations that can be achieved by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations. To explore how the conformational multiplicity of proteins might be modified in the environmental niches of biological media, we focus on the periplasmic protein FbpA from H. influenzae which is a part of the mechanism developed by bacteria to capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its unique biological environment. The collected SAXS data complemented by SEC analyses and binding assays point to multiple conformations at physiological IS while they are well-explained by single x-ray structures in a 15 mM buffer. Moreover, by fitting the SAXS data with unique conformations sampled by a series of MD simulations carried out under conditions mimicking the buffers, we quantify with high accuracy the populations of the occupied substates. Furthermore, we find the D52A mutant that we predicted by coarse-grained computational approaches to allosterically control the iron binding site in FbpA responds to the environmental changes in our experiments with varied conformational selection scenarios. We show an application of the environment dependence of the conformers by designing a genetically encoded iron biosensor as a chimera of FbpA and green fluorescent protein using the D52 region as the insertion point. This work exemplifies how unifying a range of experimental and computational techniques provides platforms for achieving the next generation of applications that put protein dynamics rather than the static structure at the centerstage. Support by Turkish Atomic Energy Authority, TUBITAK grant no 121Z329 and a scholarship to GL through 2214-A program. Measurements conducted at EMBL-Hamburg.

Incorporating in the curriculum active learning and project-based teaching, assuming minimal prior knowledge and emphasizing the real-world relevance of the covered topics result in better learning outcomes and help engage a more diverse group of students. In this Viewpoint, five educators who have been involved in reimagining undergraduate teaching in materials science and engineering share their insights and perspective.

This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.

FbpA is an iron transport protein from the pathogenic bacteria, Haemophilus Influenzae, which hijacks one iron from human Transferrin (Tf) and transports it through the periplasmic space of the organism. It has 309 residues, and contains an iron binding cleft with two tyrosines, one histidine and one glutamic acid residues. Iron is held in the cleft by the octahedral coordination of these residues and a phosphate which serves as a synergistic anion. In this study, we investigated the significance of pH and ionic strength (IS) on the iron binding and release dynamics of H. influenzae ferric binding protein (FbpA) through binding/release assays as well as from a structural perspective. FbpA was expressed, purified and characterized in large quantities. Size Exclusion Chromatography (SEC), Dynamic Light Scattering (DLS), UV-Vis Absorption Spectroscopy (AS) and Small Angle X-ray Scattering (SAXS) were used for detecting structural changes. SAXS measurements were used to compare solution structures with the crystal structures, as well as to detect the changes in protein conformations under different environmental conditions. Additionally, absorbance values at 480 nm due to metal-to-ligand charge transfer caused by the interaction of tyrosine residues with the iron were monitored for investigation of iron binding dynamics under different IS conditions. Our results show that both apo and holo FbpA have flexible structures in solution with the former being slightly more open compared to the latter and both forms are more stable at high IS. Results are consistent with the closing of the iron binding pocket, when iron is captured, resulting in a decrease in hydrodynamic radius and radius of gyration. It is proposed that low IS can lead to instabilities in the protein structure and expose the bound iron to chelators at the inner membrane.

Plant heterotrimeric G proteins are a major group of signaling molecules involved in regulation of critical processes including stress adaptation, seed size, grain quality and immune responses. Despite an abundance of in situ functional studies; purification of the individual subunits of the plant heterotrimer for biophysical and structural characterization and for studies on their interactions are lacking. In this study cloning of the genes encoding the β subunit AGB1 of A. thaliana and its γ-subunits AGG1 and AGG2 using different E. coli expression vectors and screening of expression in several strains are reported. AGB1 could be expressed albeit at very low levels and in all cases it was accompanied by overexpression of E. coli chaperone proteins. AGG1 could only be detected in inclusion body fractions, whereas AGG2 was obtained in soluble fractions and was purified. Purified AGB1 and AGG2 subunits were shown to dimerize in vitro. Further characterization of AGG2 by small angle X-ray scattering measurements and by dynamic light scattering revealed that AGG2 formed homodimers with extended shape in solution. These results are also consistent with those from circular dichroism spectroscopy which yielded 39.4% helical and 50% random coil content for AGG2. This is the first study showing heterologous expression of a plant heterotrimeric G protein β subunit individually and presenting its interaction with a plant γ-subunit in vitro. Results also show that the AGG2 subunit has a disordered structure, which would account for its role in diverse interactions for establishing selectivity in signal propagation.

Iron is a fundamental metabolite for the survival of all biological systems because of its ability to function as both electron donor and acceptor. In unbound form, Fe+3 is not soluble in water whereas; Fe+2 is highly toxic in free form. Different organisms have developed specific mechanisms to transport iron. In humans transferrin is the responsible protein for carrying iron, whereas in bacteria Ferric Binding Protein (Fbp) performs this function. Pathogenic bacteria have evolved to sequester iron from human transferrin through an exceptional mechanism, where one of the two irons of transferrin is detached, transported through the periplasmic space and captured by Fbp. In this study, our main focus is to monitor conformational changes of Fbp from Haemophilus Influenzae, during iron release and binding in alternating environmental conditions. Fbp was expressed and purified by using recombinant DNA technology. Size Exclusion Chromatography (SEC), Dynamic Light Scattering (DLS), UV-Vis Absorption Spectroscopy (AS), Circular Dichroism (CD) and Small Angle X-ray Scattering (SAXS) are used for detecting structural changes. Additionally, features of the 480 nm ligand-to-metal charge transfer absorption peak of FBP-Iron complex was monitored during iron release/binding process for both Fe+3 and Fe+2 in different pH and ionic strength conditions. Our results confirm that Fbp changes its conformation to bind iron; hydrodynamic radius decreases when it captures the ligand because of the closing up of the metal-binding cleft. Effect of changes in pH, ionic strength and the state of iron on iron affinity are also investigated. We believe that the study on the dynamics of Fbp addresses us to further biosensor applications to determine elevated levels of iron in individual cells and targeted drug design practices.

Because of the serious neurologic consequences of iron deficiency and iron excess in the brain, interest in the iron status of the central nervous system has increased significantly in the past decade. While iron plays an important role in many physiological processes, its accumulation may lead to diseases such as Huntington's, Parkinson's, and Alzheimer's. Therefore, it is important to develop methodologies that can monitor the presence of iron in a selective and sensitive manner. In this paper, we first showed the synthesis and characterization of the iron-binding protein (FBP) from Haemophilus influenzae, specific for ferrous ions. Subsequently, we employed this protein in our nanopipette platform and utilized it in functionalized nanoprobes to monitor the presence of ferrous ions. A suite of characterization techniques: absorbance spectroscopy, dynamic light scattering, and small-angle X-ray scattering were used for FBP. The functionalized Fe-nanoprobe calibrated in ferrous chloride enabled detection from 0.05 to 10 μM, and the specificity of the modified iron probe was evaluated by using various metal ion solutions.

The effect of varying levels of cadmium and correlated changes on the expression level of a type 1 metallothionein gene (dMT) were investigated in Triticum durum cv. Balçalı-85. Increasing the cadmium concentration resulted in a decrease in the dry weights of roots and shoots, and the effect was stronger in roots. Roots also showed a higher capacity to accumulate cadmium. Southern blot analyses revealed that the dMT gene, delineated by two exons and a non-coding intron region, exists at a single locus in the T. durum genome. Changes in dMT gene expression during cadmium exposure were monitored by two approaches. Northern blot analyses showed that the transcript level in roots increased upon treatment with increasing cadmium, which was quantified by qRT-PCR as 4.5 fold of the base level at 10 μM Cd. These results show a positive correlation between cadmium exposure and expression of dMT gene in durum wheat, and will provide a basis for studies on the role of type 1 metallothioneins in cadmium response.

Student-centred active learning approach in undergraduate science education has increasingly been adopted by a number of universities world-wide during the past few decades, and mounting evidence indicates positive impacts of the active learning approach on students’ learning outcomes [1,2]. Some notable active-learning models such as Student-Centred Active Learning Environment with Upside-down Pedagogies (SCALE-UP) at North Carolina State University [3] or Technology-Enabled Active Learning (TEAL) classes at MIT [4], have successfully been applied especially to large enrolment classes. Moreover, real-life challenges faced by tomorrow’s engineers in this ever-changing world demand a strong interdisciplinary foundation applicable to real-life situations. To that end some universities have designed their own integrated science courses such as the Integrated Quantitative Science at University of Richmond [5], Frontiers of Science at Columbia University [6], and What is Life at Harvard University [7].

Background: Synchrotron radiation (SR) sources provide diverse X-ray methods for the investigation of structure -function relationships in biological macromolecules.Scope of review: Recent developments in SR sources and in the X-ray tools they offer for life sciences are reviewed. Specifically, advances in macromolecular crystallography, small angle X-ray solution scattering, X-ray absorption and fluorescence spectroscopy, and imaging are discussed with examples.Major conclusions: SR sources offer a range of X-ray techniques that can be used in a complementary fashion in studies of biological systems at a wide range of resolutions from atomic to cellular scale. Emerging applications of X-ray techniques include the characterization of disordered proteins, noncrystalline and nonequilibrium systems, elemental imaging of tissues, cells and organs, and detection of time-resolved changes in molecular structures.General significance: X-ray techniques are in the center of hybrid approaches that are used to gain insight into complex problems relating to biomolecular mechanisms, disease and possible therapeutic solutions. This article is part of a Special Issue entitled "Science for Life". Guest Editors: Dr. Austen Angell, Dr. Salvatore Magazti and Dr. Federica Migliardo. (C) 2016 Elsevier B.V. All rights reserved.

In plants heterotrimeric complexes of G proteins (consisting of alpha, beta, and gamma subunits) regulate several signaling pathways including seed germination, seedling development, organ shape and size determination. The alpha subunit has GTP binding and hydrolysis activity and the beta- gamma subunits interact with downstream effectors as a heterodimer. Some structural homology among the plant and mammalian subunits have led to early assumptions about similarities in the activation and transduction mechanisms in the two systems. However, recent evidence on the lack of membrane receptors in plants, the constitutively active state of the plant alpha subunit and the existence of a large family of gamma subunits indicate that the mechanisms involving the plant proteins may be significantly different from those in their mammalian counterparts. In our group the alpha subunit from A. thaliana (AtGPA1), an N-terminal mutant (GPA1t), the gamma subunits (AGG1, AGG2), and the rice gamma subunits (RGG1 and RGG2) were expressed in yeast and bacteria. Absorbance spectroscopy, circular dichroism spectropolarimetry, and dynamic light scattering (DLS) analyses show the structural and stability differences between AtGPA1 and GPA1t as well as among all gamma subunits. DLS, native-PAGE and small angle X-ray scattering measurements reveal the stable oligomeric forms of the proteins in solution indicating possible functional roles for the oligomers. Results also demonstrate the high level of the flexibility in the structures of all subunits. Models for possible roles of different subunits in G protein signaling in plants will be presented. Supported by Turkish Atomic Energy Commission and Instruct, a Landmark ESFRI project.