Analysis of the interaction of calcitriol with the disulfide isomerase ERp57

Calcitriol, the active form of vitamin D3, can regulate the gene expression through the binding to the nuclear receptor VDR, but it can also display nongenomic actions, acting through a membrane- associated receptor, which has been discovered as the disul de isomerase ERp57. The aim of our research is to identify the binding sites for calcitriol in ERp57 and to analyze their interaction. We rst studied the interaction through bioinformatics and uorimetric analyses. Subsequently, we focused on two protein mutants containing the predicted interaction domains with calcitriol: abb’- ERp57, containing the rst three domains, and a’-ERp57, the fourth domain only. To consolidate the achievements we used the calorimetric approach to the whole protein and its mutants. Our results allow us to hypothesize that the interaction with the a’ domain contributes to a greater extent than the other potential binding sites to the dissociation constant, calculated as a Kd of about 10−9 M

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family

Predicting function from sequence is an important goal in current biological research, and although, broad functional assignment is possible when a protein is assigned to a family, predicting functional specificity with accuracy is not straightforward. If function is provided by key structural properties and the relevant properties can be computed using the sequence as the starting point, it should in principle be possible to predict function in detail. The truncated hemoglobin family presents an interesting benchmark study due to their ubiquity, sequence diversity in the context of a conserved fold and the number of characterized members. Their functions are tightly related to O2affinity and reactivity, as determined by the association and dissociation rate constants, both of which can be predicted and analyzed using in-silico based tools. In the present work we have applied a strategy, which combines homology modeling with molecular based energy calculations, to predict and analyze function of all known truncated hemoglobins in an evolutionary context. Our results show that truncated hemoglobins present conserved family features, but that its structure is flexible enough to allow the switch from high to low affinity in a few evolutionary steps. Most proteins display moderate to high oxygen affinities and multiple ligand migration paths, which, besides some minor trends, show heterogeneous distributions throughout the phylogenetic tree, again suggesting fast functional adaptation. Our data not only deepens our comprehension of the structural basis governing ligand affinity, but they also highlight some interesting functional evolutionary trends.Fil: Bustamante, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Radusky, Leandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Boechi, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; ArgentinaFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Ten Have, Arjen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; Argentin

Structure of the Helicase Domain of DNA Polymerase Theta Reveals a Possible Role in the Microhomology-Mediated End-Joining Pathway

DNA polymerase theta (Polθ) has been identified as a crucial alternative non-homologous end-joining factor in mammalian cells. Polθ is upregulated in a range of cancer cell types defective in homologous recombination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attractive target for cancer treatment. We present crystal structures of the helicase domain of human Polθ in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA-stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polθ exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polθ helicase domain in the context of the Polθ tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain

Non-Bulk-Like Solvent Behavior in the Ribosome Exit Tunnel

As nascent proteins are synthesized by the ribosome, they depart via an exit tunnel running through the center of the large subunit. The exit tunnel likely plays an important part in various aspects of translation. Although water plays a key role in many bio-molecular processes, the nature of water confined to the exit tunnel has remained unknown. Furthermore, solvent in biological cavities has traditionally been characterized as either a continuous dielectric fluid, or a discrete tightly bound molecule. Using atomistic molecular dynamics simulations, we predict that the thermodynamic and kinetic properties of water confined within the ribosome exit tunnel are quite different from this simple two-state model. We find that the tunnel creates a complex microenvironment for the solvent resulting in perturbed rotational dynamics and heterogenous dielectric behavior. This gives rise to a very rugged solvation landscape and significantly retarded solvent diffusion. We discuss how this non-bulk-like solvent is likely to affect important biophysical processes such as sequence dependent stalling, co-translational folding, and antibiotic binding. We conclude with a discussion of the general applicability of these results to other biological cavities

Ligand Migration and Cavities within Scapharca Dimeric HbI: Studies by Time-Resolved Crystallo- graphy, Xe Binding, and Computational Analysis

SummaryAs in many other hemoglobins, no direct route for migration of ligands between solvent and active site is evident from crystal structures of Scapharca inaequivalvis dimeric HbI. Xenon (Xe) and organic halide binding experiments, along with computational analysis presented here, reveal protein cavities as potential ligand migration routes. Time-resolved crystallographic experiments show that photodissociated carbon monoxide (CO) docks within 5 ns at the distal pocket B site and at more remote Xe4 and Xe2 cavities. CO rebinding is not affected by the presence of dichloroethane within the major Xe4 protein cavity, demonstrating that this cavity is not on the major exit pathway. The crystal lattice has a substantial influence on ligand migration, suggesting that significant conformational rearrangements may be required for ligand exit. Taken together, these results are consistent with a distal histidine gate as one important ligand entry and exit route, despite its participation in the dimeric interface

Moving wall, continuous flow electronphoresis apparatus

This invention relates generally to electrophoresis devices and more particularly to a moving wall, continuous flow device in which an electrophoresis chamber is angularly positionable with respect to the direction of moving belt walls. A frame with an electrophoresis chamber is rotatably supported between two synchronously driven belt walls. This allows the chamber to be angularly positionable with respect to the direction of belt travel, which compensates for electroosmotic flow within the electrophoresis chamber. Injection of a buffer solution via an opening and a homogenous sample stream via another opening is performed at the end of a chamber, and collection of buffer and the fractionated species particles is done by a conventional collection array at an opposite end of the chamber. Belts are driven at a rate which exactly matches the flow of buffer and sample through the chamber, which entrains the buffer to behave as a rigid electrophoretic medium, eliminating flow distortions (Poiseuille effect). Additionally, belt material for each belt is stored at one end of the device and is taken up by drive wheels at an opposite end. The novelty of this invention particularly lies in the electrophoresis chamber being angularly positionable between two moving belt walls in order to compensate for electroosmotic flow. Additionally, new belt material is continuously exposed within the chamber, minimizing flow distortion due to contamination of the belt material by the sample