247 research outputs found
Development and Characterization of Nonpeptidic Small Molecule Inhibitors of the XIAP/Caspase-3 Interaction
AbstractElevated expression of inhibitor of apoptosis protein (IAP) family members in various types of cancers is thought to provide a survival advantage to these cells. Thus, antiapoptotic functions of IAPs, and their potential as novel anticancer targets have attracted considerable interest. Among the IAPs, the X chromosome-linked inhibitor of apoptosis protein (XIAP) is regarded as the most potent suppressor of mammalian apoptosis through direct binding and inhibition of caspases. A high-throughput biochemical screen of a combinatorial chemical library led to the discovery of a novel nonpeptidic small molecule that has the ability to disrupt the XIAP/caspase-3 interaction. The activity of this nonpeptidic small molecule inhibitor of the XIAP/caspase-3 interaction has been characterized both in vitro and in cells. Molecules of this type can be used to conditionally inhibit the cellular function of XIAP and may provide insights into the development of therapeutic agents that act by modulating apoptotic pathways
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Golgicide A Reveals Essential Roles for GBF1 in Golgi Assembly and Function
ADP-ribosylation factor 1 (Arf1) plays a critical role in regulating secretory traffic and membrane transport within the Golgi of eukaryotic cells. Arf1 is activated by guanine nucleotide exchange factors (ArfGEFs) which confer spatial and temporal specificity to vesicular transport. We describe here the discovery and characterization of Golgicide A (GCA), a potent, highly specific, and reversible inhibitor of the cis-Golgi ArfGEF, GBF1. Inhibition of GBF1 function resulted in rapid dissociation of COPI vesicle coat from Golgi membranes and subsequent disassembly of the Golgi and trans-Golgi network (TGN). Secretion of soluble and membrane-associated proteins was arrested at the ER-Golgi intermediate compartment, whereas endocytosis and recycling of transferrin was unaffected by GBF1 inhibition. Internalized shiga toxin was arrested within the endocytic compartment and was unable to reach the dispersed TGN. Collectively, these results highlight the central role for GBF1 in coordinating bidirectional transport and maintaining structural integrity of the Golgi
Photodissociation of water in crystalline ice: a molecular dynamics study
Ultraviolet irradiation of ice is of great interest for understanding the
chemistry in both atmospheric and astrophysical environments. In interstellar
space, photodissociation of H2O molecules can be a driving force behind the
chemistry on icy dust grains in dense, cold molecular clouds even though the
flux of UV photons is extremely low. The mechanisms of such photoinduced
processes are poorly understood, however. In this work the photodissociation
dynamics of a water molecule in crystalline ice at 10 K is studied
computationally using classical molecular dynamics. Photodissociation in the
first bilayer leads mainly to H atoms desorbing (65%), while in the third
bilayer trapping of H and OH dominates (51%). The kinetic energy distribution
of the desorbing H atoms is much broader than that for the corresponding
gas-phase photodissociation. The H atoms on average move 11 Angstroms before
becoming trapped, while OH radicals typically move 2 Angstroms. In accordance
with experiments a blueshift of the absorption spectrum is obtained relative to
gas-phase water.Comment: 23 pages, 5 figure
Transition states in protein folding kinetics: Modeling Phi-values of small beta-sheet proteins
Small single-domain proteins often exhibit only a single free-energy barrier,
or transition state, between the denatured and the native state. The folding
kinetics of these proteins is usually explored via mutational analysis. A
central question is which structural information on the transition state can be
derived from the mutational data. In this article, we model and structurally
interpret mutational Phi-values for two small beta-sheet proteins, the PIN and
the FBP WW domain. The native structure of these WW domains comprises two
beta-hairpins that form a three-stranded beta-sheet. In our model, we assume
that the transition state consists of two conformations in which either one of
the hairpins is formed. Such a transition state has been recently observed in
Molecular Dynamics folding-unfolding simulations of a small designed
three-stranded beta-sheet protein. We obtain good agreement with the
experimental data (i) by splitting up the mutation-induced free-energy changes
into terms for the two hairpins and for the small hydrophobic core of the
proteins, and (ii) by fitting a single parameter, the relative degree to which
hairpin 1 and 2 are formed in the transition state. The model helps to
understand how mutations affect the folding kinetics of WW domains, and
captures also negative Phi-values that have been difficult to interpret.Comment: 27 pages, 6 pages, 3 tables; to appear in Biophys.
Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl-tRNA synthetase enzymes
Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli . Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein–ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p -benzoyl- L -phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-Α-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable. Proteins 2010. © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71368/1/22706_ftp.pd
How to do an evaluation: pitfalls and traps
The recent literature is replete with papers evaluating computational tools (often those operating on 3D structures) for their performance in a certain set of tasks. Most commonly these papers compare a number of docking tools for their performance in cognate re-docking (pose prediction) and/or virtual screening. Related papers have been published on ligand-based tools: pose prediction by conformer generators and virtual screening using a variety of ligand-based approaches. The reliability of these comparisons is critically affected by a number of factors usually ignored by the authors, including bias in the datasets used in virtual screening, the metrics used to assess performance in virtual screening and pose prediction and errors in crystal structures used
How to do an evaluation: pitfalls and traps
The recent literature is replete with papers evaluating computational tools (often those operating on 3D structures) for their performance in a certain set of tasks. Most commonly these papers compare a number of docking tools for their performance in cognate re-docking (pose prediction) and/or virtual screening. Related papers have been published on ligand-based tools: pose prediction by conformer generators and virtual screening using a variety of ligand-based approaches. The reliability of these comparisons is critically affected by a number of factors usually ignored by the authors, including bias in the datasets used in virtual screening, the metrics used to assess performance in virtual screening and pose prediction and errors in crystal structures used
Exploring the Protein Folding Dynamics of Beta3s with Two-Dimensional Ultraviolet (2DUV) Spectroscopy
Ultraviolet (UV) spectra of proteins originate from electronic excitations of their backbone chromophore and aromatic side chains and provide a sensitive probe of the secondary structure. Recently developed femtosecond lasers allow multidimensional spectroscopy to be extended into the UV regime. Two-dimensional UV (2DUV) techniques, with short pulses, provide a promising tool to study the structures and dynamics of proteins. We combined 2DUV spectroscopy and molecular dynamics generated free energy profiles to simulate the protein electronic transitions and UV photon echo signals to monitor the protein folding process of the small protein Beta3s. Near-ultraviolet (NUV) and far-ultraviolet (FUV) signals illustrate the variation of the 2D correlation plots when the protein evolves along the underlying free energy landscape. Chiral polarization configurations of the NUV and FUV pulses are sensitive to protein structural evolution. This work provides a protocol for applying multidimensional UV spectroscopy to study protein folding. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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