186 research outputs found
The designability of protein switches by chemical rescue of structure: mechanisms of inactivation and reactivation
This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ja407644b.The ability to selectively activate function of particular proteins via pharmacological agents is a longstanding goal in chemical biology. Recently, we reported an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. However, rationally identifying analogous de novo binding sites in other enzymes represents a key challenge for extending this approach to introduce allosteric control into other enzymes. Here we show that mutation sites leading to protein inactivation via tryptophan-to-glycine substitution and allowing (partial) reactivation by the subsequent addition of indole are remarkably frequent. Through a suite of methods including a cell-based reporter assay, computational structure prediction and energetic analysis, fluorescence studies, enzymology, pulse proteolysis, x-ray crystallography and hydrogen-deuterium mass spectrometry we find that these switchable proteins are most commonly modulated indirectly, through control of protein stability. Addition of indole in these cases rescues activity not by reverting a discrete conformational change, as we had observed in the sole previously reported example, but rather rescues activity by restoring protein stability. This important finding will dramatically impact the design of future switches and sensors built by this approach, since evaluating stability differences associated with cavity-forming mutations is a far more tractable task than predicting allosteric conformational changes. By analogy to natural signaling systems, the insights from this study further raise the exciting prospect of modulating stability to design optimal recognition properties into future de novo switches and sensors built through chemical rescue of structure
Identification of novel small molecule Beclin 1 mimetics activating autophagy
Anti-apoptotic proteins Bcl-2 and Bcl-xL could block autophagy by binding to Beclin 1 protein, an essential inducer of autophagy. Compounds mimicking Beclin 1 might be able to disrupt Bcl-xL/2-Beclin 1 interaction, free out Beclin 1, and thus trigger autophagy. In order to identify small molecule Beclin 1 mimetics, a fluorescence polarization-based high-throughput screening of 50,316 compounds was carried out with a Z' score of 0.82 ± 0.05, and an outcome of 58 hits. After the structure analysis, three acridine analogues were unveiled and confirmed using the fluorescence polarization assay and the surface plasmon resonance assay. Moreover, a set of 17 additional acridine analogues was prepared and tested. Compound 7 showed selectivity for Bcl-xL (KD = 6.5 ΌM) over Bcl-2 (KD = 160 ΌM) protein, and potent cytotoxicity (nanomolar scale) in PC-3, PC-3a and DU145 prostate cancer cells. Furthermore, induction of autophagy was also demonstrated in PC-3 and PC-3a cells treated with some acridine compounds by LC3 conversion immunoblotting and LC3 fluorescence microscopy. These Beclin 1 mimetics will be invaluable tools for developing novel autophagy inducers, better understanding the roles of autophagy in cancer, and will contribute to cancer therapy
Dominant Folding Pathways of a WW Domain
We investigate the folding mechanism of the WW domain Fip35 using a realistic
atomistic force field by applying the Dominant Reaction Pathways (DRP)
approach. We find evidence for the existence of two folding pathways, which
differ by the order of formation of the two hairpins. This result is consistent
with the analysis of the experimental data on the folding kinetics of WW
domains and with the results obtained from large-scale molecular dynamics (MD)
simulations of this system. Free-energy calculations performed in two
coarse-grained models support the robustness of our results and suggest that
the qualitative structure of the dominant paths are mostly shaped by the native
interactions. Computing a folding trajectory in atomistic detail only required
about one hour on 48 CPU's. The gain in computational efficiency opens the door
to a systematic investigation of the folding pathways of a large number of
globular proteins
Allostery in Its Many Disguises: From Theory to Applications.
Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used here to provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts
Mechanical Strength of 17 134 Model Proteins and Cysteine Slipknots
A new theoretical survey of proteins' resistance to constant speed stretching
is performed for a set of 17 134 proteins as described by a structure-based
model. The proteins selected have no gaps in their structure determination and
consist of no more than 250 amino acids. Our previous studies have dealt with
7510 proteins of no more than 150 amino acids. The proteins are ranked
according to the strength of the resistance. Most of the predicted top-strength
proteins have not yet been studied experimentally. Architectures and folds
which are likely to yield large forces are identified. New types of potent
force clamps are discovered. They involve disulphide bridges and, in
particular, cysteine slipknots. An effective energy parameter of the model is
estimated by comparing the theoretical data on characteristic forces to the
corresponding experimental values combined with an extrapolation of the
theoretical data to the experimental pulling speeds. These studies provide
guidance for future experiments on single molecule manipulation and should lead
to selection of proteins for applications. A new class of proteins, involving
cystein slipknots, is identified as one that is expected to lead to the
strongest force clamps known. This class is characterized through molecular
dynamics simulations.Comment: 40 pages, 13 PostScript figure
Capturing the essence of folding and functions of biomolecules using Coarse-Grained Models
The distances over which biological molecules and their complexes can
function range from a few nanometres, in the case of folded structures, to
millimetres, for example during chromosome organization. Describing phenomena
that cover such diverse length, and also time scales, requires models that
capture the underlying physics for the particular length scale of interest.
Theoretical ideas, in particular, concepts from polymer physics, have guided
the development of coarse-grained models to study folding of DNA, RNA, and
proteins. More recently, such models and their variants have been applied to
the functions of biological nanomachines. Simulations using coarse-grained
models are now poised to address a wide range of problems in biology.Comment: 37 pages, 8 figure
Manipulating Biopolymer Dynamics by Anisotropic Nanoconfinement
How the geometry of nano-sized confinement affects dynamics of biomaterials
is interesting yet poorly understood. An elucidation of structural details upon
nano-sized confinement may benefit manufacturing pharmaceuticals in biomaterial
sciences and medicine. The behavior of biopolymers in nano-sized confinement is
investigated using coarse-grained models and molecular simulations.
Particularly, we address the effects of shapes of a confinement on protein
folding dynamics by measuring folding rates and dissecting structural
properties of the transition states in nano-sized spheres and ellipsoids. We
find that when the form of a confinement resembles the geometrical properties
of the transition states, the rates of folding kinetics are most enhanced. This
knowledge of shape selectivity in identifying optimal conditions for reactions
will have a broad impact in nanotechnology and pharmaceutical sciences.Comment: to appear in Nano Letter
Natural product derivative Gossypolone inhibits Musashi family of RNA-binding proteins
Background: The Musashi (MSI) family of RNA-binding proteins is best known for the role in post-transcriptional regulation of target mRNAs. Elevated MSI1 levels in a variety of human cancer are associated with up-regulation of Notch/Wnt signaling. MSI1 binds to and negatively regulates translation of Numb and APC (adenomatous polyposis coli), negative regulators of Notch and Wnt signaling respectively. Methods: Previously, we have shown that the natural product (-)-gossypol as the first known small molecule inhibitor of MSI1 that down-regulates Notch/Wnt signaling and inhibits tumor xenograft growth in vivo. Using a fluorescence polarization (FP) competition assay, we identified gossypolone (Gn) with a > 20-fold increase in Ki value compared to (-)-gossypol. We validated Gn binding to MSI1 using surface plasmon resonance, nuclear magnetic resonance, and cellular thermal shift assay, and tested the effects of Gn on colon cancer cells and colon cancer DLD-1 xenografts in nude mice. Results: In colon cancer cells, Gn reduced Notch/Wnt signaling and induced apoptosis. Compared to (-)-gossypol, the same concentration of Gn is less active in all the cell assays tested. To increase Gn bioavailability, we used PEGylated liposomes in our in vivo studies. Gn-lip via tail vein injection inhibited the growth of human colon cancer DLD-1 xenografts in nude mice, as compared to the untreated control (P < 0.01, n = 10). Conclusion: Our data suggest that PEGylation improved the bioavailability of Gn as well as achieved tumor-targeted delivery and controlled release of Gn, which enhanced its overall biocompatibility and drug efficacy in vivo. This provides proof of concept for the development of Gn-lip as a molecular therapy for colon cancer with MSI1/MSI2 overexpression
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