Di Michele M.,VIB |
Di Michele M.,Ghent University |
Stes E.,VIB |
Stes E.,Ghent University |
And 16 more authors.
Journal of Proteome Research | Year: 2015
Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-RafWT and B-RafV600E. The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-RafWT KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-RafWT or B-RafV600E in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases. © 2015 American Chemical Society.
Zhang B.,Karolinska Institutet |
Pirmoradian M.,Karolinska Institutet |
Pirmoradian M.,Biomotif AB |
Chernobrovkin A.,Karolinska Institutet |
Zubarev R.A.,Karolinska Institutet
Molecular and Cellular Proteomics | Year: 2014
Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of "cloning" chimeric tandem spectra for cofragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted cofragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average nine peptide-spectrum matches per second on a Q-Exactive workbench, whereas the actual MS/MS acquisition rate was close to seven spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant "one MS/MS spectrum - one peptide" paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Cherry A.L.,Karolinska Institutet |
Finta C.,Karolinska Institutet |
Karlstrom M.,Karolinska Institutet |
Jin Q.,Karolinska Institutet |
And 9 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013
Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics. © 2013 International Union of Crystallography.
Biomotif Ab | Date: 2013-09-13
The present invention relates to a method tor enriching and/or separating and/or immobilizing an analyte of interest comprising bringing an analyte of interest into contact with a derivatizing agent; incubating said analyte with said derivatizing agent, thereby incorporating a sulphonic acid group or as analogue thereof into the molecular structure of mo analyte of interest; bringing the analyte of interest into contact with a molecularly imprinted polymer with selective affinity foe a sulphonic acid group or an analogue thereof; and enriching and/or separating and/or immobilizing the analyte of interest by nee of the molecularly imprinted polymer. Further disclosed is a kit comprising a derivatizing agent, which contains a sulphonic group or an analogue thereof and a reactive group for creating a covalent bond between said derivatizing agent nod an analyte of interest, and a molecularly imprinted polymer with selective affinity for a sulphonic acid group or an analogue thereof.
Biomotif Ab | Date: 2010-11-16
Apparatus and methods to perform hydrogen/deuterium exchange using semipermeable membranes are described. The system has two channels separated by a semipermeable membrane. One channel comprises a flow carrying the analyte of interest, and the second channel comprises a solution comprising a deuterated solvent (e.g. deuterium oxide). The system does not require an external electric field gradient across the membrane to perform the hydrogen-deuterium exchange procedure. The present invention facilitates sample and reagent handling as well as simplifies manufacture of devices and/or instrumentation related to deuterium exchange. Further described is a chemical analysation device for analysing chemical compositions and/or compounds, and a method for analysing chemical compounds and a computer program product for inducing a computer to perform steps in the method. Also described is a method for analyzing interactions between analytes and charged molecules, and calculating binding coefficients of the analytes with respect to the charged molecules.