| 1. |
- Altincekic, Nadide, et al.
(author)
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Targeting the Main Protease (Mpro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies
- 2024
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In: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 19:2, s. 563-574
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Journal article (peer-reviewed)abstract
- The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.
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| 2. |
- Andersson, Hanna, 1979, et al.
(author)
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Assessing the Ability of Spectroscopic Methods to Determine the Difference in the Folding Propensities of Highly Similar beta-Hairpins
- 2017
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In: Acs Omega. - : American Chemical Society (ACS). - 2470-1343. ; 2:2, s. 508-516
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Journal article (peer-reviewed)abstract
- We have evaluated the ability of nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies to describe the difference in the folding propensities of two structurally highly similar cyclic beta-hairpins, comparing the outcome to that of molecular dynamics simulations. NAMFIS-type NMR ensemble analysis and CD spectroscopy were observed to accurately describe the consequence of altering a single interaction site, whereas a single-site C-13 NMR chemical shift melting curve-based technique was not.
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| 3. |
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| 4. |
- Andersson, Hanna, 1979, et al.
(author)
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Solvent Effects on Nitrogen Chemical Shifts
- 2015
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In: Annual Reports on NMR Spectroscopy. - : Elsevier. - 0066-4103. - 9780128021231 ; 86, s. 73-210
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Research review (peer-reviewed)abstract
- Due to significant developments in cryogenic probe technology and the easy access to inverse detection pulse programmes (HSQC, HMBC), the sensitivity of nitrogen NMR has lately vastly improved. As a consequence, nitrogen NMR has turned into a useful and commonly available tool for solution studies of molecular structure and properties for small organic compounds likewise biopolymers. The high sensitivity of the nitrogen lone pair to changes in the molecular environment, alterations in intra- and intermolecular interactions, and in molecular conformation along with its wide, up to 1200 ppm chemical shift dispersion make nitrogen NMR to an exceptionally sensitive reporter tool. The nitrogen chemical shift has been applied in various fields of chemistry, including for instance the studies of transition metal complexes, chemical reactions such as N-alkylation and N-oxidation, tautomerization, protonation–deprotonation equilibria, hydrogen and halogen bonding, and elucidation of molecular conformation and configuration. The 15N NMR data observed in the investigation of these molecular properties and processes is influenced by the medium it is acquired in. This influence may be due to direct coordination of solvent molecules to transition metal complexes, alteration of tautomerization equilibria, and solvent polarity induced electron density changes of conjugated systems, for example. Thus, the solvent may significantly alter the observed nitrogen NMR shifts. This review aims to provide an overview of solvent effects of practical importance, and discusses selected experimental reports from various subfields of chemistry.
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| 5. |
- Blobel, Jascha, et al.
(author)
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Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins
- 2011
-
In: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 40:12, s. 1327-1338
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Journal article (peer-reviewed)abstract
- Addition of a 50 mM mixture of l-arginine and l-glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.
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| 6. |
- Brath, Ulrika, et al.
(author)
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Differential responses of the backbone and side-chain conformational dynamics in FKBP12 upon binding the transition-state analog FK506: implications for transition-state stabilization and target protein recognition.
- 2009
-
In: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 387:1, s. 233-244
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Journal article (peer-reviewed)abstract
- FKBP12 serves a dual role as a peptidyl-prolyl cis-trans isomerase and as a modulator of several cell signaling pathways. The macrolide FK506 is a transition-state analog of the catalyzed reaction and displaces FKBP12 from its natural target proteins. We compared the conformational exchange dynamics of the backbone and methyl-bearing side chains of FKBP12 in the free and FK506-bound states using NMR relaxation-dispersion experiments. Our results show that the free enzyme exchanges between the ground state and an excited state that resembles the ligand-bound state or Michaelis complex. In FK506-bound FKBP12, the backbone is confined to a single conformation, while conformational exchange prevails for many methyl groups. The residual side-chain dynamics in the transition-state analog-bound state suggests that the transition-state ensemble involves multiple conformations, a finding that challenges the long-standing concept of conformational restriction in the transition-state complex. Furthermore, exchange between alternative conformations is observed in the bound state for an extended network of methyl groups that includes locations remote from the active site. Several of these locations are known to be important for interactions with cellular target proteins, including calcineurin and the ryanodine receptor, suggesting that the conformational heterogeneity might play a role in the promiscuous binding of FKBP12 to different targets.
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| 7. |
- Brath, Ulrika, et al.
(author)
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Functional dynamics of human FKBP12 revealed by methyl C-13 rotating frame relaxation dispersion NMR spectroscopy
- 2006
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 128:17, s. 5718-5727
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Journal article (peer-reviewed)abstract
- Transverse relaxation dispersion NMR spectroscopy can provide atom-specific information about time scales, populations, and the extent of structural reorganization in proteins under equilibrium conditions. A method is described that uses side-chain methyl groups as local reporters for conformational transitions taking place in the microsecond regime. The experiment measures carbon nuclear spin relaxation rates in the presence of continuous wave off-resonance irradiation, in proteins uniformly enriched with C-13, and partially randomly labeled with 2 H. The method was applied to human FK-506 binding protein (FKBP12), which uses a common surface for binding substrates in its dual role as both an immunophilin and folding assistant. Conformational dynamics on a time scale of similar to 130 mu s were detected for methyl groups located in the substrate binding pocket, demonstrating its plasticity in the absence of substrate. The spatial arrangement of affected side-chain atoms suggests that substrate recognition involves the rapid relative movement of the subdomain comprising residues Ala81-Thr96 and that the observed dynamics play an important role in facilitating the interaction of this protein with its many partners, including calcineurin.
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| 8. |
- Brath, Ulrika, et al.
(author)
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Mapping the sevoflurane-binding site of calmodulin
- 2014
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In: Pharmacology Research & Perspectives. - : Wiley. - 2052-1707 .- 2052-1707. ; 2:1
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Journal article (peer-reviewed)abstract
- General anesthetics, with sevoflurane (SF) being the first choice inhalational anesthetic agent, provide reversible, broad depressor effects on the nervous system yet have a narrow margin of safety. As characterization of low-affinity binding interactions of volatile substances is exceptionally challenging with the existing methods, none of the numerous cellular targets proposed as chief protagonists in anesthesia could yet be confirmed. The recognition that most critical functions modulated by volatile anesthetics are under the control of intracellular Ca2+ concentration, which in turn is primarily regulated by calmodulin (CaM), motivated us for characterization of the SF–CaM interaction. Solution NMR (Nuclear Magnetic Resonance) spectroscopy was used to identify SF-binding sites using chemical shift displacement, NOESY and heteronuclear Overhauser enhancement spectroscopy (HOESY) experiments. Binding affinities were measured using ITC (isothermal titration calorimetry). SF binds to both lobes of (Ca2+)4-CaM with low mmol/L affinity whereas no interaction was observed in the absence of Ca2+. SF does not affect the calcium binding of CaM. The structurally closely related SF and isoflurane are shown to bind to the same clefts. The SF-binding clefts overlap with the binding sites of physiologically relevant ion channels and bioactive small molecules, but the binding affinity suggests it could only interfere with very weak CaM targets.
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| 9. |
- Brath, Ulrika
(author)
-
Molecular dynamics studied by NMR relaxation experiments. Characterization of functional dynamics in the FK506 binding protein FKBP12.
- 2008
-
Doctoral thesis (other academic/artistic)abstract
- The presented thesis work is concerned with the study of molecular dynamics using liquid nuclear magnetic resonance (NMR) spectroscopy. Spin relaxation dispersion experiments in the rotating frame (R1ρ) are employed to provide a detailed, atomic-resolution view of protein dynamics. Comprehensive investigations of 15N backbone and 13C methyl side-chain conformational exchange in the FK506 binding protein, FKBP12, were carried out. The human enzyme FKBP12 catalyzes the cis-trans isomerization of peptidyl-prolyl linkages in proteins, thereby increasing the rate of protein folding. Recombinant protein production with 13C/2H-enrichment yielded 13CHD2 isotopomers and allowed for development of novel R1ρ experiments aimed to characterize the methyl-bearing side-chains. The joint analysis of backbone and side-chain dynamics revealed a collective conformational exchange process with a correlation time of 121 ± 26 microseconds in the free enzyme. The locations of the exchanging nuclei showed close correspondence with residues previously shown to be important for the catalytic activity, which emphasize the connection between protein function and dynamics. The FKBP12 isomerase activity is strongly inhibited by the small organic molecule FK506. In addition, the bound conformation of FK506 exhibit distinct structural transition state analog characteristics. Conformational exchange dynamics studies of the FKBP12 – FK506 complex demonstrated a striking rigidity of the backbone atoms, in line with the established theory that the transition state is more constrained than the free state. Conformational exchange dynamics found for peripheral methyl groups is suggested to play a role in the subsequent binding of the FKBP12 – FK506 complex to other target molecules. A specific isotope labeling strategy using [1-13C]-glucose was shown to produce isolated 13C nuclei in the side-chains of Phe/Tyr/Trp/His. Proteins with this characteristic labeling provide nuclei amenable to R1ρ experiments, which were previously excluded from analysis due to complications arising from uniform carbon labeling. The method thus extended the collection of dynamic probes to include the frequently occurring and often functionally important aromatic side-chains. R1ρ studies of the C-terminal part of a mutant Calmodulin protein revealed microsecond time scale dynamics contributions to Tyr80Cδ, confirming previous reports of conformational exchange present for the protein backbone nuclei. Detection of conformational exchange in isotropic media requires a chemical shift difference between the exchanging states. However, such chemical shift difference can be replaced by differential residual dipolar couplings (RDC) in the exchanging states for molecules dissolved in partially aligning media. The RDC-induced contribution to the exchange-free transverse relaxation rate can be suppressed using relaxation dispersion experiments, thus revealing details of the conformational exchange characteristics. The theory was described, and the method was demonstrated using the model molecule N,N-dimethyltrichloroacetamide.
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| 10. |
- Brath, Ulrika, et al.
(author)
-
Paramagnetic ligand tagging to identify protein binding sites
- 2015
-
In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 137:35, s. 11391-11398
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Journal article (peer-reviewed)abstract
- Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity.
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| 11. |
- Carlsson, Anna-Carin, 1976, et al.
(author)
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Solvent Effects on Halogen Bond Symmetry
- 2013
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In: CrysteEngComm. - 1466-8033. ; 15:16, s. 3087-3092
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Journal article (peer-reviewed)abstract
- The symmetric arrangement of the iodine and bromine centred 3-center–4-electron halogen bond is revealed to remain preferred in a polar, aprotic solvent environment. Acetonitrile is unable to compete with pyridine for halogen bonding; however, its polarity weakly modulates the energy of the interaction and influences IPE-NMR experiments.
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| 12. |
- Carlsson, Anna-Carin, 1976, et al.
(author)
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Symmetric Halogen Bonding is Preferred in Solution
- 2012
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 134, s. 5706-5715
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Journal article (peer-reviewed)abstract
- Halogen bonding is a recently rediscovered secondary interaction that shows potential to become a complementary molecular tool to hydrogen bonding in rational drug design and in material sciences. Whereas hydrogen bond symmetry has been the subject of systematic studies for decades, the understanding of the analogous three-center halogen bonds is yet in its infancy. The isotopic perturbation of equilibrium (IPE) technique with 13C NMR detection was applied to regioselectively deuterated pyridine complexes to investigate the symmetry of [N−I−N]+ and [N−Br−N]+ halogen bonding in solution. Preference for a symmetric arrangement was observed for both a freely adjustable and for a conformationally restricted [N−X−N]+ model system, as also confirmed by computation on the DFT level. A closely attached counterion is shown to be compatible with the preferred symmetric arrangement. The experimental observations and computational predictions reveal a high energetic gain upon formation of symmetric, three-center four-electron halogen bonding. Whereas hydrogen bonds are generally asymmetric in solution and symmetric in the crystalline state, the analogous bromine and iodine centered halogen bonds prefer symmetric arrangement in solution.
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| 13. |
- Danelius, Emma, et al.
(author)
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Insight into β-Hairpin Stability: Interstrand Hydrogen Bonding
- 2013
-
In: Synlett : Accounts and Rapid Communications in Synthetic Organic Chemistry. - : Georg Thieme Verlag KG. - 0936-5214 .- 1437-2096. ; 24:18, s. 2407-2410
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Journal article (peer-reviewed)abstract
- For evaluation of the role of interstrand hydrogen bonding for β-hairpin stability, two cyclic peptides differing only in side chain hydroxy-to-methyl substitution were designed and synthesized on solid phase following the Fmoc-t-Bu-Trt protection strategy. Subsequent to cyclization in solution, combined computational and experimental ensemble analysis revealed higher conformational stability of the peptide capable of interstrand hydrogen bonding.
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| 14. |
- Gustavsson, Emil, 1987, et al.
(author)
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Modulation of Structural Heterogeneity Controls Phytochrome Photoswitching
- 2020
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 118:2, s. 415-421
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Journal article (peer-reviewed)abstract
- Phytochromes sense red/far-red light and control many biological processes in plants, fungi, and bacteria. Although the crystal structures of dark- and light-adapted states have been determined, the molecular mechanisms underlying photoactivation remain elusive. Here, we demonstrate that the conserved tongue region of the PHY domain of a 57-kDa photosensory module of Deinococcus radiodurans phytochrome changes from a structurally heterogeneous dark state to an ordered, light-activated state. The results were obtained in solution by utilizing a laser-triggered activation approach detected on the atomic level with high-resolution protein NMR spectroscopy. The data suggest that photosignaling of phytochromes relies on careful modulation of structural heterogeneity of the PHY tongue.
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| 15. |
- Igumenova, Tatyana I., et al.
(author)
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Characterization of Chemical Exchange Using Residual Dipolar Coupling
- 2007
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 129:44, s. 13396-13396
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Journal article (peer-reviewed)abstract
- Abstract in Undetermined NMR line shape analysis and relaxation dispersion measurements for N,N-dimethyltrichloroacetamide (DMTCA) weakly aligned using poly-γ-benzyl-l-glutamate, which forms a lyotropic nematic phase when dissolved in chloroform, were used to characterize chemical exchange kinetics for the rotation around the C−N amide bond. At low temperatures (<312 K), slow-exchange 1H-coupled 13C NMR spectra show two resolved 13C methyl quartets with different residual dipolar coupling constants (RDCs). At high temperatures (>312 K), a single population-averaged 13C methyl quartet is observed; in this regime, the differences in RDCs contribute to differential line broadening of the quartet components. Self-consistent measurements of the difference in RDCs are obtained from both line shape and relaxation dispersion techniques. The results show that NMR spectroscopy of weakly aligned molecules allows complete characterization of chemical exchange processes using RDCs, even if exchange broadening is absent in isotropic samples.
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| 16. |
- Isaksson, Linnéa, et al.
(author)
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Signaling Mechanism of Phytochromes in Solution.
- 2021
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In: Structure. - : Elsevier BV. - 1878-4186 .- 0969-2126. ; 29:2, s. 151-160
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Journal article (peer-reviewed)abstract
- Phytochrome proteins guide the red/far-red photoresponse of plants, fungi, and bacteria. Crystal structures suggest that the mechanism of signal transduction from the chromophore to the output domains involves refolding of the so-called PHY tongue. It is currently not clear how the two other notable structural features of the phytochrome superfamily, the so-called helical spine and a knot in the peptide chain, are involved in photoconversion. Here, we present solution NMR data of the complete photosensory core module from Deinococcus radiodurans. Photoswitching between the resting and the active states induces changes in amide chemical shifts, residual dipolar couplings, and relaxation dynamics. All observables indicate a photoinduced structural change in the knot region and lower part of the helical spine. This implies that a conformational signal is transduced from the chromophore to the helical spine through the PAS and GAF domains. The discovered pathway underpins functional studies of plant phytochromes and may explain photosensing by phytochromes under biological conditions.
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| 17. |
- Jensen, Maja, 1978, et al.
(author)
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Survivin prevents the polycomb repressor complex 2 from methylating histone 3 lysine 27
- 2023
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In: Iscience. ; 26:7
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Journal article (peer-reviewed)abstract
- This study investigates the role of survivin in epigenetic control of gene transcription through interaction with the polycomb repressive complex 2 (PRC2). PRC2 is responsible for silencing gene expression by trimethylating lysine 27 on histone 3. We observed differential expression of PRC2 subunits in CD4(+) T cells with varying levels of survivin expression, and ChIP-seq results indicated that survivin colocalizes with PRC2 along DNA. Inhibition of survivin resulted in a significant increase in H3K27 trimethylation, implying that survivin prevents PRC2 from functioning. Peptide microarray showed that survivin interacts with peptides from PRC2 subunits, and machine learning revealed that amino acid composition contains relevant information for predicting survivin interaction. NMR and BLI experiments supported the interaction of survivin with PRC2 subunit EZH2. Finally, protein-protein docking revealed that the survivin-EZH2 interaction interface overlaps with catalytic residues of EZH2, potentially inhibiting its H3K27 methylation activity. These findings suggest that survivin inhibits PRC2 function.
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| 18. |
- Niebling, Stephan, et al.
(author)
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The Impact of Interchain Hydrogen Bonding on b-Hairpin Stability is Readily Predicted by Molecular Dynamics Simulation
- 2015
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In: Peptide Science. - : Wiley. - 1097-0282 .- 0006-3525. ; 104:6, s. 703-706
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Journal article (peer-reviewed)abstract
- Peptides are frequently used model systems for protein folding. They are also gaining increased importance as therapeutics. Here, the ability of molecular dynamics (MD) simulation for describing the structure and dynamics of β-hairpin peptides was investigated, with special attention given to the impact of a single interstrand sidechain to sidechain interaction. The MD trajectories were compared to structural information gained from solution NMR. By assigning frames from restraint-free MD simulations to an intuitive hydrogen bond on/off pattern, folding ratios and folding pathways were predicted. The computed molecular model successfully reproduces the folding ratios determined by NMR, indicating that MD simulation may be straightforwardly used as a screening tool in β-hairpin design.
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| 19. |
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| 20. |
- Wallerstein, Johan, et al.
(author)
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Entropy-Entropy Compensation between the Protein, Ligand, and Solvent Degrees of Freedom Fine-Tunes Affinity in Ligand Binding to Galectin-3C
- 2021
-
In: Jacs Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:4, s. 484-500
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Journal article (peer-reviewed)abstract
- Molecular recognition is fundamental to biological signaling. A central question is how individual interactions between molecular moieties affect the thermodynamics of ligand binding to proteins and how these effects might propagate beyond the immediate neighborhood of the binding site. Here, we investigate this question by introducing minor changes in ligand structure and characterizing the effects of these on ligand affinity to the carbohydrate recognition domain of galectin-3, using a combination of isothermal titration calorimetry, X-ray crystallography, NMR relaxation, and computational approaches including molecular dynamics (MD) simulations and grid inhomogeneous solvation theory (GIST). We studied a congeneric series of ligands with a fluorophenyl-triazole moiety, where the fluorine substituent varies between the ortho, meta, and para positions (denoted O, M, and P). The M and P ligands have similar affinities, whereas the O ligand has 3-fold lower affinity, reflecting differences in binding enthalpy and entropy. The results reveal surprising differences in conformational and solvation entropy among the three complexes. NMR backbone order parameters show that the O-bound protein has reduced conformational entropy compared to the M and P complexes. By contrast, the bound ligand is more flexible in the O complex, as determined by F-19 NMR relaxation, ensemble-refined X-ray diffraction data, and MD simulations. Furthermore, GIST calculations indicate that the O-bound complex has less unfavorable solvation entropy compared to the other two complexes. Thus, the results indicate compensatory effects from ligand conformational entropy and water entropy, on the one hand, and protein conformational entropy, on the other hand. Taken together, these different contributions amount to entropy-entropy compensation among the system components involved in ligand binding to a target protein.
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| 21. |
- Weininger, Ulrich, et al.
(author)
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Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection
- 2014
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In: Journal of Biomolecular NMR. - : Kluwer Academic Publishers. - 0925-2738 .- 1573-5001. ; 59:1, s. 23-29
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Journal article (peer-reviewed)abstract
- Protein dynamics on the microsecond-millisecond time scales often play a critical role in biological function. NMR relaxation dispersion experiments are powerful approaches for investigating biologically relevant dynamics with site-specific resolution, as shown by a growing number of publications on enzyme catalysis, protein folding, ligand binding, and allostery. To date, the majority of studies has probed the backbone amides or side-chain methyl groups, while experiments targeting other sites have been used more sparingly. Aromatic side chains are useful probes of protein dynamics, because they are over-represented in protein binding interfaces, have important catalytic roles in enzymes, and form a sizable part of the protein interior. Here we present an off-resonance R1ρ experiment for measuring microsecond to millisecond conformational exchange of aromatic side chains in selectively 13C labeled proteins by means of longitudinal- and transverse-relaxation optimization. Using selective excitation and inversion of the narrow component of the 13C doublet, the experiment achieves significant sensitivity enhancement in terms of both signal intensity and the fractional contribution from exchange to transverse relaxation; additional signal enhancement is achieved by optimizing the longitudinal relaxation recovery of the covalently attached 1H spins. We validated the L-TROSY-selected R1ρ experiment by measuring exchange parameters for Y23 in bovine pancreatic trypsin inhibitor at a temperature of 328 K, where the ring flip is in the fast exchange regime with a mean waiting time between flips of 320 μs. The determined chemical shift difference matches perfectly with that measured from the NMR spectrum at lower temperatures, where separate peaks are observed for the two sites. We further show that potentially complicating effects of strong scalar coupling between protons (Weininger et al. in J Phys Chem B 117: 9241-9247, 2013b) can be accounted for using a simple expression, and provide recommendations for data acquisition when the studied system exhibits this behavior. The present method extends the repertoire of relaxation methods tailored for aromatic side chains by enabling studies of faster processes and improved control over artifacts due to strong coupling. © 2014 The Author(s).
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