| 1. |
- Blobel, Jascha, et al.
(författare)
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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
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 40:12, s. 1327-1338
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Block, Keith I., et al.
(författare)
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Designing a broad-spectrum integrative approach for cancer prevention and treatment
- 2015
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Ingår i: Seminars in Cancer Biology. - : Academic Press. - 1044-579X .- 1096-3650. ; 35, s. S276-S304
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Forskningsöversikt (refereegranskat)abstract
- Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity "broadspectrum" therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested, many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to be relatively inexpensive, it should help us address stages and types of cancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for future research is offered. (C) 2015 The Authors. Published by Elsevier Ltd.
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| 3. |
- Diehl, Carl
(författare)
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Conformational Entropy and Protein Flexibility in Drug Design Studied by NMR Spectroscopy
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are complex molecules, present in all of the vital functions of life. The function of a protein is regulated by interactions between protein and other molecules. Drug design in pharmaceutical science aims to regulate the function of a protein by the design of synthesized molecules that binds to a protein with high affinity. A change in protein flexibility upon ligand binding can provide significant effects on the affinity of the ligand, where the change in flexibility can be related to entropy, a fundamental thermodynamic parameter. The focus of this thesis has been the characterization of ligand-induced changes in conformational entropy using nuclear magnetic resonance (NMR) spectroscopy experiments in combination with other techniques. NMR relaxation experiments and molecular dynamics (MD) simulations were used to characterize changes in the dynamics of backbone and side chain amides in the carbohydrate recognition domain of Galectin-3 (Gal3) upon binding of lactose. Order parameters determined from NMR relaxation experiments and MD simulations for backbone amides agreed qualitatively and showed an increased flexibility upon binding lactose, thus giving a significant and favourable contribution from conformational entropy to the free energy of binding. Using a combination of NMR spectroscopy, isothermal titration calorimetry (ITC), and X-ray crystallography, we characterized the binding of three ligands to Gal3, where the dissociation constants ranged over 2 orders of magnitude. 15N and 2H spin relaxation measurements showed that protein backbone and side chains respond to ligand binding by, on average, increased conformational fluctuations, where the response in increased fluctuations differs for the three ligands. The results reveal an intricate interplay between structure and conformational fluctuations in the ligand-bound complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. The binding of matrix metalloprotease 12 (MMP12) was dissected using two enantiomeric inhibitors, RR and SS, with dissociation constants differing by one order of magnitude. Enantiomers have identical physical properties and chemical potentials in solvent. Thus, any differences between two enantiomers in their binding thermodynamics must be due to properties in the bound states. The inhibitors represent a novel class of MMP12 inhibitors, called hydroxy-hydantoins that comprise a weak zinc-binding group, a hydantoin, and a lipophilic biphenyl moiety, which is connected to the hydantoin via a carbinol linker. The binding was characterized using a combination of NMR spectroscopy, ITC, X-ray spectroscopy and MD-simulations. ITC shows highly separate thermodynamic patterns for the two enantiomers upon binding, due to a 180 degree flip of the zinc-coordinating hydantoin ring between RR- and SS-MMP12. The binding of the stronger inhibitor RR is driven by a favourable enthalpic contribution to the free energy of binding, whereas the binding of the weaker inhibitor SS is driven by an entropic contribution. NMR relaxation experiments and MD simulations indicate a favourable contribution to the free energy of binding from the change in protein flexibility upon binding the stronger inhibitor RR in comparison to the weaker inhibitor SS, which is supported by thermodynamic integration using MD simulations. Thus, the change in conformational entropy for binding RR is favourable, whereas the total change in entropy for binding is more favourable for binding SS, yielding conflicting results regarding the entropic contributions to the free energy of binding. The binding process of Gal3 was characterized using CPMG relaxation dispersion experiments for 3 different ligands. The apo-state of Gal3 exhibits intrinsic conformational exchange, where a minor population samples the bound state. The bound-states of Gal3 exhibited conformational exchange, where fitted chemical shifts from relaxation data correlates with chemical shift differences between ligand- and apo-states. Off-rates for the ligands correlate with the dissociation constants of the ligands, thus indicating that on-rates are identical and diffusion controlled for all ligands. The off-rates for the designed ligands are significantly slower, demonstrating that these stabilize the bound conformation, but do not affect the transition barrier of ligand binding. A 3-state binding process is proposed, where apo-Gal3 is present in a non-binding ground-state which samples a binding competent high-energy state.
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| 4. |
- Diehl, Carl, et al.
(författare)
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Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3.
- 2009
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Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 45:1-2, s. 157-169
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Tidskriftsartikel (refereegranskat)abstract
- The conformational entropy of proteins can make significant contributions to the free energy of ligand binding. NMR spin relaxation enables site-specific investigation of conformational entropy, via order parameters that parameterize local reorientational fluctuations of rank-2 tensors. Here we have probed the conformational entropy of lactose binding to the carbohydrate recognition domain of galectin-3 (Gal3), a protein that plays an important role in cell growth, cell differentiation, cell cycle regulation, and apoptosis, making it a potential target for therapeutic intervention in inflammation and cancer. We used (15)N spin relaxation experiments and molecular dynamics simulations to monitor the backbone amides and secondary amines of the tryptophan and arginine side chains in the ligand-free and lactose-bound states of Gal3. Overall, we observe good agreement between the experimental and computed order parameters of the ligand-free and lactose-bound states. Thus, the (15)N spin relaxation data indicate that the molecular dynamics simulations provide reliable information on the conformational entropy of the binding process. The molecular dynamics simulations reveal a correlation between the simulated order parameters and residue-specific backbone entropy, re-emphasizing that order parameters provide useful estimates of local conformational entropy. The present results show that the protein backbone exhibits an increase in conformational entropy upon binding lactose, without any accompanying structural changes.
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| 5. |
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| 6. |
- Diehl, Carl, et al.
(författare)
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Protein Flexibility and Conformational Entropy in Ligand Design Targeting the Carbohydrate Recognition Domain of Galectin-3.
- 2010
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 132, s. 14577-14589
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Tidskriftsartikel (refereegranskat)abstract
- Rational drug design is predicated on knowledge of the three-dimensional structure of the protein-ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. (15)N and (2)H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein-carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.
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| 7. |
- Diehl, Carl, et al.
(författare)
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Structural analysis of a complex between small ubiquitin-like modifier 1 (SUMO1) and the ZZ domain of CREB-binding protein (CBP/p300) reveals a new interaction surface on SUMO
- 2016
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 291:24, s. 12658-12672
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Tidskriftsartikel (refereegranskat)abstract
- We have recently discovered that the ZZ zinc finger domain represents a novel small ubiquitin-like modifier (SUMO) binding motif. In this study we identify the binding epitopes in the ZZ domain of CBP (CREB-binding protein) and SUMO1 using NMR spectroscopy. The binding site on SUMO1 represents a unique epitope for SUMO interaction spatially opposite to that observed for canonical SUMO interaction motifs (SIMs). HADDOCK docking simulations using chemical shift perturbations and residual dipolar couplings was employed to obtain a structural model for the ZZ domain-SUMO1 complex. Isothermal titration calorimetry experiments support this model by showing that the mutation of key residues in the binding site abolishes binding and that SUMO1 can simultaneously and noncooperatively bind both the ZZ domain and a canonical SIM motif. The binding dynamics of SUMO1 was further characterized using 15N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersions, which define the off rates for the ZZ domain and SIM motif and show that the dynamic binding process has different characteristics for the two cases. Furthermore, in the absence of bound ligands SUMO1 transiently samples a high energy conformation, which might be involved in ligand binding.
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| 8. |
- Diehl, Carl, et al.
(författare)
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Structure and Interactions of a Dimeric Variant of sHIP, a Novel Virulence Determinant of Streptococcus pyogenes.
- 2016
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes is one of the most significant bacterial pathogens in the human population mostly causing superficial and uncomplicated infections (pharyngitis and impetigo) but also invasive and life-threatening disease. We have previously identified a virulence determinant, protein sHIP, which is secreted at higher levels by an invasive compared to a non-invasive strain of S. pyogenes. The present work presents a further characterization of the structural and functional properties of this bacterial protein. Biophysical and structural studies have shown that protein sHIP forms stable tetramers both in the crystal and in solution. The tetramers are composed of four helix-loop-helix motifs with the loop regions connecting the helices displaying a high degree of flexibility. Owing to interactions at the tetramer interface, the observed tetramer can be described as a dimer of dimers. We identified three residues at the tetramer interface (Leu84, Leu88, Tyr95), which due to largely non-polar side-chains, could be important determinants for protein oligomerization. Based on these observations, we produced a sHIP variant in which these residues were mutated to alanines. Biophysical experiments clearly indicated that the sHIP mutant appear only as dimers in solution confirming the importance of the interfacial residues for protein oligomerisation. Furthermore, we could show that the sHIP mutant interacts with intact histidine-rich glycoprotein (HRG) and the histidine-rich repeats in HRG, and inhibits their antibacterial activity to the same or even higher extent as compared to the wild type protein sHIP. We determined the crystal structure of the sHIP mutant, which, as a result of the high quality of the data, allowed us to improve the existing structural model of the protein. Finally, by employing NMR spectroscopy in solution, we generated a model for the complex between the sHIP mutant and an HRG-derived heparin-binding peptide, providing further molecular details into the interactions involving protein sHIP.
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| 9. |
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| 10. |
- Genheden, Samuel, et al.
(författare)
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Starting-Condition Dependence of Order Parameters Derived from Molecular Dynamics Simulations
- 2010
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Ingår i: Journal of Chemical Theory and Computation. - : American Chemical Society (ACS). - 1549-9618 .- 1549-9626. ; 6:7, s. 2176-2190
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Tidskriftsartikel (refereegranskat)abstract
- We have studied how backbone N-H S-2 order parameters calculated from molecular dynamics simulations depend on the method used to calculate them, the starting conditions, and the length of the simulations. Using the carbohydrate binding domain of galectin-3 in the free and lactose-bound states as a test case, we compared the calculated order parameters with experimental data from NMR relaxation. The results indicate that the sampling can be improved by using several starting structures, taking into account conformational heterogeneity reported in crystal structures. However, the improvement is rather limited, and for 93% of the dihedrals that have alternative conformations in the crystal structures, the conformational space is well sampled even if a single conformation is used as the starting structure. Moreover, the agreement with experimental data is improved when using several short simulations, rather than a single long simulation. In the present case, we find that similar to 10 independent simulations provide sufficient sampling, and the ideal length of the simulations is similar to 10 ns, which is similar to 25% longer than the global correlation time for rotational diffusion. On the other hand, the equilibration time appears to be less important, and our results suggest that an equilibration time of 0.25 ns is sufficient. We have also compared four different methods to extract the order parameters from the simulations, namely, the autocorrelation function and isotropic reorientational eigenmode dynamics using three different window sizes. Overall, the four methods yield comparable results, but large differences between the methods may serve to pinpoint cases for which the calculated parameters are unreliable.
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