| 1. |
- Bernado, Pau, et al.
(författare)
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Structure and Dynamics of Ribosomal Protein L12: An Ensemble Model Based on SAXS and NMR Relaxation
- 2010
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 98:10, s. 2374-2382
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the N-15 relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome.
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| 2. |
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| 3. |
- 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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| 4. |
- 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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| 5. |
- Dreydoppel, Matthias, et al.
(författare)
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Transition-State Compressibility and Activation Volume of Transient Protein Conformational Fluctuations
- 2021
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Ingår i: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:6, s. 833-842
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Tidskriftsartikel (refereegranskat)abstract
- Proteins are dynamic entities that intermittently depart from their ground-state structures and undergo conformational transitions as a critical part of their functions. Central to understanding such transitions are the structural rearrangements along the connecting pathway, where the transition state plays a special role. Using NMR relaxation at variable temperature and pressure to measure aromatic ring flips inside a protein core, we obtain information on the structure and thermodynamics of the transition state. We show that the isothermal compressibility coefficient of the transition state is similar to that of short-chain hydrocarbon liquids, implying extensive local unfolding of the protein. Our results further indicate that the required local volume expansions of the protein can occur not only with a net positive activation volume of the protein, as expected from previous studies, but also with zero activation volume by compaction of remote void volume, when averaged over the ensemble of states.
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| 6. |
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| 7. |
- Kumar, S, et al.
(författare)
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Trifluoroethanol-induced beta -> alpha transition in beta-lactoglobulin: hydration and cosolvent binding studied by 2H, 17O, and 19F magnetic relaxation dispersion.
- 2003
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 42:46, s. 13708-13716
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Tidskriftsartikel (refereegranskat)abstract
- Alcohols, such as 2,2,2-trifluoroethanol (TFE), have been shown to induce a cooperative transition to an open helical structure in many proteins, but the underlying molecular mechanism has not been identified. Here, we employ the technique of magnetic relaxation dispersion (MRD) to study the TFE-induced transition of -lactoglobulin at pH 2.4. Unlike traditional techniques that focus on protein secondary structure, the MRD method directly monitors the solvent, providing quantitative information about preferential solvation and solvent penetration and about the overall size and structural integrity of the protein. In this multinuclear MRD study, we use the 2H and 17O resonances to examine hydration and the 19F resonance to study TFE. The transformation from the native to the helical state via an intermediate state at 300 K is found to be accompanied by a progressive expansion of the protein and loss of specific long-lived hydration sites. The observation of 17O and 19F dispersions from the helical state shows that water and TFE penetrate the protein. The MRD data indicate a strong accumulation of TFE at the surface as well as in the interior of the protein. At 277 K, BLG is much less affected by TFE, remaining in the native state at 16% TFE, but adopting a nonnative structure at 30% TFE. This nonnative structure is not penetrated by long-lived water molecules. The implications of these findings for the mechanism of TFE-induced structural transformations are discussed.
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| 8. |
- Modig, Kristofer, et al.
(författare)
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Dynamics of protein and peptide hydration
- 2004
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 126:1, s. 102-114
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Tidskriftsartikel (refereegranskat)abstract
- Biological processes often involve the surfaces of proteins, where the structural and dynamic properties of the aqueous solvent are modified. Information about the dynamics of protein hydration can be obtained by measuring the magnetic relaxation dispersion (MRD) of the water 2 H and 17 0 nuclei or by recording the nuclear Overhauser effect (NOE) between water and protein protons. Here, we use the MRD method to study the hydration of the cyclic peptide oxytocin and the globular protein BPTI in deeply supercooled solutions. The results provide a detailed characterization of water dynamics in the hydration layer at the surface of these biomolecules. More than 95% of the water molecules in contact with the biomolecular surface are found to be no more than two-fold motionally retarded as compared to bulk water. In contrast to small nonpolar molecules, the retardation factor for BPTI showed little or no temperature dependence, suggesting that the exposed nonpolar residues do not induce clathrate-like hydrophobic hydration structures. New NOE data for oxytocin and published NOE data for BPTI were analyzed, and a mutually consistent interpretation of MRD and NOE results was achieved with the aid of a new theory of intermolecular dipolar relaxation that accounts explicitly for the dynamic perturbation at the biomolecular surface. The analysis indicates that water-protein NOES are dominated by long-range dipolar couplings to bulk water, unless the monitored protein proton is near a partly or fully buried hydration site where the water molecule has a long residence time.
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| 9. |
- Modig, Kristofer, et al.
(författare)
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High water mobility on the ice-binding surface of a hyperactive antifreeze protein.
- 2010
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Ingår i: Physical chemistry chemical physics : PCCP. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 12:Online 29th July 2010, s. 10189-10197
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Tidskriftsartikel (refereegranskat)abstract
- Antifreeze proteins (AFPs) prevent uncontrolled ice formation in organisms exposed to subzero temperatures by binding irreversibly to specific planes of nascent ice crystals. To understand the thermodynamic driving forces and kinetic mechanism of AFP activity, it is necessary to characterize the hydration behavior of these proteins in solution. With this aim, we have studied the hyperactive insect AFP from Tenebrio molitor (TmAFP) with the (17)O magnetic relaxation dispersion (MRD) method, which selectively monitors the rotational motion and exchange kinetics of water molecules on picosecond-microsecond time scales. The global hydration behavior of TmAFP is found to be similar to non-antifreeze proteins, with no evidence of ice-like or long-ranged modifications of the solvent. However, two sets of structural water molecules, located within the core and on the ice-binding face in the crystal structure of TmAFP, may have functional significance. We find that 2 of the 5 internal water molecules exchange with a residence time of 8 +/- 1 mus at 300 K and a large activation energy of approximately 50 kJ mol(-1), reflecting intermittent large-scale conformational fluctuations in this exceptionally dense and rigid protein. Six water molecules arrayed with ice-like spacing in the central trough on the ice-binding face exchange with bulk water on a sub-nanosecond time scale. The combination of high order and fast exchange may allow these water molecules to contribute entropically to the ice-binding affinity without limiting the absorption rate.
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| 10. |
- Modig, Kristofer, et al.
(författare)
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Model-independent interpretation of NMR relaxation data for unfolded proteins: the acid-denatured state of ACBP
- 2008
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Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 42:3, s. 163-177
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the acid-unfolded state of acyl-coenzyme A binding protein (ACBP) using N-15 laboratory frame nuclear magnetic resonance (NMR) relaxation experiments at three magnetic field strengths. The data have been analyzed using standard model-free fitting and models involving distribution of correlation times. In particular, a model-independent method of analysis that does not assume any analytical form for the correlation time distribution is proposed. This method explains correlations between model-free parameters and the analytical distribution parameters found by other authors. The analysis also shows that the relaxation data are consistent with and complementary to information obtained from other parameters, especially secondary chemical shifts and residual dipolar couplings, and strengthens the conclusions of previous observations that three out of the four regions that form helices in the native structure appear to contain residual secondary structure also in the acid-denatured state.
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