| 1. |
- Blobel, Jascha, et al.
(författare)
-
Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins
- 2011
-
Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 40:12, s. 1327-1338
-
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.
|
|
| 2. |
- Block, Keith I., et al.
(författare)
-
Designing a broad-spectrum integrative approach for cancer prevention and treatment
- 2015
-
Ingår i: Seminars in Cancer Biology. - : Academic Press. - 1044-579X .- 1096-3650. ; 35, s. S276-S304
-
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.
|
|
| 3. |
- Diehl, Carl
(författare)
-
Conformational Entropy and Protein Flexibility in Drug Design Studied by NMR Spectroscopy
- 2010
-
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.
|
|
| 4. |
- Diehl, Carl, et al.
(författare)
-
Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3.
- 2009
-
Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 45:1-2, s. 157-169
-
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.
|
|
| 5. |
|
|
| 6. |
- Diehl, Carl, et al.
(författare)
-
Protein Flexibility and Conformational Entropy in Ligand Design Targeting the Carbohydrate Recognition Domain of Galectin-3.
- 2010
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 132, s. 14577-14589
-
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.
|
|
| 7. |
- Diehl, Carl, et al.
(författare)
-
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
-
Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 291:24, s. 12658-12672
-
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.
|
|
| 8. |
- Diehl, Carl, et al.
(författare)
-
Structure and Interactions of a Dimeric Variant of sHIP, a Novel Virulence Determinant of Streptococcus pyogenes.
- 2016
-
Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 7
-
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.
|
|
| 9. |
|
|
| 10. |
- Genheden, Samuel, et al.
(författare)
-
Starting-Condition Dependence of Order Parameters Derived from Molecular Dynamics Simulations
- 2010
-
Ingår i: Journal of Chemical Theory and Computation. - : American Chemical Society (ACS). - 1549-9618 .- 1549-9626. ; 6:7, s. 2176-2190
-
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.
|
|
| 11. |
- Hassan, Mujtaba, et al.
(författare)
-
Benzimidazole–galactosides bind selectively to the Galectin-8 N-Terminal domain : Structure-based design and optimisation
- 2021
-
Ingår i: European Journal of Medicinal Chemistry. - : Elsevier BV. - 0223-5234. ; 223
-
Tidskriftsartikel (refereegranskat)abstract
- We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a previously reported quinoline–galactoside ligand at a resolution of 1.6 Å. Based on this X-ray structure, a collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole moieties were designed and synthesised. This led to the discovery of a 3-O-(N-methylbenzimidazolylmethyl)–galactoside with a Kd of 1.8 μM for galectin-8N, the most potent selective synthetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole–galactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-derivatised galactosides represent promising compounds for studies of the pathological implications of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory therapeutics targeting galectin-8.
|
|
| 12. |
- Hjalte, Johanna, et al.
(författare)
-
Modulating protein unfolding and refolding via the synergistic association of an anionic and a nonionic surfactant
- 2024
-
Ingår i: Journal of Colloid and Interface Science. - 0021-9797. ; 672, s. 244-255
-
Tidskriftsartikel (refereegranskat)abstract
- Hypothesis: Nonionic surfactants can counter the deleterious effect that anionic surfactants have on proteins, where the folded states are retrieved from a previously unfolded state. However, further studies are required to refine our understanding of the underlying mechanism of the refolding process. While interactions between nonionic surfactants and tightly folded proteins are not anticipated, we hypothesized that intermediate stages of surfactant-induced unfolding could define new interaction mechanisms by which nonionic surfactants can further alter protein conformation. Experiments: In this work, the behavior of three model proteins (human growth hormone, bovine serum albumin, and β-lactoglobulin) was investigated in the presence of the anionic surfactant sodium dodecylsulfate, the nonionic surfactant β-dodecylmaltoside, and mixtures of both surfactants. The transitions occurring to the proteins were determined using intrinsic fluorescence spectroscopy and far-UV circular dichroism. Based on these results, we developed a detailed interaction model for human growth hormone. Using nuclear magnetic resonance and contrast-variation small-angle neutron scattering, we studied the amino acid environment and the conformational state of the protein. Findings: The results demonstrate the key role of surfactant cooperation in defining the conformational state of the proteins, which can shift away or toward the folded state depending on the nonionic-to-ionic surfactant ratio. Dodecylmaltoside, initially a non-interacting surfactant, can unexpectedly associate with sodium dodecylsulfate-unfolded proteins to further impact their conformation at low nonionic-to-ionic surfactant ratio. When this ratio increases, the protein begins to retrieve the folded state. However, the native conformation cannot be fully recovered due to remnant surfactant molecules still adsorbed to the protein. This study demonstrates that the conformational landscape of the protein depends on a delicate interplay between the surfactants, ultimately controlled by the ratio between them, resulting in unpredictable changes in the protein conformation.
|
|
| 13. |
- Kadhirvel, Saraboji, et al.
(författare)
-
The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics
- 2012
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:1, s. 296-306
-
Tidskriftsartikel (refereegranskat)abstract
- The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultrahigh-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 angstrom at 100 K, 1.25 angstrom at 298 K) and in complex with lactose (0.86 angstrom) or glycerol (0.9 angstrom). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of beta-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.
|
|
| 14. |
|
|
| 15. |
- Lindman, Stina, et al.
(författare)
-
pK(a) Values for the Unfolded State under Native Conditions Explain the pH-Dependent Stability of PGB1.
- 2010
-
Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 99:10, s. 3365-3373
-
Tidskriftsartikel (refereegranskat)abstract
- Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pK(a) values. However, pK(a) values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pK(a) values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pK(a) values were determined for carboxyl groups by monitoring their pH-dependent (13)C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pK(a) values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pK(a) values of the folded and unfolded states and compared to experimental stability data. The use of experimental pK(a) values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone.
|
|
| 16. |
- Petruk, Ganna, et al.
(författare)
-
Targeting Toll-like receptor-driven systemic inflammation by engineering an innate structural fold into drugs
- 2023
-
Ingår i: Nature Communications. - : Springer. - 2041-1723. ; 14, s. 1-20
-
Tidskriftsartikel (refereegranskat)abstract
- There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.
|
|
| 17. |
- Sanchez-Fernandez, Adrian, et al.
(författare)
-
An integrative toolbox to unlock the structure and dynamics of protein-surfactant complexes
- 2020
-
Ingår i: Nanoscale Advances. - : Royal Society of Chemistry (RSC). - 2516-0230. ; 2:9, s. 4011-4023
-
Tidskriftsartikel (refereegranskat)abstract
- The interactions between protein and surfactants play an important role in the stability and performance of formulated products. Due to the high complexity of such interactions, multi-technique approaches are required to study these systems. Here, an integrative approach is used to investigate the various interactions in a model system composed of human growth hormone and sodium dodecyl sulfate. Contrast variation small-angle neutron scattering was used to obtain information on the structure of the protein, surfactant aggregates and surfactant-protein complexes. 1H and 1H-13C HSQC nuclear magnetic resonance spectroscopy was employed to probe the local structure and dynamics of specific amino acids upon surfactant addition. Through the combination of these advanced methods with fluorescence spectroscopy, circular dichroism and isothermal titration calorimetry, it was possible to identify the interaction mechanisms between the surfactant and the protein in the pre- and post-micellar regimes, and interconnect the results from different techniques. As such, the protein was revealed to evolve from a partially unfolded conformation at low SDS concentration to a molten globule at intermediate concentrations, where the protein conformation and local dynamics of hydrophobic amino acids are partially affected compared to the native state. At higher surfactant concentrations the local structure of the protein appears disrupted, and a decorated micelle structure is observed, where the protein is wrapped around a surfactant assembly. Importantly, this integrative approach allows for the identification of the characteristic fingerprints of complex transitions as seen by each technique, and establishes a methodology for an in-detail study of surfactant-protein systems. This journal is
|
|
| 18. |
- Solomentsev, Gleb, et al.
(författare)
-
Conformational Entropy of FK506 Binding to FKBP12 Determined by Nuclear Magnetic Resonance Relaxation and Molecular Dynamics Simulations
- 2018
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 57:9, s. 1451-1461
-
Tidskriftsartikel (refereegranskat)abstract
- FKBP12 (FK506 binding protein 12 kDa) is an important drug target. Nuclear magnetic resonance (NMR) order parameters, describing amplitudes of motion on the pico- to nanosecond time scale, can provide estimates of changes in conformational entropy upon ligand binding. Here we report backbone and methyl-axis order parameters of the apo and FK506-bound forms of FKBP12, based on 15N and 2H NMR relaxation. Binding of FK506 to FKBP12 results in localized changes in order parameters, notably for the backbone of residues E54 and I56 and the side chains of I56, I90, and I91, all positioned in the binding site. The order parameters increase slightly upon FK506 binding, indicating an unfavorable entropic contribution to binding of TΔS = -18 ± 2 kJ/mol at 293 K. Molecular dynamics simulations indicate a change in conformational entropy, associated with all dihedral angles, of TΔS = -26 ± 9 kJ/mol. Both these values are significant compared to the total entropy of binding determined by isothermal titration calorimetry and referenced to a reactant concentration of 1 mM (TΔS = -29 ± 1 kJ/mol). Our results reveal subtle differences in the response to ligand binding compared to that of the previously studied rapamycin-FKBP12 complex, despite the high degree of structural homology between the two complexes and their nearly identical ligand-FKBP12 interactions. These results highlight the delicate dependence of protein dynamics on drug interactions, which goes beyond the view provided by static structures, and reinforce the notion that protein conformational entropy can make important contributions to the free energy of ligand binding.
|
|
| 19. |
- Stenström, Olof, et al.
(författare)
-
Ligand-induced protein transition state stabilization switches the binding pathway from conformational selection to induced fit
- 2024
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 1091-6490. ; 121:14, s. 2317747121-2317747121
-
Tidskriftsartikel (refereegranskat)abstract
- Protein-ligand complex formation is fundamental to biological function. A central question is whether proteins spontaneously adopt binding-competent conformations to which ligands bind conformational selection (CS) or whether ligands induce the binding-competent conformation induced fit (IF). Here, we resolve the CS and IF binding pathways by characterizing protein conformational dynamics over a wide range of ligand concentrations using NMR relaxation dispersion. We determined the relative flux through the two pathways using a four-state binding model that includes both CS and IF. Experiments conducted without ligand show that galectin-3 exchanges between the ground-state conformation and a high-energy conformation similar to the ligand-bound conformation, demonstrating that CS is a plausible pathway. Near-identical crystal structures of the apo and ligand-bound states suggest that the high-energy conformation in solution corresponds to the apo crystal structure. Stepwise additions of the ligand lactose induce progressive changes in the relaxation dispersions that we fit collectively to the four-state model, yielding all microscopic rate constants and binding affinities. The ligand affinity is higher for the bound-like conformation than for the ground state, as expected for CS. Nonetheless, the IF pathway contributes greater than 70% of the total flux even at low ligand concentrations. The higher flux through the IF pathway is explained by considerably higher rates of exchange between the two protein conformations in the ligand-associated state. Thus, the ligand acts to decrease the activation barrier between protein conformations in a manner reciprocal to enzymatic transition-state stabilization of reactions involving ligand transformation.
|
|
| 20. |
- Stenström, Olof, et al.
(författare)
-
Mapping the energy landscape of protein-ligand binding via linear free energy relationships determined by protein NMR relaxation dispersion
- 2021
-
Ingår i: RSC Chemical Biology. - : Royal Society of Chemistry (RSC). - 2633-0679. ; 2:1, s. 259-265
-
Tidskriftsartikel (refereegranskat)abstract
- Biochemical signaling is mediated by complexes between macromolecular receptors and their ligands, with the duration of the signal being directly related to the lifetime of the ligand-receptor complex. In the field of drug design, the recognition that drug efficacy in vivo depends on the lifetime of the drug-protein complex has spawned the concept of designing drugs with particular binding kinetics. To advance this field it is critical to investigate how the molecular details of designed ligands might affect the binding kinetics, as well as the equilibrium binding constant. Here we use protein NMR relaxation dispersion to determine linear free energy relationships involving the on- and off-rates and the affinity for a series of congeneric ligands targeting the carbohydrate recognition domain of galectin-3. Using this approach we determine the energy landscape and the position of the transition state along the reaction coordinate of protein-ligand binding. The results show that ligands exhibiting reduced off-rates achieve this by primarily stabilizing the bound state, but do not affect the transition state to any greater extent. The transition state forms early, that is, it is located significantly closer to the free state than to the bound state, suggesting a critical role of desolvation. Furthermore, the data suggest that different subclasses of ligands show different behavior with respect to these characteristics.
|
|
| 21. |
- Weininger, Ulrich, et al.
(författare)
-
(13)C relaxation experiments for aromatic side chains employing longitudinal- and transverse-relaxation optimized NMR spectroscopy.
- 2012
-
Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 53:3, s. 181-190
-
Tidskriftsartikel (refereegranskat)abstract
- Aromatic side chains are prevalent in protein binding sites, perform functional roles in enzymatic catalysis, and form an integral part of the hydrophobic core of proteins. Thus, it is of great interest to probe the conformational dynamics of aromatic side chains and its response to biologically relevant events. Indeed, measurements of (13)C relaxation rates in aromatic moieties have a long history in biomolecular NMR, primarily in the context of samples without isotope enrichment that avoid complications due to the strong coupling between neighboring (13)C spins present in uniformly enriched proteins. Recently established protocols for specific (13)C labeling of aromatic side chains enable measurement of (13)C relaxation that can be analyzed in a straightforward manner. Here we present longitudinal- and transverse-relaxation optimized pulse sequences for measuring R (1), R (2), and {(1)H}-(13)C NOE in specifically (13)C-labeled aromatic side chains. The optimized R (1) and R (2) experiments offer an increase in sensitivity of up to 35 % for medium-sized proteins, and increasingly greater gains are expected with increasing molecular weight and higher static magnetic field strengths. Our results highlight the importance of controlling the magnetizations of water and aliphatic protons during the relaxation period in order to obtain accurate relaxation rate measurements and achieve full sensitivity enhancement. We further demonstrate that potential complications due to residual two-bond (13)C-(13)C scalar couplings or dipolar interactions with neighboring (1)H spins do not significantly affect the experiments. The approach presented here should serve as a valuable complement to methods developed for other types of protein side chains.
|
|
| 22. |
- Zetterberg, Fredrik R, et al.
(författare)
-
Discovery of Selective and Orally Available Galectin-1 Inhibitors
- 2023
-
Ingår i: Journal of Medicinal Chemistry. - 1520-4804. ; 66:24, s. 16980-16990
-
Tidskriftsartikel (refereegranskat)abstract
- A new series of orally available α-d-galactopyranosides with high affinity and specificity toward galectin-1 have been discovered. High affinity and specificity were achieved by changing six-membered aryl-triazolyl substituents in a series of recently published galectin-3-selective α-d-thiogalactosides (e.g., GB1107 Kd galectin-1/3 3.7/0.037 μM) for five-membered heterocycles such as thiazoles. The in vitro pharmacokinetic properties were optimized, resulting in several galectin-1 inhibitors with favorable properties. One compound, GB1490 (Kd galectin-1/3 0.4/2.7 μM), was selected for further characterization toward a panel of galectins showing a selectivity of 6- to 320-fold dependent on galectin. The X-ray structure of GB1490 bound to galectin-1 reveals the compound bound in a single conformation in the carbohydrate binding site. GB1490 was shown to reverse galectin-1-induced apoptosis of Jurkat cells at low μM concentrations. No cell cytotoxicity was observed for GB1490 up to 90 μM in the A549 cells. In pharmacokinetic studies in mice, GB1490 showed high oral bioavailability (F% > 99%).
|
|
| 23. |
- Zetterberg, Fredrik R., et al.
(författare)
-
Discovery of the Selective and Orally Available Galectin-1 Inhibitor GB1908 as a Potential Treatment for Lung Cancer
-
Ingår i: Journal of Medicinal Chemistry. - 1520-4804.
-
Tidskriftsartikel (refereegranskat)abstract
- We have previously described a new series of selective and orally available galectin-1 inhibitors resulting in the thiazole-containing glycomimetic GB1490. Here, we show that the introduction of polar substituents to the thiazole ring results in galectin-1-specific compounds with low nM affinities. X-ray structural analysis of a new ligand-galectin-1 complex shows changes in the binding mode and ligand-protein hydrogen bond interactions compared to the GB1490-galectin-1 complex. These new high affinity ligands were further optimized with respect to affinity and ADME properties resulting in the galectin-1-selective GB1908 (Kd galectin-1/3 0.057/6.0 μM). In vitro GB1908 inhibited galectin-1-induced apoptosis in Jurkat cells (IC50 = 850 nM). Pharmacokinetic experiments in mice revealed that a dose of 30 mg/kg b.i.d. results in free levels of GB1908 in plasma over galectin-1 Kd for 24 h. GB1908 dosed with this regimen reduced the growth of primary lung tumor LL/2 in a syngeneic mouse model.
|
|
