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- Wallerstein, Johan, et al.
(författare)
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Minute Additions of DMSO Affect Protein Dynamics Measurements by NMR Relaxation Experiments through Significant Changes in Solvent Viscosity
- 2019
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Ingår i: ChemPhysChem. - : Wiley. - 1439-4235. ; 20:2, s. 326-332
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Tidskriftsartikel (refereegranskat)abstract
- Studies of protein−ligand binding often rely on dissolving the ligand in dimethyl sulfoxide (DMSO) to achieve sufficient solubility, and then titrating the ligand solution into the protein solution. As a result, the final protein−ligand solution contains small amounts of DMSO in the buffer. Here we report how the addition of DMSO impacts studies of protein conformational dynamics. We used 15N NMR relaxation to compare the rotational diffusion correlation time (τC) of proteins in aqueous buffer with and without DMSO. We found that τC scales with the viscosity of the water−DMSO mixture, which depends sensitively on the amount of DMSO and varies by a factor of 2 across the relevant concentration range. NMR relaxation studies of side chains dynamics are commonly interpreted using τC as a fixed parameter, obtained from backbone 15N relaxation data acquired on a separate sample. Model-free calculations show that errors in τC, arising from mismatched DMSO concentration between samples, lead to significant errors in order parameters. Our results highlight the importance of determining τC for each sample or carefully matching the DMSO concentrations between samples.
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| 2. |
- Wallerstein, Johan
(författare)
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Molecular recognition and dynamics in proteins studied by NMR
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Knowledge of dynamics in protein is very important in the description of protein function and molecular recognition. The thesis investigates protein dynamics on time-scales from milli- to sub-nanosecond, with focus on the latter, using NMR spin relaxation experiments on two proteins, the 138-residue carbohydrate recognition domain of galectin-3 (Gal3C) and the 56-residue B1 domain of bacterial protein G (PGB1). Fives studies are presented.By measuring the exchange contribution to R2-relaxation, as a function of pH, using 13C-CPMG-experiments directed on the side-chain carbonyls of glutamic and aspartic acid in PGB1, we could determine site specific protonation rate constants k(on), and deprotonation rate constants k(off). A linear free-energy relationship between log k(on) and pKa is found, which provides information on the free-energy landscape of the protonation reaction, showing that the variability among residues in these rates arises from charge stabilization of the deprotonated state.Dimethyl sulfoxide (DMSO) is often used for dissolving nonpolar ligands in protein-ligand studies. DMSO change the viscosity, which is proportional to rotational correlation time. The correlation time of PGB1 and Gal3C was determined for different DMSO-concentrations. Effects of minute additions of DMSO in samples used in spin-relaxation experiments were examined for the case of 2H-methyl side chain model-free studies. Chemical shift perturbations studies on apo-Gal3C show uniform changes of chemical shifts, indicating DMSO has a minor impact on the hydration layer.15N-backbone and 2H-methyl side chain NMR order parameters (S2) are determined for three Gal3C complexes using the Lipari-Szabo model-free formalism developed in the 80s. Minor changes in ligand structure generate differences in the conformational entropy. Specifically, the radial distribution of conformational entropy, with ligand in the centre reveals how entropy varies between consecutive shells within the protein. The study combines NMR relaxation with isothermal titration calorimetry (ITC), X-ray crystallography, and computational approaches.The structure–thermodynamic relationship for halogen bonds C–X (X=F, Cl, Br, and I) between the ligand substituents and the backbone C=O of a glycine in Gal3C was studied with NMR, ITC, X-ray crystallography and computational approaches. The sigma-hole associated with the halogen bond affects the electron density of surrounding 15N nuclei in amides, and consequently the chemical shift of these nuclei. There is a correlation between ITC-determined enthalpy of binding vs amide chemical shift.In Gal3C bound to natural substrate lactose, the chemical shifts changes of the 13C-E1 in histidine side chains are traced as a function of pH using 1H-13C HSQC-experiments. The experiment helps in determination of the tautomeric state in the proteins four histidines. The histidine in the binding site is in tautomer ND1, another is in tautomer NE2 and two are partly charged. Neutron diffraction studies at pH 7.5 confirms the NMR-studies.
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