| 1. |
- Ahlner, Alexandra, 1984-, et al.
(författare)
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Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity
- 2015
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Ingår i: Journal of Biomolecular NMR. - : Springer Netherlands. - 0925-2738 .- 1573-5001. ; 62:3, s. 341-351
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Tidskriftsartikel (refereegranskat)abstract
- A selective isotope labeling scheme based on the utilization of [2-13C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13Cα chemical shifts using Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-13C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state 13Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s−1, despite the small fraction of 13Cα–13Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using 13Cα spin probes.
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| 2. |
- Ahlner, Alexandra, et al.
(författare)
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PINT: a software for integration of peak volumes and extraction of relaxation rates
- 2013
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Ingår i: Journal of Biomolecular NMR. - : Springer Verlag (Germany). - 0925-2738 .- 1573-5001. ; 56:3, s. 191-202
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Tidskriftsartikel (refereegranskat)abstract
- We present the software Peak INTegration (PINT), designed to perform integration of peaks in NMR spectra. The program is very simple to run, yet powerful enough to handle complicated spectra. Peaks are integrated by fitting predefined line shapes to experimental data and the fitting can be customized to deal with, for instance, heavily overlapped peaks. The results can be inspected visually, which facilitates systematic optimization of the line shape fitting. Finally, integrated peak volumes can be used to extract parameters such as relaxation rates and information about low populated states. The utility of PINT is demonstrated by applications to the 59 residue SH3 domain of the yeast protein Abp1p and the 289 residue kinase domain of murine EphB2.
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| 3. |
- Billeter, Martin, 1955
(författare)
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Non-uniform sampling in biomolecular NMR
- 2017
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Ingår i: Journal of Biomolecular Nmr. - : Springer Science and Business Media LLC. - 0925-2738 .- 1573-5001. ; 68:2, s. 65-66
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Tidskriftsartikel (refereegranskat)
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| 4. |
- Björnerås, Johannes, et al.
(författare)
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Resolving complex mixtures : trilinear diffusion data
- 2014
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Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 0925-2738 .- 1573-5001. ; 58:4, s. 251-257
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Tidskriftsartikel (refereegranskat)abstract
- Complex mixtures are at the heart of biology, and biomacromolecules almost always exhibit their function in a mixture, e.g., the mode of action for a spider venom is typically dependent on a cocktail of compounds, not just the protein. Information about diseases is encoded in body fluids such as urine and plasma in the form of metabolite concentrations determined by the actions of enzymes. To understand better what is happening in real living systems we urgently need better methods to characterize such mixtures. In this paper we describe a potent way to disentangle the NMR spectra of mixture components, by exploiting data that vary independently in three or more dimensions, allowing the use of powerful algorithms to decompose the data to extract the information sought. The particular focus of this paper is on NMR diffusion data, which are typically bilinear but can be extended by a third dimension to give the desired data structure.
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| 5. |
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| 6. |
- Chi N, Celestine, 1978-, et al.
(författare)
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Extending the eNOE data set of large proteins by evaluation of NOEs with unresolved diagonals.
- 2015
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Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 0925-2738 .- 1573-5001. ; 62:1, s. 63-9
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Tidskriftsartikel (refereegranskat)abstract
- The representation of a protein's spatial sampling at atomic resolution is fundamental for understanding its function. NMR has been established as the best-suited technique toward this goal for small proteins. However, the accessible information content rapidly deteriorates with increasing protein size. We have recently demonstrated that for small proteins distance restraints with an accuracy smaller than 0.1 Å can be obtained by replacing traditional semi-quantitative Nuclear Overhauser Effects (NOEs) with exact NOEs (eNOE). The high quality of the data allowed us to calculate structural ensembles of the small model protein GB3 consisting of multiple rather than a single state. The analysis has been limited to small proteins because NOEs of spins with unresolved diagonal peaks cannot be used. Here we propose a simple approach to translate such NOEs into correct upper distance restraints, which opens access to larger biomolecules. We demonstrate that for 16 kDa cyclophilin A the collection of such restraints extends the original 1254 eNOEs to 3471.
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| 7. |
- 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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| 8. |
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| 9. |
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| 10. |
- Dreydoppel, Matthias, et al.
(författare)
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1H R1ρ relaxation dispersion experiments in aromatic side chains
- 2021
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Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 0925-2738 .- 1573-5001. ; 75:44846, s. 383-392
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Tidskriftsartikel (refereegranskat)abstract
- Aromatic side chains are attractive probes of protein dynamic, since they are often key residues in enzyme active sites and protein binding sites. Dynamic processes on microsecond to millisecond timescales can be studied by relaxation dispersion experiments that attenuate conformational exchange contributions to the transverse relaxation rate by varying the refocusing frequency of applied radio-frequency fields implemented as either CPMG pulse trains or continuous spin-lock periods. Here we present an aromatic 1H R1ρ relaxation dispersion experiment enabling studies of two to three times faster exchange processes than achievable by existing experiments for aromatic side chains. We show that site-specific isotope labeling schemes generating isolated 1H–13C spin pairs with vicinal 2H–12C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann–Hahn matching due to the 3JHH couplings and limited chemical shift differences among 1H spins in phenylalanine, tyrosine and the six-ring moiety of tryptophan. This labeling pattern is sufficient in that remote protons do not cause additional complications. We validated the approach by measuring ring-flip kinetics in the small protein GB1. The determined rate constants, kflip, agree well with previous results from 13C R1ρ relaxation dispersion experiments, and yield 1H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic1H R1ρ relaxation dispersion experiment in combination with the site-selective 1H–13C/2H–12C labeling scheme enable measurement of exchange rates up to kex = 2kflip = 80,000 s–1, and serve as a useful complement to previously developed 13C-based methods.
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