| 1. |
- Brath, Ulrika, et al.
(författare)
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Differential responses of the backbone and side-chain conformational dynamics in FKBP12 upon binding the transition-state analog FK506: implications for transition-state stabilization and target protein recognition.
- 2009
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 387:1, s. 233-244
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Tidskriftsartikel (refereegranskat)abstract
- FKBP12 serves a dual role as a peptidyl-prolyl cis-trans isomerase and as a modulator of several cell signaling pathways. The macrolide FK506 is a transition-state analog of the catalyzed reaction and displaces FKBP12 from its natural target proteins. We compared the conformational exchange dynamics of the backbone and methyl-bearing side chains of FKBP12 in the free and FK506-bound states using NMR relaxation-dispersion experiments. Our results show that the free enzyme exchanges between the ground state and an excited state that resembles the ligand-bound state or Michaelis complex. In FK506-bound FKBP12, the backbone is confined to a single conformation, while conformational exchange prevails for many methyl groups. The residual side-chain dynamics in the transition-state analog-bound state suggests that the transition-state ensemble involves multiple conformations, a finding that challenges the long-standing concept of conformational restriction in the transition-state complex. Furthermore, exchange between alternative conformations is observed in the bound state for an extended network of methyl groups that includes locations remote from the active site. Several of these locations are known to be important for interactions with cellular target proteins, including calcineurin and the ryanodine receptor, suggesting that the conformational heterogeneity might play a role in the promiscuous binding of FKBP12 to different targets.
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| 2. |
- Brath, Ulrika, et al.
(författare)
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Functional dynamics of human FKBP12 revealed by methyl C-13 rotating frame relaxation dispersion NMR spectroscopy
- 2006
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 128:17, s. 5718-5727
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Tidskriftsartikel (refereegranskat)abstract
- Transverse relaxation dispersion NMR spectroscopy can provide atom-specific information about time scales, populations, and the extent of structural reorganization in proteins under equilibrium conditions. A method is described that uses side-chain methyl groups as local reporters for conformational transitions taking place in the microsecond regime. The experiment measures carbon nuclear spin relaxation rates in the presence of continuous wave off-resonance irradiation, in proteins uniformly enriched with C-13, and partially randomly labeled with 2 H. The method was applied to human FK-506 binding protein (FKBP12), which uses a common surface for binding substrates in its dual role as both an immunophilin and folding assistant. Conformational dynamics on a time scale of similar to 130 mu s were detected for methyl groups located in the substrate binding pocket, demonstrating its plasticity in the absence of substrate. The spatial arrangement of affected side-chain atoms suggests that substrate recognition involves the rapid relative movement of the subdomain comprising residues Ala81-Thr96 and that the observed dynamics play an important role in facilitating the interaction of this protein with its many partners, including calcineurin.
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| 3. |
- Brath, Ulrika
(författare)
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Molecular dynamics studied by NMR relaxation experiments. Characterization of functional dynamics in the FK506 binding protein FKBP12.
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The presented thesis work is concerned with the study of molecular dynamics using liquid nuclear magnetic resonance (NMR) spectroscopy. Spin relaxation dispersion experiments in the rotating frame (R1ρ) are employed to provide a detailed, atomic-resolution view of protein dynamics. Comprehensive investigations of 15N backbone and 13C methyl side-chain conformational exchange in the FK506 binding protein, FKBP12, were carried out. The human enzyme FKBP12 catalyzes the cis-trans isomerization of peptidyl-prolyl linkages in proteins, thereby increasing the rate of protein folding. Recombinant protein production with 13C/2H-enrichment yielded 13CHD2 isotopomers and allowed for development of novel R1ρ experiments aimed to characterize the methyl-bearing side-chains. The joint analysis of backbone and side-chain dynamics revealed a collective conformational exchange process with a correlation time of 121 ± 26 microseconds in the free enzyme. The locations of the exchanging nuclei showed close correspondence with residues previously shown to be important for the catalytic activity, which emphasize the connection between protein function and dynamics. The FKBP12 isomerase activity is strongly inhibited by the small organic molecule FK506. In addition, the bound conformation of FK506 exhibit distinct structural transition state analog characteristics. Conformational exchange dynamics studies of the FKBP12 – FK506 complex demonstrated a striking rigidity of the backbone atoms, in line with the established theory that the transition state is more constrained than the free state. Conformational exchange dynamics found for peripheral methyl groups is suggested to play a role in the subsequent binding of the FKBP12 – FK506 complex to other target molecules. A specific isotope labeling strategy using [1-13C]-glucose was shown to produce isolated 13C nuclei in the side-chains of Phe/Tyr/Trp/His. Proteins with this characteristic labeling provide nuclei amenable to R1ρ experiments, which were previously excluded from analysis due to complications arising from uniform carbon labeling. The method thus extended the collection of dynamic probes to include the frequently occurring and often functionally important aromatic side-chains. R1ρ studies of the C-terminal part of a mutant Calmodulin protein revealed microsecond time scale dynamics contributions to Tyr80Cδ, confirming previous reports of conformational exchange present for the protein backbone nuclei. Detection of conformational exchange in isotropic media requires a chemical shift difference between the exchanging states. However, such chemical shift difference can be replaced by differential residual dipolar couplings (RDC) in the exchanging states for molecules dissolved in partially aligning media. The RDC-induced contribution to the exchange-free transverse relaxation rate can be suppressed using relaxation dispersion experiments, thus revealing details of the conformational exchange characteristics. The theory was described, and the method was demonstrated using the model molecule N,N-dimethyltrichloroacetamide.
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| 4. |
- Igumenova, Tatyana I., et al.
(författare)
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Characterization of Chemical Exchange Using Residual Dipolar Coupling
- 2007
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 129:44, s. 13396-13396
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Tidskriftsartikel (refereegranskat)abstract
- Abstract in Undetermined NMR line shape analysis and relaxation dispersion measurements for N,N-dimethyltrichloroacetamide (DMTCA) weakly aligned using poly-γ-benzyl-l-glutamate, which forms a lyotropic nematic phase when dissolved in chloroform, were used to characterize chemical exchange kinetics for the rotation around the C−N amide bond. At low temperatures (<312 K), slow-exchange 1H-coupled 13C NMR spectra show two resolved 13C methyl quartets with different residual dipolar coupling constants (RDCs). At high temperatures (>312 K), a single population-averaged 13C methyl quartet is observed; in this regime, the differences in RDCs contribute to differential line broadening of the quartet components. Self-consistent measurements of the difference in RDCs are obtained from both line shape and relaxation dispersion techniques. The results show that NMR spectroscopy of weakly aligned molecules allows complete characterization of chemical exchange processes using RDCs, even if exchange broadening is absent in isotropic samples.
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| 5. |
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