| 1. |
- Niemi, MEK, et al.
(författare)
-
- 2021
-
swepub:Mat__t
|
|
| 2. |
|
|
| 3. |
- Damberg, Peter, 1973-
(författare)
-
Development of NMR methods for studies of dynamics and structure : applications to motilin, Alzheimer β-peptide and ubiquitin
- 2001
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- NMR spin relaxation was used to study structure and dynamics of two biologically important peptides.The first case concerns the three-dimensional structure of the 22 amino acid residue peptide hormone motilin in the presence of SDS micelles, which mimic a biomembrane environment. Motilin was found to have a well-defined structure when associated with the micelle, with a central amphiphatic helix and two turns in the biologically active N-terminal end. Residues 3-5 are buried in the interior of the micelle as evident from experiments where spin-labels were inserted into the micelles at certain depths. 13C NMR relaxation on a selectively isotope labeled motilin sample showed that the rotational correlation time is in agreement with the peptide being associated with a spherical micelle.The peptide corresponding to residues 12-28 of the Alzheimer related peptide Ab was studied in solution by NMR spectroscopy, circular dichroism spectroscopy, molecular weight cut-off filtering and NMR diffusion measurements. Both increased salt concentration and increased temperature induced reversible random-coil to b-sheet transitions and aggregation, but with certain differences between the structures induced by salt and temperature.Development of NMR methods used for studying molecular motions, such as diffusion and local dynamics is discussed. The methodology for estimation of translational diffusion coefficients by NMR was improved by taking into account the inhomogeneity of the magnetic field gradient pulses. A practical way to correct for the inhomogeniety was suggested.Parameters describing the chemical shift anisotropies of 13C and 15N nuclei have been estimated from the relaxation rates of the nuclear spins measured at several magnetic field strengths. New NMR methodology was developed where only longitudinal rates need to be measured. The site-specific chemical shift anisotropies for 15N nuclei in the backbone of ubiquitin were estimated. It was found that the variations in the effective shift anisotropy along the polypeptide backbone for 15N nuclei in ubiquitin are of the same order as the variations in isotropic chemical shift, i.e. a 90% confidence region for the site-to-site variability spans from 3.7 ppm to 10.5 ppm. The average orientation parameter for the CSA tensors was found to be -0.84, which corresponds to an angle between the symmetry axis of the CSA tensor and the N-H bond vector of 19° in the approximation of an axially symmetric CSA tensor. Significant variations between the orientation parameters of different sites were observed with a 90% confidence interval spanning from 0.027 to 0.066.Fluorescence polarization anisotropy decay data and NMR relaxation data to describe the dynamics of a 13C-labeled tyrosine ring in motilin were compared. The two methods showed very similar results in terms of correlation times and order parameters. This study indicates that the previously observed differences between the results of the two methods applied to the same protein are mainly due to either influences of internal probe dynamics or varying sample conditions, in particular peptide or protein concentrations.
|
|
| 4. |
- Robertson, Angus J., et al.
(författare)
-
NMR Observation of Sulfhydryl Signals in SARS-CoV-2 Main Protease Aids Structural Studies
- 2022
-
Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 23:19
-
Tidskriftsartikel (refereegranskat)abstract
- The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro/Nsp5), is a key antiviral drug target. NMR spectroscopy of this large system proved challenging and resonance assignments have remained incomplete. Here we present the near-complete (>97 %) backbone assignments of a C145A variant of Mpro (MproC145A) both with, and without, the N-terminal auto-cleavage substrate sequence, in its native homodimeric state. We also present SILLY (Selective Inversion of thioL and Ligand for NOESY), a simple yet effective pseudo-3D NMR experiment that utilizes NOEs to identify interactions between Cys-thiol or aliphatic protons, and their spatially proximate backbone amides in a perdeuterated protein background. High protection against hydrogen exchange is observed for 10 of the 11 thiol groups in MproC145A, even those that are partially accessible to solvent. A combination of SILLY methods and high-resolution triple-resonance NMR experiments reveals site-specific interactions between Mpro, its substrate peptides, and other ligands, which present opportunities for competitive binding studies in future drug design efforts.
|
|
| 5. |
- Sgourakis, Nikolaos G., et al.
(författare)
-
Determination of the Structures of Symmetric Protein Oligomers from NMR Chemical Shifts and Residual Dipolar Couplings
- 2011
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 133:16, s. 6288-6298
-
Tidskriftsartikel (refereegranskat)abstract
- Symmetric protein dimers, trimers, and higher-order cyclic oligomers play key roles in many biological processes. However, structural studies of oligomeric systems by solution NMR can be difficult due to slow tumbling of the system and the difficulty in identifying NOE interactions across protein interfaces. Here, we present an automated method (RosettaOligomers) for determining the solution structures of oligomeric systems using only chemical shifts, sparse NOEs, and domain orientation restraints from residual dipolar couplings (RDCs) without a need for a previously determined structure of the monomeric subunit. The method integrates previously developed Rosetta protocols for solving the structures of monomeric proteins using sparse NMR data and for predicting the structures of both nonintertwined and intertwined symmetric oligomers. We illustrated the performance of the method using a benchmark set of nine protein dimers, one trimer, and one tetramer with available experimental data and various interface topologies. The final converged structures are found to be in good agreement with both experimental data and previously published high-resolution structures. The new approach is more readily applicable to large oligomeric systems than conventional structure-determination protocols, which often require a large number of NOEs, and will likely become increasingly relevant as more high-molecular weight systems are studied by NMR
|
|
