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Development of NMR methods for studies of dynamics and structure: applications to motilin, Alzheimer β-peptide and ubiquitin
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Stockholm University , 2001. , p. 56
National Category
Biological Sciences
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-142233ISBN: 91-7265-382-5 (print)OAI: oai:DiVA.org:su-142233DiVA, id: diva2:1091808
Public defence
2001-11-30, 14:00 (English)
Opponent
Note

Härtill 6 uppsatser

Available from: 2017-04-28 Created: 2017-04-28 Last updated: 2017-09-28Bibliographically approved

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