Investigation of the kinetics of protein folding and the ensemble of conformations in non-native states of proteins by liquid NMR spectroscopy

For a complete description of protein folding dynamics and the structure of the folded state, of unfolded and of non-native states of proteins and the kinetics of protein folding from the unfolded state to the folded state have to be determined. The focus of this PhD thesis was the development of novel NMR methodologies to study protein folding using NMR spectroscopy. This has been achieved by studying three model proteins ubiquitin, [alpha]-lactalbumin and lysozyme in their folded and especially in their unfolded states. The proteins were chosen, because [alpha]-lactalbumin and lysozyme are two proteins with nearly identical fold but different function and ubiquitin is a very stable protein without disulfide bonds. Methodologies A new NMR pulse sequence for the determination of ... and ... coupling constants in proteins was developped. The method is based on J-modulated HSQCs and can be applied to folded as well as unfolded proteins. The new coupling constants report on backbone [phi] and [psi] angles. . Residual structure and long-range interactions in unfolded proteins can be detected by a new combination of site directed non-conservative mutagenesis and NMR analysis. Identification of long-range interactions is a) based on the analysis and interpretation of R₂ relaxation rates, for which models have been derived and b) based on NMR diffusion data which directly correlate to the compactness of a given protein. A method to study laser triggered kinetics of protein folding by time-resolved photo- CIDNP NMR was developed. Two lasers were coupled into an NMR spectrometer:

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2005

Author dc:creator

• Wirmer, Julia

Advisor dc:contributor.advisor

• Harold Schwalbe.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/32425

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/32425