Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.Vita.Includes bibliographical references (p. 187-202).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:(cont.) one for initiation of folding by releasing ions from photo-labile chelators with dead times as low as 200ms, and one for induction of photo-CINDP NMR. The method can not only be applied to ion induced kinetics of protein folding, but is generally applicable to kinetics of biomacromolecules such as proteins and RNA that involve photo-protected cofactors as could be shown in our laboratory. Investigations on ubiquitin The newly developed NMR method for the determination of ... and ... coupling constants was applied to folded ubiquitin. Analysis revealed the dependence of the coupling constants on the backbone conformation, predominantly on [psi], making the pulse sequence and the parameterization developed here a valuable new tool for the determination of the backbone conformation in folded as well as in unfolded proteins. Unfolded ubiquitin was investigated using scalar coupling constants and ¹H,¹⁵N relaxation data. The experimental data agree well with models proposed to describe unfolded states of proteins as a statistical coil where dynamics are governed solely by segmental motions. Unfolded ubiquitin is thus a good model for a protein without detectable residual structure in its unfolded state. Investigations on lysozyme Residual structure in the unfolded states of non conservative single point mutants (A9G, W62G, W62Y, W 1 1G and W123G) of hen lysoszyme was monitored by chemical shift measurements, ¹⁵N transverse relaxation rates and particularly diffusion constants. Long- range interactions between hydrophobic clusters of unfolded lysozyme were observed. Single point mutations dramatically alter the overall compactness of the unfolded state.(cont.) Investigations on [alpha]-lactalbumin · Isotope labeled bovine [alpha]-lactalbumin (BLA) was expressed heterologously using a new construct with a His-tag and a trypsin cleavage site. The sequence and stability of the obtained BLA is identical to the wild type protein. This makes it a perfect construct to study kinetics of folding and unfolded states of BLA. ¹³C,¹⁵N isotope labeled unfolded BLA was assigned applying standard and non-standard NMR assignment experiments. Residual secondary structure was identified near the N- and the C-terminus of unfolded BLA, in regions belonging to the -domain in the folded state, suggesting a possible folding nucleus. · This unfolded state of BLA was furthermore compared to the unfolded state of human lactalbumin (HLA) and lysozyme based on residual structure and ¹⁵N relaxation data. The unfolded states vary considerably for the three proteins, which possess very similar structures in their native state. The structural ensemble in the unfolded states of proteins are determined by the primary sequence of the protein and even smallest single point mutations as found between HLA and BLA can change the conformation of the unfolded state considerably. · The Ca²⁺-triggered folding kinetics of BLA under constant denaturant (4M urea) has been investigated by laser induced release of Ca²⁺-ions from a photolabile chelator within the NMR spectrometer and subsequent photo-CIDNP signal detection. A folding intermediate possessing a tyrosine residue in a non-native conformation was detected 200ms after initiation of folding. Therefore, parts of the polypeptide chain in the [beta]-domain of BLA sample non-native conformations, while a hydrophobic core is formed.(cont.) The findings in the kinetic investigations are in line with the detected residual structure. Refolding of amino acids involved in non-native clusters in the intermediates has to proceed the correct folding and therefore constitutes a rate limiting step on the Ca²⁺-induced refolding of [alpha]- lactalbumin.by Julia Wirmer.Ph.D
