Study of protein folding and misfolding by NMR spectroscopy

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

In prior years, close collaboration with Philip Anfinrud resulted in novel hardware that makes it possible possible to monitor by NMR spectroscopy the folding of a protein in a residue-specific manner upon jumping the applied hydrostatic pressure. Pressure changes of up to 2.5 kbar, requiring 1-2 ms, are feasible and compatible with the recording of high quality NMR data. For proteins with a substantial volume difference between the folded and unfolded states, the thermodynamic equilibrium between folded and unfolded states can be altered by varying the hydrostatic pressure. Hydrostatic pressure can also monomerize oligomeric species, and aggregates of Abeta rapidly form at atmospheric pressure but dissolve into monomeric species at high pressure. The aggregated species has been implicated in the etiology of Alzheimer's disease, but no structural information is currently available on the type of interactions formed in the oligomer. Much of the current work is focused at answering this question. Melittin is a 26-residue peptide that rapidly forms tetramers at atmospheric pressure but dissociates at 2.5 kbar. We are using this peptide as a model system to develop the NMR technology needed to study Abeta. Its native C-terminal amide significantly affects its equilibrium structure and dynamics in solution and its presence is a prerequisite for studying the tetramerization. We successfully developed a method for producing triply isotopically labeled (2H, 13C, and 15N) native melittin through recombinant expression followed by chemical amidation. We showed that structural models produced with AlphaFold-Multimer for tetrameric melittin are in even better agreement with experimental residual dipolar couplings (RDCs) than the 2.0- resolution X-ray crystal structure, and are currently using this model system to investigate whether the dependence of the oligomerization kinetics as a function of concentration provides a reliable indicator for the oligomer stoichiometry of the transition state species. In a separate study of protein folding, we are investigating the pathways by which a ubiquitin mutant (L50A) switches from its unfolded state to the folded state, which is virtually indistinguishable from that of the wild-type protein. Multiple meta-stable intermediates in the folding pathway of this protein have been identified on the basis of the NMR spectra. Whereas for one of the highly populated (up to 35%) intermediates, the structure can be identified as that of a "slipped" out-of-register C-terminal beta strand, a second intermediate (populated at up to ca 15%) is also present. Whereas this second intermediate appears to be also on-pathway, its structure has not yet been identified. The L50A ubiquitin mutant represents the first case of a protein for which multiple, on pathway, meta-stable folding intermediates have been identified.