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 used this peptide as a model system to develop the NMR technology needed to study Abeta. By integrating non-equilibrium pressure-jump and equilibrium Carr-Purcell-Meiboom-Gill relaxation dispersion data we fully mapped the kinetic landscape of melittin tetramerization. While monomeric peptides weakly form dimers (Kd,D/M ≈ 26 mM) whose population never exceeds 1.6% at 288 K, dimers associate tightly to form stable tetrameric species (Kd,T/D ≈ 740 nM). Exchange between the monomer and dimer, along with exchange between the dimer and tetramer, occurs on the millisecond time scale. The new NMR strategy used for revealing the steps in this oligomerization process can be readily applied to studying the folding and misfolding of a wide range of oligomeric assemblies. 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. Work to identify the structure of this second intermediate currently relies on the development of mutants of ubiquitin where the structure is stably occupied, such that it can be studied in detail by conventional NMR methods. The rates and Arrhenius activation energies for proline peptide bond isomerization under native, folding conditions were measured for the three Pro residues in ubiquitin. It was observed that for Pro-38, isomerization under conditions of protein folding are accelerated by nearly 10-fold relative to unfolding conditions, with a smaller ca 2-fold increase for Pro-19 and Pro-37. These observations suggest that proline isomerization is a lower kinetic barrier during protein folding than often assumed.