title: Dynamics in the unfolded state of beta2-microglobulin studied by NMR.
abstract: Many proteins form amyloid-like fibrils in vitro under conditions that favour the population of partially folded conformations or denatured state ensembles. Characterising the structural and dynamic properties of these states is crucial towards understanding the mechanisms of self-assembly in amyloidosis. The aggregation of beta2-microglobulin (beta2m) into amyloid fibrils in vivo occurs in the condition known as dialysis-related amyloidosis (DRA) and the protein has been shown to form amyloid-like fibrils under acidic conditions in vitro. We have used a number of 1H-15N nuclear magnetic resonance (NMR) experiments in conjunction with site-directed mutagenesis to study the acid-unfolded state of beta2m. 15N NMR transverse relaxation experiments reveal that the acid-denatured ensemble, although predominantly unfolded at the N and C termini, contains substantial non-native structure in the central region of the polypeptide chain, stabilised by long-range interactions between aromatic residues and by the single disulphide bond. Relaxation dispersion studies indicate that the acid-unfolded ensemble involves two or more distinct species in conformational equilibrium on the micro- to millisecond time-scale. One of these species appears to be hydrophobically collapsed, as mutations in an aromatic-rich region of the protein, including residues that are solvent-exposed in the native protein, disrupt this structure and cause a consequent decrease in the population of this conformer. Thus, acid-unfolded beta2m consists of a heterogeneous ensemble of rapidly fluctuating species, some of which contain stable, non-native hydrophobic clusters. Given that amyloid assembly of beta2m proceeds with lag kinetics under the conditions of this study, a rarely populated species such as a conformer with non-native aromatic clustering could be key to the initiation of amyloidosis.
authors.affiliation: School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: Platt
authors.forename: Victoria J
authors.initials: VJ
authors.lastname: McParland
authors.forename: Arnout P
authors.initials: AP
authors.lastname: Kalverda
authors.forename: Steve W
authors.initials: SW
authors.lastname: Homans
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D000143:Acids / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013696:Temperature; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000143:Acids; D001613:beta 2-Microglobulin