title: The Significance of the Location of Mutations for the Native-State Dynamics of Human Lysozyme.
abstract: The conversion of human lysozyme into amyloid fibrils is associated with a rare but fatal hereditary form of nonneuropathic systemic amyloidosis. The accumulation of large amounts of aggregated protein is thought to be initiated by the formation of transient intermediate species of disease-related lysozyme variants, essentially due to the loss of global cooperativity under physiologically relevant conditions. Interestingly, all five naturally occurring, amyloidogenic, single-point mutations are located in the β-domain of lysozyme, the region that is predominantly unfolded during the formation of the transient intermediate species. Given the lack of known naturally occurring, amyloidogenic, single-point mutations in the α-domain, we chose three specific mutations to address the effects that location may have on native-state dynamics, as studied by hydrogen-deuterium (HD) exchange experiments analyzed by NMR spectroscopy, and mass spectrometry. We compared the effect of a destabilizing α-domain mutation (I23A) with that of the well-characterized I59T β-domain variant. We also investigated the effect of a mutation that has minor effects on native-state stability at the domain interface (I56V) and compared it with that of a variant with similar stability within the C-helix (I89V). We show that when variants have similar reduced native-state stabilities, the location of the mutation (I23A versus I59T) is crucial to the native-state dynamics, with the α-domain mutation having a significantly lower ability to populate transient intermediate species under physiologically relevant conditions. Interestingly, the mutation at the interface (I56V) has a greater effect in facilitating the formation of transient intermediate species at elevated temperatures compared with the variants containing α-domain mutations, even though this mutation results in only minor changes to the native-state stability of lysozyme. These findings reveal that the location of specific mutations is an important factor in determining the native-state dynamical properties of human lysozyme in the context of its propensity to populate the aggregation-prone transient intermediate species associated with pathogenic amyloid formation.
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Minkoo
authors.initials: M
authors.lastname: Ahn
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Christine L
authors.initials: CL
authors.lastname: Hagan
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Ana
authors.initials: A
authors.lastname: Bernardo-Gancedo
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Erwin
authors.initials: E
authors.lastname: De Genst
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Francisco N
authors.initials: FN
authors.lastname: Newby
authors.affiliation: Institute of Structural and Molecular Biology, University College London and Birkbeck College, London, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Christodoulou
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Anne
authors.initials: A
authors.lastname: Dhulesia
authors.affiliation: Laboratory of Enzymology and Protein Folding, Centre for Protein Engineering, InBios, Institute of Chemistry, University of Liege, Liege (Sart Tilman), Belgium.
authors.forename: Mireille
authors.initials: M
authors.lastname: Dumoulin
authors.affiliation: Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, United Kingdom.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. Electronic address: jrk38@cam.ac.uk.
authors.forename: Janet R
authors.initials: JR
authors.lastname: Kumita
mesh_terms: D000682:Amyloid / Q000737:chemistry; D004795:Enzyme Stability; D006801:Humans; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D000072417:Protein Domains; D055503:Protein Multimerization; D017433:Protein Structure, Secondary
chemical_list: D000682:Amyloid; D009113:Muramidase