An engineered transthyretin monomer that is nonamyloidogenic, unless it is partially denatured.
Transthyretin (TTR) is a soluble human plasma protein that can be converted into amyloid by acid-mediated dissociation of the homotetramer into monomers. The pH required for disassembly also results in tertiary structural changes within the monomeric subunits. To understand whether these tertiary structural changes are required for amyloidogenicity, we created the Phe87Met/Leu110Met TTR variant (M-TTR) that is monomeric according to analytical ultracentrifugation and gel filtration analyses and nonamyloidogenic at neutral pH. Results from far- and near-UV circular dichroism spectroscopy, one-dimensional proton NMR spectroscopy, and X-ray crystallography, as well as the ability of M-TTR to form a complex with retinol binding protein, indicate that M-TTR forms a tertiary structure at pH 7 that is very similar if not identical to that found within the tetramer. Reducing the pH results in tertiary structural changes within the M-TTR monomer, rendering it amyloidogenic, demonstrating the requirement for partial denaturation. M-TTR exhibits stability toward acid and urea denaturation that is nearly identical to that characterizing wild-type (WT) TTR at low concentrations (0.01-0.1 mg/mL), where monomeric WT TTR is significantly populated at intermediate urea concentrations prior to the tertiary structural transition. However, the kinetics of denaturation and fibril formation are much faster for M-TTR than for tetrameric WT TTR, particularly at near-physiological concentrations, because of the barrier associated with the tetramer to folded monomer preequilibrium. These results demonstrate that the tetramer to folded monomer transition is insufficient for fibril formation; further tertiary structural changes within the monomer are required.
Jiang
X
X
Department of Chemistry and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Smith
C S
CS
Petrassi
H M
HM
Hammarström
P
P
White
J T
JT
Sacchettini
J C
JC
Kelly
J W
JW
D019943:Amino Acid Substitution; D000682:Amyloid / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D019281:Dimerization; D004355:Drug Stability; D005456:Fluorescent Dyes; D019791:Guanidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011228:Prealbumin / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis; D014461:Ultracentrifugation / Q000379:methods; D014508:Urea
D000682:Amyloid; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D011228:Prealbumin; D011994:Recombinant Proteins; D014364:Tryptophan; D014508:Urea; D019791:Guanidine