title: Denaturation and solvent effect on the conformation and fibril formation of TGFBIp.
abstract: OBJECTIVE
  Transforming growth factor beta-induced protein (TGFBIp) aggregates into the phenotypic amyloid fibrils and/or non-amyloid deposits in corneal dystrophies and other disorders. While significant progress has been made in molecular genetics to successfully establish the link between the missense mutations of TGFBI and TGFBIp-related corneal dystrophies, the underlying mechanism for the abnormal aggregation remains elusive due to the lack of insights into the conformational perturbations induced by mutations. In the present study, we examined the effects of denaturants and a co-solvent on recombinant TGFBIp, with a focus on protein conformational changes and amyloid fibril formation.
  
  
  METHODS
  Recombinant TGFBIp was subjected to various spectroscopic studies, such as far-ultraviolet circular dichroism (far-UV CD), intrinsic tryptophan fluorescence and quenching, and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence, under various denaturing conditions (urea and guanidine hydrochloride [GndHCl], acidic pH, and trifluoroethanol [TFE, co-solvent]). A thioflavin T (ThT) fluorescence assay was used to determine the fibril formation of TGFBIp. In addition, a rabbit polyclonal antibody against the oligomer precursors that initiate the formation of amyloid fibrils was also used in dot blot experiments to detect the formation of prefibrillar precursors.
  
  
  RESULTS
  The purified recombinant TGFBIp is in the folded state according to its intrinsic tryptophan fluorescence analyses. A single-step unfolding process was observed in the GndHCl denaturation experiment. Results from far-UV CD, intrinsic tryptophan fluorescence, and ANS fluorescence experiments showed that TFE exerted its solvent effects by initially unfolding and transforming TGFBIp to a beta-sheet-enriched conformer at 20%. When increased to 40%, TFE changed TGFBIp into a non-native alpha-helix conformer. Although GndHCl and TFE led to protein unfolding, enhanced fibril formation could only be observed in the presence of TFE and at acidic pH, according to the ThT fluorescence assays. The paradigmatic protofibrillar TGFBIp oligomers were also detected during the fibril formation by the dot blot experiment.
  
  
  CONCLUSIONS
  Our results suggest that protein unfolding may serve as the prerequisite but is not sufficient for the fibrillogenesis. Other factors, such as the solvent used, fragmentation, or pH, may also be crucial for the formation of TGFBIp fibrils.
authors.affiliation: Department of Ophthalmology, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Grothe
authors.forename: Morgan R
authors.initials: MR
authors.lastname: Little
authors.forename: Angela S
authors.initials: AS
authors.lastname: Cho
authors.forename: Andrew J W
authors.initials: AJ
authors.lastname: Huang
authors.forename: Ching
authors.initials: C
authors.lastname: Yuan
mesh_terms: D000682:Amyloid / Q000378:metabolism*; D002460:Cell Line; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis; D012997:Solvents / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000378:metabolism*; D014270:Trifluoroethanol / Q000494:pharmacology; D014364:Tryptophan / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D000099222:betaIG-H3 Protein
chemical_list: D000682:Amyloid; D016326:Extracellular Matrix Proteins; D019791:Guanidine; D011994:Recombinant Proteins; D012997:Solvents; D016212:Transforming Growth Factor beta; D014270:Trifluoroethanol; D014364:Tryptophan; D014508:Urea; D000099222:betaIG-H3 Protein