title: Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis.
abstract: The amyloidogenic homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to partially denatured monomers in order to aggregate. TTR contains two distinct quaternary interfaces, one of which defines the binding sites for thyroxine and small-molecule amyloidogenesis inhibitors. Kinetic stabilization of the tetramer can be accomplished either by the binding of amyloidogenesis inhibitors selectively to the native state over the dissociative transition state or by the introduction of trans-suppressor subunits (T119M) into heterotetramers to destabilize the dissociative transition state. In each case, increasing the dissociation activation barrier prevents tetramer dissociation. Herein, we demonstrate that tethering two subunits whose quaternary interface defines the thyroxine binding site also dramatically increases the barrier for tetramer dissociation, apparently by destabilization of the dissociative transition state. The tethered construct (TTR-L-TTR)2 is structurally and functionally equivalent to wild-type TTR. Urea is unable to denature (TTR-L-TTR)2, yet it is able to maintain the denatured state once denaturation is achieved by GdnHCl treatment, suggesting that (TTR-L-TTR)2 is kinetically rather than thermodynamically stabilized, consistent with the identical wild-type TTR and (TTR-L-TTR)2 GdnHCl denaturation curves. Studies focused on a construct containing a single TTR-L-TTR chain and two normal monomer subunits establish that alteration of only one quaternary structural interface is sufficient to impose kinetic stabilization on the entire quaternary structure.
authors.affiliation: Department of Chemistry and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Ted R
authors.initials: TR
authors.lastname: Foss
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Kelker
authors.forename: R Luke
authors.initials: RL
authors.lastname: Wiseman
authors.forename: Ian A
authors.initials: IA
authors.lastname: Wilson
authors.forename: Jeffery W
authors.initials: JW
authors.lastname: Kelly
mesh_terms: D000682:Amyloid / Q000096:biosynthesis / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation / Q000187:drug effects; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
chemical_list: D000682:Amyloid; D008024:Ligands; D011228:Prealbumin; D021122:Protein Subunits; D014508:Urea; D019791:Guanidine