Synthesis of an Analog of the Thyroid Hormone-binding Protein Transthyretin via Regioselective Chemical Ligation

Abstract

Transthyretin is an essential protein responsible for the transport of thyroid hormones and retinol in human serum and is also implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. Here we report the solid phase synthesis of the monomeric unit of a transthyretin analog (equivalent to 127 amino acids) using t-Boc chemistry and peptide ligation and its folding to form a functional 54-kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts (positions 1-51, 54 -99, and 102-127) and ligated using a chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of transthyretin's native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, transthyretin antibody recognition, and thyroid hormone binding. Other folding products included a high molecular weight aggregate as well as a transient dimeric species. This represents one of the largest macromolecules chemically synthesized to date and demonstrates the potential of protein chemical synthesis for investigations of protein-ligand interactions.

Synthesis and Structural Analysis of the N-Terminal Domain of the Thyroid Hormone-Binding Protein Transthyretin

“…The purified chloroacetylated 54 -127 peptide was then exchanged to the iodoacetylated form and ligated with C-thiolated 1 -51 (peptide III) to produce fully ligated 1 -127 synthetic TTR. The ligated 1 -127 peptide was purified by RP-HPLC in excellent yield, and its identity confirmed by ESMS (22).…”

“…The synthetic TTR spontaneously folded to its tetramer complex in the presence of the ligand T4, in 0.075 M NH 4 HCO 3 (22). Western analysis was employed to determine the subunit molecular mass and confirm the recognition of the sTTR by anti-TTR antiserum.…”

“…The focus of that presentation was on the chemical ligation strategy used to produce synthetic TTR. Accordingly, the current article provides a brief overview of that methodology, which has been described in full elsewhere (22), but focuses mainly on new experimental data on the solution properties of the N-terminal fragment peptide, TTR(1 -51). It was of interest to determine whether this peptide would adopt a stable monomeric β-sheet structure or indeed any stable secondary structure in the absence of the remainder of the protein, and whether it may form fibrils by itself.…”

“…In a recent report (22) we demonstrated the total chemical synthesis of an analogue of human TTR using a thioether-based strategy for the sequential ligation of three peptide fragments of TTR. We showed that this synthetic TTR (henceforth referred to as sTTR) could be successfully refolded and reconstituted to form a 55 kDa tetrameric structure able to bind the thyroid hormone T4.…”

“…The TTR fragment was synthesized as described previously (22). It comprises the N-terminal 51 amino acids of TTR linked to an alkyl thiol, with Cys10 replaced by Ala.…”