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We have studied regular acting, wild-type human insulin at potency of 100 U/mL from four different pharmaceutical products directly from their final finished formulation by the combined use of mass spectrometry (MS), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), and single-crystal protein crystallography (PX). All products showed similar oligomeric assembly in solution as judged by DLS and SAXS measurements. The NMR spectra were compatible with well folded proteins, showing close conformational identity for the human insulin in the four products. Crystallographic assays conducted with the final formulated products resulted in all insulin crystals belonging to the R3 space group with two a dimer in the asymmetric unit, both with the B-chain in the T configuration. Meta-analysis of the 24 crystal structures solved from the four distinct insulin products revealed close similarity between them regardless of variables such as biological origin, product batch, country origin of the product, and analytical approach, revealing a low conformational variability for the converging insulin structural ensemble. We propose the use of MS, SAXS, NMR fingerprint, and PX as a precise chemical and structural proof of folding identity of regular insulin in the final, formulated product.

Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn2؉ enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn 2؉ at pH 4.6 -7. Human transthyretin (TTR)5 is a 55 kDa, ␤-sheet-rich homotetramer found in serum and cerebrospinal fluid. It participates in thyroxine (T4) transport (1) and in the binding of holo retinol-binding protein (holo-RBP) and is believed to reduce the glomerular filtration of the relatively small (21 kDa) holo-RBP (2). In humans, TTR and its more than 100 variants are involved in amyloid diseases such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP), among others (3). In SSA, deposits composed of the wild-type TTR (WT-TTR) are found in the heart. Autopsies show that at least 25% of all individuals over 80 years have such deposits, which prove fatal in 10% of these cases (4). FAP is caused by the accumulation of amyloid fibrils formed by TTR mutants, mainly around basal membranes of Schwann cells, inside the endoneurium of peripheral nerves (5, 6). Onset typically occurs in the third decade, with a life expectancy of ϳ10 years (6). Several drugs are being tested against TTR amyloidosis (3), but so far, the only treatment for FAP is liver transplantation (7).A widely accepted hypothesis for TTR amyloidogenesis presupposes tetramer dissociation into a misfolded monomer which aggregates, giving rise to amyloid fibrils. In vitro these events can be triggered by mild acidification (pH 5.0 -4.0), although amyloid formation takes several days for completion (8). In vivo these events might occur inside lysosomes during TTR turnover (8). Recently, J. W. Kelly and co-workers (9) described an engineered monomer of TTR (M-TTR) in which the introduction of two bulky amino acid residues in the protein-protein interface (F87M/L110M) avoids oligomerization into dimers or tetramers. M-TTR aggregates in solution in the same pH range as WT-TTR, although for M-TTR this occurs on a much faster time scale because tetramer dissociation into monomers is the rate-limiting step in TTR aggregation (9).Monomers (A-D) of TTR are composed of eight anti-parallel ␤-strands (A-H) arranged as a ␤-barrel (Greek key) with a short ␣-helix following strand E. The dimer (AB) is held together by hydrogen bonds involving the H and F strands located along the edge of each monomer. Two dimers (AB, CD) associate backto-back via hydrophobic interactions between residues of loops connecting strands AB and GH (10) (Fig. 1, A and B).Numerous studies with WT and variants of TTR have attempted to unravel the structural rearrangements that precede amyloid formation (10 -13). Only minor global and local changes are observed in most of these structures (10) R. L.).The atomic coordinates and structure factors (codes 3GRG, 3GRB, 3GPS, and 3DGD)

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