From free to free market: cost recovery in federally funded clinical research.

In a climate of increased expectation for the translation of research, academic clinical research units are looking at new ways to streamline their operation and maintain effective translational support services. Clinical research, although undeniably expensive, is an essential step in the translation of any medical breakthrough, and as a result, many academic clinical research units are actively looking to expand their clinical services despite financial pressures. We examine some of the hybrid academic-business models in 19 clinical research centers within the Clinical and Translational Science Award consortium that are emerging to address the issue of cost recovery of clinical research that is supported by the United States federal government. We identify initiatives that have succeeded or failed, essential supporting and regulatory components, and lessons learned from experience to design an optimal cost recovery model and a timeline for its implementation.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation.

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.

Mutating the tight-dimer interface of dihydrodipicolinate synthase disrupts the enzyme quaternary structure: toward a monomeric enzyme.

Dihydrodipicolinate synthase (DHDPS) is a tetrameric enzyme that is the first enzyme unique to the ( S)-lysine biosynthetic pathway in plants and bacteria. Previous studies have looked at the important role of Tyr107, an amino acid residue located at the tight-dimer interface between two monomers, in participating in a catalytic triad of residues during catalysis. In this study, we examine the importance of this residue in determining the quaternary structure of the DHDPS enzyme. The Tyr107 residue was mutated to tryptophan, and structural, biophysical, and kinetic studies were carried out on the mutant enzyme. These revealed that while the solid-state structure of the mutant enzyme was largely unchanged, as judged by X-ray crystallography, it exists as a mixture of primarily monomer and tetramer in solution, as determined by analytical ultracentrifugation, size-exclusion chromatography, and mass spectrometry. The catalytic ability of the DHDPS enzyme was reduced by the mutation, which also allowed the adventitious binding of alpha-ketoglutarate to the active site. A reduction in the apparent melting temperature of the mutant enzyme was observed. Thus, the tetrameric quaternary structure of DHDPS is critical to controlling specificity, heat stability, and intrinsic activity.

The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner.

Amyloid fibrils are typically rigid, unbranched structures with diameters of approximately 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low pH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underlying protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression.

Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris.

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200- and 30-fold lower, respectively, than that of wild-type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post-transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease-associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native-state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease.

L55P transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers.

Transthyretin is a tetrameric protein associated with the commonest form of systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry, we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, L55P. Wild-type subunit exchange occurs via both monomers and dimers, whereas exchange via dimers is the dominant mechanism for the L55P variant. Because patients with the L55P mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and L55P tetramers. We found that hybrid tetramers containing two or three L55P subunits dominate in the early stages of the reaction. Surprisingly, we also found that, in the presence of L55P transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidosis.

The flight of macromolecular complexes in a mass spectrometer.

The discovery that conditions can be found such that non-covalent macromolecular complexes can survive the transition from solution to gas phase and remain intact during their flight in a mass spectrometer is an intriguing observation. While the nature of the interaction between the components, either ionic, hydrophobic or van der Waals, undoubtedly has an effect on the stability of these gas phase species, the role of small molecules in conferring additional stability is often overlooked. Here we review historical aspects of the development of mass spectrometry for macromolecular complexes with particular focus on the role of small molecules in stabilizing gas-phase complexes. Moreover, we demonstrate how the dissociation of small molecules from subunits within a macromolecular complex can be used to probe the topological arrangement. Overall, therefore, we show that mass spectrometry used in this way is capable of addressing features of the energy landscape not readily accessed by traditional structural biology approaches.

FTIR reveals structural differences between native beta-sheet proteins and amyloid fibrils.

The presence of beta-sheets in the core of amyloid fibrils raised questions as to whether or not beta-sheet-containing proteins, such as transthyretin, are predisposed to form such fibrils. However, we show here that the molecular structure of amyloid fibrils differs more generally from the beta-sheets in native proteins. This difference is evident from the amide I region of the infrared spectrum and relates to the distribution of the phi/psi dihedral angles within the Ramachandran plot, the average number of strands per sheet, and possibly, the beta-sheet twist. These data imply that amyloid fibril formation from native beta-sheet proteins can involve a substantial structural reorganization.

Tandem mass spectrometry defines the stoichiometry and quaternary structural arrangement of tryptophan molecules in the multiprotein complex TRAP.

We have used tandem mass spectrometry to examine the stoichiometry and binding sites of trp molecules in various assemblies of the protein complex TRAP. The results show that TRAP forms oligomers containing 11 and 12 subunits. MS/MS experiments show that up to 11 trp molecules bind to the 12-mer but that during gas-phase dissociation 5 then 6 trp molecules are released reflecting the different gas-phase stabilities of the partially ligated forms. At high trp concentrations, the protein assembles to form a double ring structure. Tandem mass spectrometry reveals that it is composed of 24 subunits with up to 22 molecules of trp. Dissociation of the complex reveals the same dissociation pathway as for the single ring structure, allowing us to propose a model for the assembly of the TRAP 24-mer based on the different environments of trp molecules. More generally, these results demonstrate the power of tandem mass spectrometry for defining the stoichiometry and quaternary structural arrangement of subunits and ligands within a 46-component multiprotein multiligand complex.

Structural change in response to ligand binding.

The minutiae of subtle changes that occur in response to ligand binding in multiprotein complexes are often difficult to assess without resource to high resolution X-ray analysis. Recent developments in mass spectrometry, however, are providing insight into dynamic changes within components. In this article we review recent applications of MS for selection of ligands and definition of their binding characteristics for individual protein targets through to macromolecular complexes such as ribosomes.

Screening transthyretin amyloid fibril inhibitors: characterization of novel multiprotein, multiligand complexes by mass spectrometry.

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein.

A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies.

We report the design and first applications of a tandem mass spectrometer (a quadrupole time-of-flight mass spectrometer) optimized for the transmission and analysis of large macromolecular assemblies. Careful control of the pressure gradient in the different pumping stages of the instrument has been found to be essential for the detection of macromolecular particles. Such assemblies are, however, difficult to analyze by tandem-MS approaches, because they give rise to signals above m/z 3,000-4,000, the limit for commercial quadrupoles. By reducing the frequency of the quadrupole to 300 kHz and using it as a narrow-band mass filter, we show that it is possible to isolate ions from a single peak at m/z 22,000 in a window as narrow as 22 m/z units. Using cesium iodide cluster signals, we show that the mass range in the time-of-flight (TOF) analyzer extends beyond m/z 90,000, in theory to more than m/z 150,000. We also demonstrate that the resolution of the instrument is greater than 3,000 at m/z 44,500. Tandem-MS capabilities are illustrated by separating components from heterooligomeric assemblies formed between tetrameric transthyretin, thyroxine, retinol-binding protein, and retinol. Isolation of a single charge state at m/z 5,340 in the quadrupole and subsequent collision-induced dissociation (CID) in the gas-filled collision cell leads to the formation of ions from individual subunits and subcomplexes, identified by their mass and charge in the TOF analyzer.