Margaret G. McCammon

FTIR reveals structural differences between native β‐sheet proteins and amyloid fibrils

The presence of β‐sheets in the core of amyloid fibrils raised questions as to whether or not β‐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 β‐sheets in native proteins. This difference is evident from the amide I region of the infrared spectrum and relates to the distribution of the ϕ/ψ dihedral angles within the Ramachandran plot, the average number of strands per sheet, and possibly, the β‐sheet twist. These data imply that amyloid fibril formation from native β‐sheet proteins can involve a substantial structural reorganization.

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.

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.

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.

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.

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.

Me, My Cell, and I: The Role of the Collision Cell in the Tandem Mass Spectrometry of Macromolecules

The molecular apparatus of the cell comprises a multitude of large protein assemblies that carry out the majority of biological activities. These cellular machines are often heterogeneous in nature, with transient noncovalent interactions playing a vital role in their function. Such dynamic characteristics make investigation by traditional structural biology approaches challenging, but in recent years mass spectrometry (MS), a relative newcomer to the field, has developed at a spectacular pace to offer a novel molecular viewpoint. Today MS provides far more than simple mass measurement, contributing in multiple ways to our understanding of these cellular assemblies. Much of this new ability is attributable to the flexible application of collisioninduced dissociation (CID).