A kinetic study of ovalbumin fibril formation: the importance of fragmentation and end-joining.

The ability to control the morphologies of biomolecular aggregates is a central objective in the study of self-assembly processes. The development of predictive models offers the surest route for gaining such control. Under the right conditions, proteins will self-assemble into fibers that may rearrange themselves even further to form diverse structures, including the formation of closed loops. In this study, chicken egg white ovalbumin is used as a model for the study of fibril loops. By monitoring the kinetics of self-assembly, we demonstrate that loop formation is a consequence of end-to-end association between protein fibrils. A model of fibril formation kinetics, including end-joining, is developed and solved, showing that end-joining has a distinct effect on the growth of fibrillar mass density (which can be measured experimentally), establishing a link between self-assembly kinetics and the underlying growth mechanism. These results will enable experimentalists to infer fibrillar morphologies from an appropriate analysis of self-assembly kinetic data.

A mass-spectrometry-based framework to define the extent of disorder in proteins.

In the past decade, mass spectrometry (MS) coupled with electrospray ionization (ESI) has been extensively applied to the study of intact proteins and their complexes, often without the requirement of labels. Solvent conditions (for example, pH, ionic strength, and concentration) affect the observed desolvated species; the ease of altering such extrinsic factors renders ESI-MS an appropriate method by which to consider the range of conformational states that proteins may occupy, including natively folded, disordered and amyloid. Rotationally averaged collision cross sections of the ionized forms of proteins, provided by the combination of mass spectrometry and ion mobility (IM-MS), are also instructive in exploring conformational landscapes in the absence of solvent. Here, we ask the following question: "If the only technique you had was ESI-IM-MS, what information would it provide on the structural preferences of an unknown protein?" We have selected 20 different proteins, both monomeric and multimeric, ranging in mass from 2846 Da (melittin) to 150 kDa (Immunoglobulin G), and we consider how they are presented to a mass spectrometer under different solvent conditions. Mass spectrometery allows us to distinguish which of these proteins are structured (melittin, human beta defensin 1, truncated human lymphotactin, Cytochrome C, holo hemoglobin-α, ovalbumin, human transthyretin, avidin, bovine serum albumin, concanavalin, human serum amyloid protein, and Immunoglobulin G) from those that contain at least some regions of disorder (human lymphotactin, N-terminal p53, α-Synuclein, N-terminal MDM2, and p53 DNA binding domain) or denatured due to solvent conditions (ubiquitin, apo hemoglobin-α, apo hemoglobin-ß) by considering two experimental parameters: the range of charge states occupied by the protein (Δz) and the range of collision cross sections in which the protein is observed (ΔCCS). We also provide a simple model to predict the difference between the collision cross sections of the most compact and the most extended form of a given protein, based on the volume of the amino acids it contains. We compare these calculated parameters with experimental values. In addition, we consider the occupancy of conformations based on the intensities of ions in the mass spectra. This allows us to qualitatively predict the potential energy landscape of each protein. Our empirical approach to assess order or disorder is shown to be more accurate than the use of charge hydropathy plots, which are frequently used to predict disorder, and could provide an initial route to characterization. Finally, we present an ESI-IM-MS methodology to determine if a given protein is structured or disordered.