Interpretation of mass spectra is challenging because they report a ratio of two physical quantities, mass and charge, which may each have multiple components that overlap in m/z. Previous approaches to disentangling the two have focused on peak assignment or fitting. However, the former struggle with complex spectra, and the latter are generally computationally intensive and may require substantial manual intervention. We propose a new data analysis approach that employs a Bayesian framework to separate the mass and charge dimensions. On the basis of this approach, we developed UniDec (Universal Deconvolution), software that provides a rapid, robust, and flexible deconvolution of mass spectra and ion mobility-mass spectra with minimal user intervention. Incorporation of the charge-state distribution in the Bayesian prior probabilities provides separation of the m/z spectrum into its physical mass and charge components. We have evaluated our approach using systems of increasing complexity, enabling us to deduce lipid binding to membrane proteins, to probe the dynamics of subunit exchange reactions, and to characterize polydispersity in both protein assemblies and lipoprotein Nanodiscs. The general utility of our approach will greatly facilitate analysis of ion mobility and mass spectra.

Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and dynamic complexes with target proteins, serving as a first line of defense in preventing their aggregation into either amorphous deposits or amyloid fibrils. Their apparently broad target specificity makes sHSPs attractive for investigating ways to tackle disorders of protein aggregation. The two most abundant sHSPs in human tissue are αB-crystallin (ABC) and HSP27; here we present high-resolution structures of their core domains (cABC, cHSP27), each in complex with a segment of their respective C-terminal regions. We find that both truncated proteins dimerize, and although this interface is labile in the case of cABC, in cHSP27 the dimer can be cross-linked by an intermonomer disulfide linkage. Using cHSP27 as a template, we have designed an equivalently locked cABC to enable us to investigate the functional role played by oligomerization, disordered N and C termini, subunit exchange, and variable dimer interfaces in ABC. We have assayed the ability of the different forms of ABC to prevent protein aggregation in vitro. Remarkably, we find that cABC has chaperone activity comparable to that of the full-length protein, even when monomer dissociation is restricted through disulfide linkage. Furthermore, cABC is a potent inhibitor of amyloid fibril formation and, by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-β peptide to cells. Overall we present a small chaperone unit together with its atomic coordinates that potentially enables the rational design of more effective chaperones and amyloid inhibitors.X-ray crystallography | ion mobility mass spectrometry | nuclear magnetic resonance spectroscopy

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A central goal in biochemistry is to explain the causes of protein sequence, structure, and function. Mainstream approaches seek to rationalize sequence and structure in terms of their effects on function and to identify function’s underlying determinants by comparing related proteins to each other. Although productive, both strategies suffer from intrinsic limitations that have left important aspects of many proteins unexplained. These limits can be overcome by reconstructing ancient proteins, experimentally characterizing their properties, and retracing their evolution through time. This approach has proven to be a powerful means for discovering how historical changes in sequence produced the functions, structures, and other physical/chemical characteristics of modern proteins. It has also illuminated whether protein features evolved because of functional optimization, historical constraint, or blind chance. Here we review recent studies employing ancestral protein reconstruction and show how they have produced new knowledge not only of molecular evolutionary processes but also of the underlying determinants of modern proteins’ physical, chemical, and biological properties.