Engineering the kinetic stability of a ß-trefoil protein by tuning its topological complexity.

Kinetic stability, defined as the rate of protein unfolding, is central to determining the functional lifetime of proteins, both in nature and in wide-ranging medical and biotechnological applications. Further, high kinetic stability is generally correlated with high resistance against chemical and thermal denaturation, as well as proteolytic degradation. Despite its significance, specific mechanisms governing kinetic stability remain largely unknown, and few studies address the rational design of kinetic stability. Here, we describe a method for designing protein kinetic stability that uses protein long-range order, absolute contact order, and simulated free energy barriers of unfolding to quantitatively analyze and predict unfolding kinetics. We analyze two ß-trefoil proteins: hisactophilin, a quasi-three-fold symmetric natural protein with moderate stability, and ThreeFoil, a designed three-fold symmetric protein with extremely high kinetic stability. The quantitative analysis identifies marked differences in long-range interactions across the protein hydrophobic cores that partially account for the differences in kinetic stability. Swapping the core interactions of ThreeFoil into hisactophilin increases kinetic stability with close agreement between predicted and experimentally measured unfolding rates. These results demonstrate the predictive power of readily applied measures of protein topology for altering kinetic stability and recommend core engineering as a tractable target for rationally designing kinetic stability that may be widely applicable.

Immature ALS-associated mutant superoxide dismutases form variable aggregate structures through distinct oligomerization processes.

Protein misfolding and aggregation are hallmarks of many diseases, including amyotrophic lateral sclerosis (ALS). In familial ALS, aberrant self-association of mutant Cu,Zn-superoxide dismutase (SOD1) is implicated as a key contributor to disease. Mutations have the largest impacts on the stability of the most immature form of SOD1, the unmetallated, disulfide-reduced monomer (apoSH SOD1). Here we demonstrate that, despite the marginal stability of apoSH SOD1, aggregation is little correlated with the degree of protein unfolding, and multiple modes of aggregation occur, depending on the mutation and solution conditions. Light scattering and atomic force microscopy reveal two distinct mutant SOD1 behaviours: high aggregator mutants form abundant small assemblies, while low aggregator mutants form fewer, more fibre-like aggregates. Attenuated total reflectance-Fourier transform infrared spectroscopy and Thioflavin T binding show the aggregates maintain native-like anti-parallel beta structure. These results provide new evidence that ALS-associated mutations promote the aggregation of apoSH SOD1 through multiple pathways, with broad implications for understanding mechanisms of protein self-association in disease and biotechnology.

A fine balance of hydrophobic-electrostatic communication pathways in a pH-switching protein.

Allostery is the phenomenon of coupling between distal binding sites in a protein. Such coupling is at the crux of protein function and regulation in a myriad of scenarios, yet determining the molecular mechanisms of coupling networks in proteins remains a major challenge. Here, we report mechanisms governing pH-dependent myristoyl switching in monomeric hisactophilin, whereby the myristoyl moves between a sequestered state, i.e., buried within the core of the protein, to an accessible state, in which the myristoyl has increased accessibility for membrane binding. Measurements of the pH and temperature dependence of amide chemical shifts reveal protein local structural stability and conformational heterogeneity that accompany switching. An analysis of these measurements using a thermodynamic cycle framework shows that myristoyl-proton coupling at the single-residue level exists in a fine balance and extends throughout the protein. Strikingly, small changes in the stereochemistry or size of core and surface hydrophobic residues by point mutations readily break, restore, or tune myristoyl switch energetics. Synthesizing the experimental results with those of molecular dynamics simulations illuminates atomistic details of coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions. The simulations were critical for discerning which of the many ionizable residues in hisactophilin are important for switching and identifying the contributions of nonnative interactions in switching. The strategy of using temperature-dependent NMR presented here offers a powerful, widely applicable way to elucidate the molecular mechanisms of allostery in proteins at high resolution.

Methodological advances and strategies for high resolution structure determination of cellular protein aggregates.

Aggregation of proteins is at the nexus of molecular processes crucial to aging, disease, and employing proteins for biotechnology and medical applications. There has been much recent progress in determining the structural features of protein aggregates that form in cells; yet, owing to prevalent heterogeneity in aggregation, many aspects remain obscure and often experimentally intractable to define. Here, we review recent results of structural studies for cell-derived aggregates of normally globular proteins, with a focus on high-resolution methods for their analysis and prediction. Complementary results obtained by solid-state NMR spectroscopy, FTIR spectroscopy and microspectroscopy, cryo-EM, and amide hydrogen/deuterium exchange measured by NMR and mass spectrometry, applied to bacterial inclusion bodies and disease inclusions, are uncovering novel information on in-cell aggregation patterns as well as great diversity in the structural features of useful and aberrant protein aggregates. Using these advances as a guide, this review aims to advise the reader on which combination of approaches may be the most appropriate to apply to their unique system.

Quenched hydrogen-deuterium amide exchange optimization for high-resolution structural analysis of cellular protein aggregates.

Inclusion bodies (IBs) are large, insoluble aggregates that often form during the overexpression of proteins in bacteria. These aggregates are of broad fundamental and practical significance, for recombinant protein preparation and due to their relevance to aggregation-related medical conditions and their recent emergence as promising functional nanomaterials. Despite their significance, high resolution knowledge of IB structure remains very limited. Such knowledge will advance understanding and control of IB formation and properties in myriad practical applications. Here, we report a detailed quenched hydrogen-deuterium amide exchange (qHDX) method with NMR readout to define the structure of IBs at the level of individual residues throughout the protein. Applying proper control of experimental conditions, such as sample pH, water content, temperature, and intrinsic rate of amide exchange, yields in depth results for these cellular protein aggregates. qHDX results illustrated for Cu, Zn superoxide dismutase 1 (SOD1) and Adnectins show their IBs include native-like structure and some but not all mutations alter IB structure.

High-Resolution NMR H/D Exchange of Human Superoxide Dismutase Inclusion Bodies Reveals Significant Native Features Despite Structural Heterogeneity.

Protein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high-resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native-like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation-prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.

A Descriptor Set for Quantitative Structure-property Relationship Prediction in Biologics.

There has been a remarkable increase in the number of biologics, especially monoclonal antibodies, in the market over the last decade. In addition to attaining the desired binding to their targets, a crucial aspect is the 'developability' of these drugs, which includes several desirable properties such as high solubility, low viscosity and aggregation, physico-chemical stability, low immunogenicity and low poly-specificity. The lack of any of these desirable properties can lead to significant hurdles in advancing them to the clinic and are often discovered only during late stages of drug development. Hence, in silico methods for early detection of these properties, particularly the ones that affect aggregation and solubility in the earlier stages can be highly beneficial. We have developed a computational framework based on a large and diverse set of protein specific descriptors that is ideal for making liability predictions using a QSPR (quantitative structure-property relationship) approach. This set offers a high degree of feature diversity that may coarsely be classified based on (1) sequence (2) structure and (3) surface patches. We assess the sensitivity and applicability of these descriptors in four dedicated case studies that are believed to be representative of biophysical characterizations commonly employed during the development process of a biologics drug candidate. In addition to data sets obtained from public sources, we have validated the descriptors on novel experimental data sets in order to address antibody developability and to generate prospective predictions on Adnectins. The results show that the descriptors are well suited to assist in the improvement of protein properties of systems that exhibit poor solubility or aggregation.

Design for Solubility May Reveal Induction of Amide Hydrogen/Deuterium Exchange by Protein Self-Association.

Structural heterogeneity often constrains the characterization of aggregating proteins to indirect or low-resolution methods, obscuring mechanistic details of association. Here, we report progress in understanding the aggregation of Adnectins, engineered binding proteins with an immunoglobulin-like fold. We rationally design Adnectin solubility and measure amide hydrogen/deuterium exchange (HDX) under conditions that permit transient protein self-association. Protein-protein binding commonly slows rates of HDX; in contrast, we find that Adnectin association may induce faster HDX for certain amides, particularly in the C-terminal ß-strand. In aggregation-prone proteins, we identify a pattern of very different rates of amide HDX for residues linked by reciprocal hydrogen bonds in the native structure. These results may be explained by local loss of native structure and formation of an inter-protein interface. Amide HDX induced by self-association, detected here by deliberate modulation of propensity for such interactions, may be a general phenomenon with the potential to expose mechanisms of aggregation by diverse proteins.

Wild-type and mutant SOD1 localizes to RNA-rich structures in cells and mice but does not bind RNA.

Many of the genes whose mutation causes Amyotrophic Lateral Sclerosis (ALS) are RNA-binding proteins which localize to stress granules, while others impact the assembly, stability, and elimination of stress granules. This has led to the hypothesis that alterations in the dynamics of stress granules and RNA biology cause ALS. Genetic mutations in Superoxide Dismutase 1 (SOD1) also cause ALS. Evidence demonstrates that SOD1 harboring ALS-linked mutations is recruited to stress granules, induces changes in alternative splicing, and could be an RNA-binding protein. Whether SOD1 inclusions contain RNA in disease models and whether SOD1 directly binds RNA remains uncertain. We applied methods including cross-linking immunoprecipitation and in vitro gel shift assays to detect binding of SOD1 to RNA in vitro, in cells with and without stress granules, and in mice expressing human SOD1 G93A. We find that SOD1 localizes to RNA-rich structures including stress granules, and SOD1 inclusions in mice contain mRNA. However, we find no evidence that SOD1 directly binds RNA. This suggests that SOD1 may impact stress granules, alternative splicing and RNA biology without binding directly to RNA.

Insight into the Autosomal-Dominant Inheritance Pattern of SOD1-Associated ALS from Native Mass Spectrometry.

About 20% of all familial amyotrophic lateral sclerosis (ALS) cases are associated with mutations in superoxide dismutase (SOD1), a homodimeric protein. The disease has an autosomal-dominant inheritance pattern. It is, therefore, important to determine whether wild-type and mutant SOD1 subunits self-associate randomly or preferentially. A measure for the extent of bias in subunit association is the coupling constant determined in a double-mutant cycle type analysis. Here, cell lysates containing co-expressed wild-type and mutant SOD1 subunits were analyzed by native mass spectrometry to determine these coupling constants. Strikingly, we find a linear positive correlation between the coupling constant and the reported average duration of the disease. Our results indicate that inter-subunit communication and a preference for heterodimerization greatly increase the disease severity.