Engineering the kinetic stability of a β-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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

Computational Modeling of Protein Stability: Quantitative Analysis Reveals Solutions to Pervasive Problems.

Accurate modeling of the effects of mutations on protein stability is central to understanding and controlling proteins in myriad natural and applied contexts. Here, we reveal through rigorous quantitative analysis that stability prediction tools often favor mutations that increase stability at the expense of solubility. Moreover, while these tools may accurately identify strongly destabilizing mutations, the experimental effect of mutations predicted to stabilize is actually near neutral on average.

TNF receptor-associated factor 6 interacts with ALS-linked misfolded superoxide dismutase 1 and promotes aggregation.

Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1 rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations.

Temperature dependence of NMR chemical shifts: Tracking and statistical analysis.

Isotropic chemical shifts measured by solution nuclear magnetic resonance (NMR) spectroscopy offer extensive insights into protein structure and dynamics. Temperature dependences add a valuable dimension; notably, the temperature dependences of amide proton chemical shifts are valuable probes of hydrogen bonding, temperature-dependent loss of structure, and exchange between distinct protein conformations. Accordingly, their uses include structural analysis of both folded and disordered proteins, and determination of the effects of mutations, binding, or solution conditions on protein energetics.

Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol.

Bound metals are observed in a great many natural proteins, where they perform diverse roles in determining protein folding, stability and function. Due to the broad impact of bound metals on biophysical and biochemical properties of proteins, it is valuable to have accurate and facile methods for determining the metal content of proteins. Here we describe an optimized methodology using 4-(2-pyridylazo)resorcinol (PAR) to simultaneously quantify two metal ions in solution.

Effects of maturation on the conformational free-energy landscape of SOD1.

Amyotrophic lateral sclerosis (ALS) is a devastating fatal syndrome characterized by very rapid degeneration of motor neurons. A leading hypothesis is that ALS is caused by toxic protein misfolding and aggregation, as also occurs in many other neurodegenerative disorders, such as prion, Alzheimer's, Parkinson's, and Huntington's diseases. A prominent cause of familial ALS is mutations in the protein superoxide dismutase (SOD1), which promote the formation of misfolded SOD1 conformers that are prone to aberrant interactions both with each other and with other cellular components.

Computational tools help improve protein stability but with a solubility tradeoff.

Accurately predicting changes in protein stability upon amino acid substitution is a much sought after goal. Destabilizing mutations are often implicated in disease, whereas stabilizing mutations are of great value for industrial and therapeutic biotechnology. Increasing protein stability is an especially challenging task, with random substitution yielding stabilizing mutations in only ∼2% of cases.

Asymmetric Anchoring Is Required for Efficient Ω-Loop Opening and Closing in Cytosolic Phosphoenolpyruvate Carboxykinase.

Mobile Ω-loops play essential roles in the function of many enzymes. Here we investigated the importance of a residue lying outside of the mobile Ω-loop element in the catalytic function of an H477R variant of cytosolic phosphoenolpyruvate carboxykinase using crystallographic, kinetic, and computational analysis. The crystallographic data suggest that the efficient transition of the Ω-loop to the closed conformation requires stabilization of the N-terminus of the loop through contacts between R461 and E588.

Exploring the relationships between protein sequence, structure and solubility.

Aggregation can be thought of as a form of protein folding in which intermolecular associations lead to the formation of large, insoluble assemblies. Various types of aggregates can be differentiated by their internal structures and gross morphologies (e.g.

Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that, in some cases, has been linked with mutations to the antioxidant metalloenzyme superoxide dismutase (SOD1). Although the mature form of this enzyme is highly stable and resistant to aggregation, the most immature form, lacking metal and a stabilizing intrasubunit disulfide bond, apoSOD1, is dynamic and hypothesized to be a major cause of toxicity in vivo. Previous solution NMR studies of wild-type apoSOD1 have shown that the ground state interconverts with a series of sparsely populated and transiently formed conformers, some of which have aberrant nonnative structures.

Using natural sequences and modularity to design common and novel protein topologies.

Protein design is still a challenging undertaking, often requiring multiple attempts or iterations for success. Typically, the source of failure is unclear, and scoring metrics appear similar between successful and failed cases. Nevertheless, the use of sequence statistics, modularity and symmetry from natural proteins, combined with computational design both at the coarse-grained and atomistic levels is propelling a new wave of design efforts to success.

ALS-linked misfolded SOD1 species have divergent impacts on mitochondria.

Approximately 20 % of familial Amyotrophic Lateral Sclerosis (ALS) is caused by mutations in superoxide dismutase (SOD1), which leads to misfolding of the SOD1 protein, resulting in a toxic gain of function. Several conformation-restricted antibodies have been generated that specifically recognize misfolded SOD1 protein, and have been used as therapeutics in pre-clinical models. Misfolded SOD1 selectively associates with spinal cord mitochondria in SOD1 rodent models.

Ensemble Modeling and Intracellular Aggregation of an Engineered Immunoglobulin-Like Domain.

The production of recombinant proteins in Escherichia coli frequently results in the formation of insoluble protein aggregates called inclusion bodies (IBs). The determinants of IB formation remain poorly understood and are of much interest for biotechnological and research applications, as well as offering insight into disease-related in vivo protein aggregation. Here we investigate a set of engineered target-binding proteins based upon the fibronectin type III domain, and we find that variations in sequence at just three positions in a solvent-exposed loop greatly alter the extent of IB formation.

Concurrent Increases and Decreases in Local Stability and Conformational Heterogeneity in Cu, Zn Superoxide Dismutase Variants Revealed by Temperature-Dependence of Amide Chemical Shifts.

The chemical shifts of backbone amide protons in proteins are sensitive reporters of local structural stability and conformational heterogeneity, which can be determined from their readily measured linear and nonlinear temperature-dependences, respectively. Here we report analyses of amide proton temperature-dependences for native dimeric Cu, Zn superoxide dismutase (holo pWT SOD1) and structurally diverse mutant SOD1s associated with amyotrophic lateral sclerosis (ALS). Holo pWT SOD1 loses structure with temperature first at its periphery and, while having extremely high global stability, nevertheless exhibits extensive conformational heterogeneity, with ∼1 in 5 residues showing evidence for population of low energy alternative states.

Combined Isothermal Titration and Differential Scanning Calorimetry Define Three-State Thermodynamics of fALS-Associated Mutant Apo SOD1 Dimers and an Increased Population of Folded Monomer.

Many proteins are naturally homooligomers, homodimers most frequently. The overall stability of oligomeric proteins may be described in terms of the stability of the constituent monomers and the stability of their association; together, these stabilities determine the populations of different monomer and associated species, which generally have different roles in the function or dysfunction of the protein. Here we show how a new combined calorimetry approach, using isothermal titration calorimetry to define monomer association energetics together with differential scanning calorimetry to measure total energetics of oligomer unfolding, can be used to analyze homodimeric unmetalated (apo) superoxide dismutase (SOD1) and determine the effects on the stability of structurally diverse mutations associated with amyotrophic lateral sclerosis (ALS).