Ligand Binding Kinetics to Evaluate Function and Stability of AR in Nanodiscs.

G-Protein coupled receptors (GPCRs) represent one of the largest classes of therapeutic targets. However, developing successful therapeutics to target GPCRs is a challenging endeavor with many molecules failing during in vivo clinical trials due to a lack of efficacy. The in vitro identification of drug targeted residence time (1/k) has been suggested to improve prediction of in vivo success.

The Interplay between GSK3β and Tau Ser262 Phosphorylation during the Progression of Tau Pathology.

Tau hyperphosphorylation has been linked directly to the formation of toxic neurofibrillary tangles (NFTs) in tauopathies, however, prior to NFT formation, the sequence of pathological events involving tau phosphorylation remains unclear. Here, the effect of glycogen synthase kinase 3β (GSK3β) on tau pathology was examined independently for each step of transcellular propagation; namely, tau intracellular aggregation, release, cellular uptake and seeding activity. We find that overexpression of GSK3β-induced phosphorylated 0N4R tau led to a higher level of tau oligomerization in SH-SY5Y neuroblastoma cells than wild type 0N4R, as determined by several orthogonal assays.

Cholesterol Dependent Activity of the Adenosine A Receptor Is Modulated via the Cholesterol Consensus Motif.

Background: Membrane cholesterol dysregulation has been shown to alter the activity of the adenosine A receptor (AR), a G protein-coupled receptor, thereby implicating cholesterol levels in diseases such as Alzheimer's and Parkinson's. A limited number of AR crystal structures show the receptor interacting with cholesterol, as such molecular simulations are often used to predict cholesterol interaction sites.

Methods: Here, we use experimental methods to determine whether a specific interaction between amino acid side chains in the cholesterol consensus motif (CCM) of full length, wild-type human AR, and cholesterol modulates activity of the receptor by testing the effects of mutational changes on functional consequences, including ligand binding, G protein coupling, and downstream activation of cyclic AMP.

Critical Molecular and Cellular Contributors to Tau Pathology.

Tauopathies represent a group of neurodegenerative diseases including Alzheimer's disease (AD) that are characterized by the deposition of filamentous tau aggregates in the brain. The pathogenesis of tauopathies starts from the formation of toxic 'tau seeds' from hyperphosphorylated tau monomers. The presence of specific phosphorylation sites and heat shock protein 90 facilitates soluble tau protein aggregation.

Membrane cholesterol depletion reduces downstream signaling activity of the adenosine A receptor.

Cholesterol has been shown to modulate the activity of multiple G Protein-coupled receptors (GPCRs), yet whether cholesterol acts through specific interactions, indirectly via modifications to the membrane, or via both mechanisms is not well understood. High-resolution crystal structures of GPCRs have identified bound cholesterols; based on a β-adrenergic receptor (βAR) structure bound to cholesterol and the presence of conserved amino acids in class A receptors, the cholesterol consensus motif (CCM) was identified. Here in mammalian cells expressing the adenosine A receptor (AR), ligand dependent production of cAMP is reduced following membrane cholesterol depletion with methyl-beta-cyclodextrin (MβCD), indicating that AR signaling is dependent on cholesterol.

Cellular engineering for therapeutic protein production: product quality, host modification, and process improvement.

Recombinant proteins offer many therapeutic advantages unavailable in traditional small molecule drugs, but the need for cellular versus chemical synthesis complicates production. Avenues for producing therapeutic biologics are continuously expanding, and developments in biochemistry, cell biology, and bioengineering fuel new discoveries that promise safer, more efficient, and cheaper drugs for consumers. Numerous approaches to express recombinant proteins exist, but Escherichia coli, Saccharomyces cerevisiae, and mammalian systems (e.

AR Binding Kinetics in the Ligand Depletion Regime.

Ligand binding plays a fundamental role in stimulating the downstream signaling of membrane receptors. Here, ligand-binding kinetics of the full-length human adenosine A receptor (AR) reconstituted in detergent micelles were measured using a fluorescently labeled ligand via fluorescence anisotropy. Importantly, to optimize the signal-to-noise ratio, these experiments were conducted in the ligand depletion regime.

Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids.

The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR.

Conformational stability as a design target to control protein aggregation.

Non-native protein aggregation is a prevalent problem occurring in many biotechnological manufacturing processes and can compromise the biological activity of the target molecule or induce an undesired immune response. Additionally, some non-native aggregation mechanisms lead to amyloid fibril formation, which can be associated with debilitating diseases. For natively folded proteins, partial or complete unfolding is often required to populate aggregation-prone conformational states, and therefore one proposed strategy to mitigate aggregation is to increase the free energy for unfolding (ΔGunf) prior to aggregation.

Computational design and biophysical characterization of aggregation-resistant point mutations for γD crystallin illustrate a balance of conformational stability and intrinsic aggregation propensity.

γD crystallin is a natively monomeric eye-lens protein that is associated with hereditary juvenile cataract formation. It is an attractive model system as a multidomain Greek-key protein that aggregates through partially folded intermediates. Point mutations M69Q and S130P were used to test (1) whether the protein-design algorithm RosettaDesign would successfully predict mutants that are resistant to aggregation when combined with informatic sequence-based predictors of peptide aggregation propensity and (2) how the mutations affected relative unfolding free energies (ΔΔG(un)) and intrinsic aggregation propensity (IAP).

Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast.

Heterologous protein expression can easily overwhelm a cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. In the current model of the UPR, the chaperone BiP modulates the activation of the UPR due to its interactions with the signaling protein Ire1p and newly synthesized proteins. In this research, 4-4-20 scFv variants were generated by rational design to alter BiP binding to newly synthesized scFv proteins or via directed evolution aimed at improved secretion.

The C-terminus of the P22 tailspike protein acts as an independent oligomerization domain for monomeric proteins.

TSP (P22 tailspike protein) is a well-established model system for studying the folding and assembly of oligomeric proteins, and previous studies have documented both in vivo and in vitro folding intermediates using this protein. Especially important is the C-terminus of TSP, which plays a critical role in the assembly and maturation of the protrimer intermediate to its final trimeric form. In the present study, we show that by grafting the C-terminus of TSP on to the monomeric MBP (maltose-binding protein), the resulting chimaera (MBP-537) is a trimeric protein.

A top-down approach to mechanistic biological modeling: application to the single-chain antibody folding pathway.

A top-down approach to mechanistic modeling of biological systems is presented and exemplified with the development of a hypothesis-driven mathematical model for single-chain antibody fragment (scFv) folding in Saccharomyces cerevisiae by mediators BiP and PDI. In this approach, model development starts with construction of the most basic mathematical model--typically consisting of predetermined or newly-elucidated biological behavior motifs--capable of reproducing desired biological behaviors. From this point, mechanistic detail is added incrementally and systematically, and the effects of each addition are evaluated.

Refolding of G protein alpha subunits from inclusion bodies expressed in Escherichia coli.

Heterotrimeric G proteins relay signals from G protein-coupled receptors (GPCRs) to the interior of the cell. The signaling cascades induced by G protein activation control a wide range of cellular processes. The alpha subunit is believed to determine which G protein couples to each GPCR, and is the primary determinant of the type of signal transmitted.

Heterologous GPCR expression: a bottleneck to obtaining crystal structures.

G protein-coupled receptors (GPCRs) are an important, medically relevant class of integral membrane proteins. Laboratories throughout all disciplines of science devote time and energy into developing practical methods for the discovery, isolation, and characterization of these proteins. Since the crystal structure of rhodopsin was solved 6 years ago, the race to determine high-resolution structures of more GPCRs has gained momentum.

PDI improves secretion of redox-inactive beta-glucosidase.

Although manipulation of the endoplasmic reticulum (ER) folding environment in the yeast Saccharomyces cerevisiae has been shown to increase the secretory productivity of recombinant proteins, the cellular interactions and processes of native enzymes and chaperones such as protein disulfide isomerase (PDI) are still unclear. Previously, we reported that overexpression of the ER chaperone PDI enabled up to a 3-fold increase in secretion levels of the Pyrococcus furiosus beta-glucosidase in the yeast S. cerevisiae.

Expression and purification of milligram levels of inactive G-protein coupled receptors in E. coli.

G-protein coupled receptors (GPCRs) are seven transmembrane helical proteins involved in cell signaling and response. They are targets for many existing therapeutic agents, and numerous drug discovery efforts. Production of large quantities of these receptors for drug screening and structural biology remains challenging.

Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate.

To elucidate determinants of thermostability and folding pathways of the intrinsically stable proteins from extremophilic organisms, we are studying beta-glucosidase from Pyrococcus furiosus. Using fluorescence and circular dichroism spectroscopy, we have characterized the thermostability of beta-glucosidase at 90 degrees C, the lowest temperature where full unfolding is achieved with urea. The chemical denaturation profile reveals that this homotetrameric protein unfolds at 90 degrees C with an overall DeltaG degrees of approximately 20 kcal mol(-1).

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS.

Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates.

Exceptional total and functional yields of the human adenosine (A2a) receptor expressed in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was used to express a medically relevant G-protein coupled receptor (GPCR), the human adenosine (A2a) receptor, with a C-terminal green fluorescent protein (GFP) fusion tag. In prior studies, we established an expression system for A2a-GFP. Here, we quantified the total A2a-GFP expression levels by correlating GFP levels as detected by fluorescence and densitometry to A2a-GFP molecules overexpressed in the system.

Three amino acids that are critical to formation and stability of the P22 tailspike trimer.

The P22 tailspike protein folds by forming a folding competent monomer species that forms a dimeric, then a non-native trimeric (protrimer) species by addition of folding competent monomers. We have found three residues, R549, R563, and D572, which play a critical role in both the stability of the native tailspike protein and assembly and maturation of the protrimer. King and colleagues reported previously that substitution of R563 to glutamine inhibited protrimer formation.

Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding.

The production of recombinant proteins is a critical technology for biotechnology and biomedical research. Heterologous expression of secreted proteins can saturate the cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. The overexpression of chaperone binding protein (BiP) and disulfide bond isomerase (PDI) in Saccaromyces cerevisiae can effectively increase protein production levels of single-chain antibody (scFv) 4-4-20.

Decreases in yeast expression yields of the human adenosine A2a receptor are a result of translational or post-translational events.

The human adenosine receptor (A2a), a G-protein-coupled receptor (GPCR), was C-terminally tagged with the green fluorescent protein (GFP) and expressed in the yeast Saccharomyces cerevisiae to gain an understanding of the expression limitations of this medically relevant class of membrane proteins. The A2a-GFP protein was able to bind adenosine analogs indicating that the GFP tag did not alter the ligand binding activity of the receptor. A screen based on whole cell fluorescence was developed and a library of clones with various gene copy numbers was screened via flow cytometry to isolate clones with the highest protein expression levels.

Pressure dissociation studies provide insight into oligomerization competence of temperature-sensitive folding mutants of P22 tailspike.

Several temperature-sensitive folding (tsf) mutants of the tailspike protein from bacteriophage P22 have been found to fold with lower efficiency than the wild-type sequence, even at lowered temperatures. Previous refolding studies initiated from the unfolded monomer have indicated that the tsf mutations decrease the rate of structured monomer formation. We demonstrate that pressure treatment of the tailspike aggregates provides a useful tool to explore the effects of tsf mutants on the assembly pathway of the P22 tailspike trimer.

Maximizing recovery of native protein from aggregates by optimizing pressure treatment.

Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions.