Profiling Hinge Plasticity in Intact Monoclonal Antibodies for Antigen Recognition.

Monoclonal antibodies (mAbs) are multidomain glycosylated proteins that mediate antigen binding, among other protein-protein interactions, which makes them successful therapeutics. However, in many cases, adverse physicochemical properties can affect their antigen-binding abilities, compromising therapeutic potential. Hence, structural characterization of these biotherapeutics is strongly desired to predict and possibly redesign better variants.

Stable RNA G-Quadruplex in the 5'-UTR of Human mRNA Promotes Translation in an IRES-Independent Manner.

RNA G-quadruplex (rG4) structures can influence the fate and functions of mRNAs, especially the translation process. The presence of rG4 structures in 5'-untranslated regions (5'-UTRs) of mRNAs generally represses translation. However, rG4 structures can also promote internal ribosome entry site (IRES)-mediated translation as one of its determinants.

Profiling Enzyme Activity of l-Asparaginase II by NMR-Based Methyl Fingerprinting at Natural Abundance.

l-asparaginase II (MW 135 kDa) from is an FDA-approved protein drug used for the treatment of childhood leukemia. Despite its long history as a chemotherapeutic, the structural basis of enzyme action, in solution, remains widely contested. In this work, methyl-based 2D [H-C]-heteronuclear single-quantum correlation (HSQC) NMR, at natural abundance, has been used to profile the enzymatic activity of the commercially available enzyme drug.

Utility of High Resolution 2D NMR Fingerprinting in Assessing Viscosity of Therapeutic Monoclonal Antibodies.

Purpose: The viscosity of highly concentrated therapeutic monoclonal antibody (mAb) formulations at concentrations ≥ 100 mg/mL can significantly affect the stability, processing, and drug product development for subcutaneous delivery. An early identification of a viscosity prone mAb during candidate selection stages are often beneficial for downstream processes. Higher order structure of mAbs may often dictate their viscosity behavior at high concentration.

Utility of High Resolution NMR Methods to Probe the Impact of Chemical Modifications on Higher Order Structure of Monoclonal Antibodies in Relation to Antigen Binding.

Purpose: An understanding of higher order structure (HOS) of monoclonal antibodies (mAbs) could be critical to predicting its function. Amongst the various factors that can potentially affect HOS of mAbs, chemical modifications that are routinely encountered during production and long-term storage are of significant interest.

Methods: To this end, two Pfizer mAbs were subjected to forced deamidation stress for a period of eight weeks.

Impact of Buffers on Colloidal Property and Aggregation Propensities of a Bispecific Antibody.

Bispecific antibodies represent a promising avenue whereby 2 different binding specificities of a single-chain antibody can be grafted into a common Fc fragment to generate one antibody-like molecule. Despite the promising efficacy of such modalities, they may lack manufacturability because of stability and aggregation issues. Herein, we performed a systematic buffer screening for an aggregation-prone therapeutic bispecific antibody (BsAb) during early stage development.

Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies.

Purpose: Fc domains are an integral component of monoclonal antibodies (mAbs) and Fc-based fusion proteins. Engineering mutations in the Fc domain is a common approach to achieve desired effector function and clinical efficacy of therapeutic mAbs. It remains debatable, however, whether molecular engineering either by changing glycosylation patterns or by amino acid mutation in Fc domain could impact the higher order structure of Fc domain potentially leading to increased aggregation propensities in mAbs.

Ribosome Mediated Quinary Interactions Modulate In-Cell Protein Activities.

Ribosomes are present inside bacterial cells at micromolar concentrations and occupy up to 20% of the cell volume. Under these conditions, even weak quinary interactions between ribosomes and cytosolic proteins can affect protein activity. By using in-cell and in vitro NMR spectroscopy, and biophysical techniques, we show that the enzymes, adenylate kinase and dihydrofolate reductase, and the respective coenzymes, ATP and NADPH, bind to ribosomes with micromolar affinity, and that this interaction suppresses the enzymatic activities of both enzymes.

Total Cellular RNA Modulates Protein Activity.

RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source.

Design of a MCoTI-Based Cyclotide with Angiotensin (1-7)-Like Activity.

We report for the first time the design and synthesis of a novel cyclotide able to activate the unique receptor of angiotensin (1-7) (AT1-7), the MAS1 receptor. This was accomplished by grafting an AT1-7 peptide analog onto loop 6 of cyclotide MCoTI-I using isopeptide bonds to preserve the α-amino and C-terminal carboxylate groups of AT1-7, which are required for activity. The resulting cyclotide construct was able to adopt a cyclotide-like conformation and showed similar activity to that of AT1-7.

Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α-Synuclein-Induced Cytotoxicity in Baker's Yeast.

We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with a highly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human α-synuclein in live yeast cells.

Probing protein quinary interactions by in-cell nuclear magnetic resonance spectroscopy.

Historically introduced by McConkey to explain the slow mutation rate of highly abundant proteins, weak protein (quinary) interactions are an emergent property of living cells. The protein complexes that result from quinary interactions are transient and thus difficult to study biochemically in vitro. Cross-correlated relaxation-induced polarization transfer-based in-cell nuclear magnetic resonance allows the characterization of protein quinary interactions with atomic resolution inside live prokaryotic and eukaryotic cells.

Caught in action: selecting peptide aptamers against intrinsically disordered proteins in live cells.

Intrinsically disordered proteins (IDPs) or unstructured segments within proteins play an important role in cellular physiology and pathology. Low cellular concentration, multiple binding partners, frequent post-translational modifications and the presence of multiple conformations make it difficult to characterize IDP interactions in intact cells. We used peptide aptamers selected by using the yeast-two-hybrid scheme and in-cell NMR to identify high affinity binders to transiently structured IDP and unstructured segments at atomic resolution.

Using singular value decomposition to characterize protein-protein interactions by in-cell NMR spectroscopy.

Distinct differences between how model proteins interact in-cell and in vitro suggest that the cytosol might have a profound effect in modulating protein-protein and/or protein-ligand interactions that are not observed in vitro. Analyses of in-cell NMR spectra of target proteins interacting with physiological partners are further complicated by low signal-to-noise ratios, and the long overexpression times used in protein-protein interaction studies may lead to changes in the in-cell spectra over the course of the experiment. To unambiguously resolve the principal binding mode between two interacting species against the dynamic cellular background, we analyzed in-cell spectral data of a target protein over the time course of overexpression of its interacting partner by using single-value decomposition (SVD).

In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide.

The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists.

Design of a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity.

Herein, we report for the first time the design and synthesis of a novel cyclotide able to efficiently inhibit HIV-1 viral replication by selectively targeting cytokine receptor CXCR4. This was accomplished by grafting a series of topologically modified CVX15 based peptides onto the loop 6 of cyclotide MCoTI-I. The most active compound produced in this study was a potent CXCR4 antagonist (EC50≈20 nM) and an efficient HIV-1 cell-entry blocker (EC50≈2 nM).

Efficient one-pot cyclization/folding of Rhesus θ-defensin-1 (RTD-1).

We report an efficient approach for the chemical synthesis of Rhesus θ-defensin-1 (RTD-1) using Fmoc-based solid-phase peptide synthesis in combination with an intramolecular version of native chemical ligation. The corresponding linear thioester precursor was cyclized and folded in a one-pot reaction using reduced glutathione. The reaction was extremely efficiently yielding natively folded RTD-1 with minimal or no purification at all.

Recombinant production of rhesus θ-defensin-1 (RTD-1) using a bacterial expression system.

Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. In contrast to mammalian α- and β-defensins, rhesus θ-defensin-1 (RTD-1) comprises only 18 amino acids stabilized by three disulfide bonds and an unusual backbone cyclic topology. In this work we report for the first time the recombinant expression of the fully folded θ-defensin RTD-1 using a bacterial expression system.