Extrapolating differential scanning calorimetry data for monoclonal antibodies to low temperatures.

For irreversible denaturation transitions such as those exhibited by monoclonal antibodies, differential scanning calorimetry provides the denaturation temperature, T, the rate of denaturation at T, and the activation energy at T. These three quantities are essential but not sufficient for an accurate extrapolation of the rate of denaturation to temperatures of 25 °C and below. We have observed that the activation energy is not constant but temperature dependent due to the existence of an activation heat capacity, C.

Development of high-affinity nanobodies specific for Na1.4 and Na1.5 voltage-gated sodium channel isoforms.

Voltage-gated sodium channels, Nas, are responsible for the rapid rise of action potentials in excitable tissues. Na channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-Na nanobodies (Nbs), Nb17 and Nb82, that recognize the Na1.

Reversibility and irreversibility in the temperature denaturation of monoclonal antibodies.

There have been numerous studies of the temperature denaturation of monoclonal antibodies (mAbs) using differential scanning calorimetry (DSC). In general, mAbs are characterized by complex temperature denaturation transitions in which the various domains (CH2, CH3, Fab) give rise to different peaks in the heat capacity function. The complexity and overall irreversibility of the temperature denaturation transition is well known and has limited the number of publications with an in-depth analysis of the data.

Binding Thermodynamics to Intrinsically Disordered Protein Domains.

Many proteins are intrinsically disordered or contain one or more disordered domains. These domains can participate in binding interactions with other proteins or small ligands. Binding to intrinsically disordered protein domains requires the folding or structuring of those regions such that they can establish well-defined stoichiometric interactions.

Author Correction: A human monoclonal antibody prevents malaria infection by targeting a new site of vulnerability on the parasite.

In the version of this article originally published, data were incorrectly ascribed to monoclonal antibody CIS34 because of a labeling error. The data were generated with monoclonal antibody CIS04. Full details can be found in the correction notice.

Isothermal calorimetry of a monoclonal antibody using a conventional differential scanning calorimeter.

It has been shown that isothermal calorimetry is able to provide critical information regarding the kinetics of denaturation/aggregation of monoclonal antibodies at temperatures below T. Those measurements, however, required sophisticated specialized instrumentation. Here, we demonstrate that similar measurements can be performed using widely available conventional differential scanning calorimeters (DSC) when operated in isothermal scan mode.

SOSIP Changes Affect Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Conformation and CD4 Engagement.

The entry of human immunodeficiency virus into host cells is mediated by the envelope glycoprotein (Env) trimeric spike, which consists of three exterior gp120 subunits and three transmembrane gp41 subunits. The trimeric Env undergoes extensive conformational rearrangement upon interaction with the CD4 receptor, transitioning from the unliganded, "closed" State 1 to more-open downstream State 2 and State 3 conformations. Changes in "restraining" amino acid residues, such as leucine 193 and isoleucine 423, destabilize State 1 Env, which then assumes entry-competent, downstream conformations.

Long term stability of a HIV-1 neutralizing monoclonal antibody using isothermal calorimetry.

Different factors affect the long term stability of monoclonal antibodies, among them denaturation or partial denaturation that is often followed by aggregation. Isothermal calorimetry is capable of quantifying the kinetics of denaturation/aggregation of an antibody by measuring the heat that is released or absorbed by the process over a period of days or weeks, at temperatures below its denaturation temperature, T. The denaturation/aggregation kinetics of the anti-HIV monoclonal antibody VRC07-523LS was measured by isothermal calorimetry at different concentrations in four different formulation buffers.

Bioinspired supramolecular engineering of self-assembling immunofibers for high affinity binding of immunoglobulin G.

Many one-dimensional (1D) nanostructures are constructed by self-assembly of peptides or peptide conjugates containing a short β-sheet sequence as the core building motif essential for the intermolecular hydrogen bonding that promotes directional, anisotropic growth of the resultant assemblies. While this molecular engineering strategy has led to the successful production of a plethora of bioactive filamentous β-sheet assemblies for interfacing with biomolecules and cells, concerns associated with effective presentation of α-helical epitopes and their function preservation have yet to be resolved. In this context, we report on the direct conjugation of the protein A mimicking peptide Z33, a motif containing two α-helices, to linear hydrocarbons to create self-assembling immuno-amphiphiles (IAs).

A human monoclonal antibody prevents malaria infection by targeting a new site of vulnerability on the parasite.

Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed. Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) from several subjects immunized with an attenuated Pf whole-sporozoite (SPZ) vaccine (Sanaria PfSPZ Vaccine). Passive transfer of one of these antibodies, monoclonal antibody CIS43, conferred high-level, sterile protection in two different mouse models of malaria infection.

Publisher Correction: The β20-β21 of gp120 is a regulatory switch for HIV-1 Env conformational transitions.

The original version of this Article contained an error in the spelling of the author Amos B. Smith, III, which was incorrectly given as Amos B. SmithIII.

The β20-β21 of gp120 is a regulatory switch for HIV-1 Env conformational transitions.

The entry of HIV-1 into target cells is mediated by the viral envelope glycoproteins (Env). Binding to the CD4 receptor triggers a cascade of conformational changes in distant domains that move Env from a functionally "closed" State 1 to more "open" conformations, but the molecular mechanisms underlying allosteric regulation of these transitions are still elusive. Here, we develop chemical probes that block CD4-induced conformational changes in Env and use them to identify a potential control switch for Env structural rearrangements.

A novel lipoate attachment enzyme is shared by Plasmodium and Chlamydia species.

Lipoate is an essential cofactor for enzymes that are important for central metabolism and other processes. In malaria parasites, scavenged lipoate from the human host is required for survival. The Plasmodium falciparum mitochondrion contains two enzymes (PfLipL1 and PfLipL2) that are responsible for activating mitochondrial proteins through the covalent attachment of lipoate (lipoylation).

Crystal structures of trimeric HIV envelope with entry inhibitors BMS-378806 and BMS-626529.

The HIV-1 envelope (Env) spike is a conformational machine that transitions between prefusion (closed, CD4- and CCR5-bound) and postfusion states to facilitate HIV-1 entry into cells. Although the prefusion closed conformation is a potential target for inhibition, development of small-molecule leads has been stymied by difficulties in obtaining structural information. Here, we report crystal structures at 3.

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry.

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency.

Enthalpy screen of drug candidates.

The enthalpic and entropic contributions to the binding affinity of drug candidates have been acknowledged to be important determinants of the quality of a drug molecule. These quantities, usually summarized in the thermodynamic signature, provide a rapid assessment of the forces that drive the binding of a ligand. Having access to the thermodynamic signature in the early stages of the drug discovery process will provide critical information towards the selection of the best drug candidates for development.

Small-Molecule CD4-Mimics: Structure-Based Optimization of HIV-1 Entry Inhibition.

The optimization, based on computational, thermodynamic, and crystallographic data, of a series of small-molecule ligands of the Phe43 cavity of the envelope glycoprotein gp120 of human immunodeficiency virus (HIV) has been achieved. Importantly, biological evaluation revealed that the small-molecule CD4 mimics (4-7) inhibit HIV-1 entry into target cells with both significantly higher potency and neutralization breadth than previous congeners, while maintaining high selectivity for the target virus. Their binding mode was characterized via thermodynamic and crystallographic studies.

Three easy pieces.

Background: Differential scanning calorimetry is a powerful method that provides a complete thermodynamic characterization of the stability of a protein as a function of temperature. There are, however, circumstances that preclude a complete analysis of DSC data. The most common ones are irreversible denaturation transitions or transitions that take place at temperatures that are beyond the temperature limit of the instrument.

Conformational stability and self-association equilibrium in biologics.

Biologics exist in equilibrium between native, partially denatured, and denatured conformational states. The population of any of these states is dictated by their Gibbs energy and can be altered by changes in physical and solution conditions. Some conformations have a tendency to self-associate and aggregate, an undesirable phenomenon in protein therapeutics.

Synthetic, structural mimetics of the β-hairpin flap of HIV-1 protease inhibit enzyme function.

Small-molecule mimetics of the β-hairpin flap of HIV-1 protease (HIV-1 PR) were designed based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap-flap protein-protein interaction, which functions as a gated mechanism to control access to the active site. Michaelis-Menten kinetics suggested our small-molecules are competitive inhibitors, which indicates the mode of inhibition is through binding the active site or sterically blocking access to the active site and preventing flap closure, as designed. More generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most potent compound inhibiting the protease with a modest K(i) of 11 μM.

Impact of Surface Polyethylene Glycol (PEG) Density on Biodegradable Nanoparticle Transport in Mucus ex Vivo and Distribution in Vivo.

Achieving sustained drug delivery to mucosal surfaces is a major challenge due to the presence of the protective mucus layer that serves to trap and rapidly remove foreign particulates. Nanoparticles engineered to rapidly penetrate mucosal barriers (mucus-penetrating particles, "MPP") have shown promise for improving drug distribution, retention and efficacy at mucosal surfaces. MPP are densely coated with polyethylene glycol (PEG), which shields the nanoparticle core from adhesive interactions with mucus.

Denatured state aggregation parameters derived from concentration dependence of protein stability.

Protein aggregation is a major issue affecting the long-term stability of protein preparations. Proteins exist in equilibrium between the native and denatured or partially denatured conformations. Often denatured or partially denatured conformations are prone to aggregate because they expose to solvent the hydrophobic core of the protein.

Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env.

As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones.

CD4 mimetics sensitize HIV-1-infected cells to ADCC.

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals.

Characterization of protein-protein interactions by isothermal titration calorimetry.

The analysis of protein-protein interactions has attracted the attention of many researchers from both a fundamental point of view and a practical point of view. From a fundamental point of view, the development of an understanding of the signaling events triggered by the interaction of two or more proteins provides key information to elucidate the functioning of many cell processes. From a practical point of view, understanding protein-protein interactions at a quantitative level provides the foundation for the development of antagonists or agonists of those interactions.