Propensity For Self-association Research Articles

Elucidating the Mechanisms of R248Q Mutation-Enhanced p53 Aggregation and Its Inhibition by Resveratrol.

Aggregation of p53 mutants can result in loss-of-function, gain-of-function, and dominant-negative effects that contribute to tumor growth. Revealing the mechanisms underlying mutation-enhanced p53 aggregation and dissecting how small molecule inhibitors prevent the conversion of p53 into aggregation-primed conformations are fundamentally important for the development of novel therapeutics for p53 aggregation-associated cancers. A recent experimental study shows that resveratrol (RSV) has an inhibitory effect on the aggregation of hot-spot R248Q mutant of the p53 core domain (p53C), while pterostilbene (PT) exhibits a relatively poor inhibitory efficacy. However, the conformational properties of the R248Q mutant leading to its enhanced aggregation propensity and the inhibitory mechanism of RSV against p53C aggregation are not well understood. Herein, we performed extensive all-atom molecular dynamics simulations on R248Q p53C in the absence and presence of RSV/PT, as well as wild-type (WT) p53C. Our simulations reveal that loop L3, where the mutation resides, remains compact in WT p53C, while it becomes extended in the R248Q mutant. The extension of loop L3 weakens the interactions between loop L3 and two crucial aggregation-prone regions (APRs) of p53C, leading to impaired interactions within the APRs and their structural destabilization as well as p53C. The destabilized APRs in the R248Q mutant are more exposed than in WT p53C, which is conducive to p53C aggregation. RSV has a higher preference to bind to R248Q p53C than PT. This binding not only stabilizes loop L3 of R248Q mutant to its WT-like conformation, preventing L3-extension-caused APRs' destabilization but also reduces APRs' solvent exposure, thereby inhibiting R248Q p53C aggregation. However, PT exhibits a lower hydrogen-bonding capability and a higher self-association propensity, which would lead to a reduced p53C binding and a weakened inhibitory effect on R248Q mutant aggregation. Our study provides mechanistic insights into the R248Q mutation-enhanced aggregation propensity and RSV's potent inhibition against R248Q p53C aggregation.

Phosphorylation of Influenza A Virus Matrix Protein 1 at Threonine 108 Controls Its Multimerization State and Functional Association with the STRIPAK Complex.

The influenza A virus (IAV)-encoded matrix protein 1 (M1) acts as a master regulator of virus replication and fulfills multiple structural and regulatory functions in different cell compartments. Therefore, the spatiotemporal regulation of M1 is achieved by different mechanisms, including its structural and pH-dependent flexibility, differential association with cellular factors, and posttranslational modifications. Here, we investigated the function of M1 phosphorylation at the evolutionarily conserved threonine 108 (T108) and found that its mutation to a nonphosphorylatable alanine prohibited virus replication. Absent T108, phosphorylation led to strongly increased self-association of M1 at the cell membrane and consequently prohibited its ability to enter the nucleus and to contribute to viral ribonucleoprotein nuclear export. M1 T108 phosphorylation also controls the binding affinity to the cellular STRIPAK (striatin-interacting phosphatases and kinases) complex, which contains different kinases and the phosphatase PP2A to shape phosphorylation-dependent signaling networks. IAV infection led to the redistribution of the STRIPAK scaffolding subunits STRN and STRN3 from the cell membrane to cytosolic and perinuclear clusters, where it colocalized with M1. Inactivation of the STRIPAK complex resulted in compromised M1 polymerization and IAV replication. IMPORTANCE Influenza viruses pose a major threat to human health and cause annual epidemics and occasional pandemics. Many virus-encoded proteins exert various functions in different subcellular compartments, as exemplified by the M1 protein, but the molecular mechanisms endowing the multiplicity of functions remain incompletely understood. Here, we report that phosphorylation of M1 at T108 is essential for virus replication and controls its propensity for self-association and nuclear localization. This phosphorylation also controls binding affinity of the M1 protein to the STRIPAK complex, which contributes to M1 polymerization and virus replication.

A subtle interplay between hydrophilic and hydrophobic hydration governs butanol (de)mixing in water

A possible molecular origin of the contorting water-solubility of butanol isomers lies in the self-association propensity of alcohols as well as on the specific interaction between the hydrophilic and hydrophobic moiety of butanol(s) with water. We have made a synergistic approach using far-IR (50–600 cm−1)-FTIR and MD simulation measurements to probe the extent of alteration of HBond structure of water-network in water-alcohol mixtures.The experimental and computational observations infer that extent of hydration of the non-polar moiety of the alcohol self-aggregates plays a decisive role in the (de)mixing of butanol(s) in water.

Formation of transient highly-charged mAb clusters strengthens interactions with host cell proteins and results in poor clearance of host cell proteins by protein A chromatography

Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.

Deciphering the High Viscosity of a Therapeutic Monoclonal Antibody in High Concentration Formulations by Microdialysis-Hydrogen/Deuterium Exchange Mass Spectrometry

High concentration formulations of therapeutic monoclonal antibodies (mAbs) are highly desired for subcutaneous injection. However, high concentration formulations can exhibit unusual molecular behaviors, such as high viscosity or aggregation, that present challenges for manufacturing and administration. To understand the molecular mechanism of the high viscosity exhibited by high concentration protein formulations, we analyzed a human IgG4 (mAb1) at high protein concentrations using sedimentation velocity analytical ultracentrifugation (SV-AUC), X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein surface patches analysis. Particularly, we developed a microdialysis HDX-MS method to determine intermolecular interactions at different protein concentrations. SV-AUC revealed that mAb1 displayed a propensity for self-association of Fab-Fab, Fab-Fc, and Fc-Fc. mAb1 crystal structure and HDX-MS results demonstrated self-association between complementarity-determining regions (CDRs) and Fc through electrostatic interactions. HDX-MS also indicated Fab-Fab interactions through hydrophobic surface patches constructed by mAb1 CDRs. Our multi-method approach, including fast screening of SV-AUC as well as interface analysis by X-ray crystallography and HDX-MS, helped to elucidate the high viscosity of mAb1 at high concentrations as induced by self-associations of Fab-Fc and Fab-Fab.

Fluorinated Bisphenol Sorbent Materials for Spectroscopic Chemical Threat Sensing and Photonics Applications

Alcoholic and phenolic hydrogen-bond (HB) acidic absorbents activated by fluorine chemistries have been previously developed at the U.S. Naval Research Laboratory and elsewhere to augment sorbent HB acidity, reduce sorbent HB basicity and target complementary HB basicity of a wide range of hazardous target chemicals. A significant limitation of HB sorbents developed to date has been the propensity for sorbent self-association. This intermolecular bonding between sorbent molecules hinders sorbate access to active HB acid sites in the sorbent, limiting its overall efficacy. Sorbent self-association is evidenced by broadened hydroxyl peaks in the mid-infrared (MIR) region, which can obscure important absorption frequencies that appear upon absorption of an analyte into the sorbent. In this current work our aim is to develop improved HB acidic sorbent compounds which minimize undesired sorbent self-association in order to apply these sorbents to infrared- and Raman-based sensing devices for chemical threat detection.A series of new HB acidic sorbents have been synthesized and the subsequent sorbent-sorbate interactions have been characterized by a number of methods. MIR spectroscopy has been used to help elucidate sorbent-analyte vapor interactions. The newly synthesized sorbents have been challenged with various analyte vapors, including toxic industrial chemicals, chemical warfare agent simulants, and background interferents. A particular focus of these characterization efforts has been to observe the spectral changes that occur in the hydroxyl region of the MIR upon exposure of a sorbent material to an analyte vapor. Analyte binding of an HB base occurs principally at the sorbent hydroxyl site. The resulting redshift of the hydroxyl stretching frequency is characteristic of the basicity of the analyte.These sorbent materials are specifically designed to be selective toward hazardous chemicals through complementary hydrogen-bonding interactions between sorbent and analyte molecules. Generally, common interferents, such as hydrocarbons, have little or no hydrogen-bond basicity, while hazardous chemicals of interest have moderate to high basicity. More strongly HB basic analytes trigger larger redshifts of the hydroxyl absorption frequency. Benchtop FTIR characterization has confirmed that these newly designed sorbent materials are responsive to threat chemicals of interest at low concentrations and largely unresponsive to interferent chemicals, even at relatively high concentrations.Based on the strong affinity of these sorbents to threat chemicals of interest and the significant spectral changes that occur in the MIR upon formation of the hydrogen-bonded complex, these sorbent materials make useful candidates for MIR sensing applications. A frequency shift of the hydroxyl stretch indicates a sorbate has formed a HB with the sorbent. The magnitude of the frequency shift correlates with the basicity of the analyte, which is indicative of the class of compound to which the newly bound chemical belongs. In a sensing application, this feature can provide an alert that a hazardous chemical is present, even if it is an unknown threat. While the hydroxyl region allows for class specificity of unknown compounds, the fingerprint region complexity may facilitate specific analyte recognition. At present, this work has been focused on analysis of the hydroxyl region and distinction of different classes of compounds, but future efforts will turn to the fingerprint region to provide an avenue for specific chemical identification.Raman spectroscopy has also been used to characterize these sorbent materials. Specifically, a technique known as waveguide-enhanced Raman spectroscopy (WERS) has been used, which features the use of highly evanescent, low-loss waveguides with the sorbent material as a top cladding.2 WERS can be achieved using an incredibly small footprint with a sorbent-functionalized nanophotonic waveguide that is approximately a few centimeters long. Using WERS, the differential Raman spectra of the sorbent material interacting with different chemical warfare agent simulants has been measured at parts-per-billion detection levels. The spectra exhibit extrapolated three-sigma detection limits as low as 3 ppb. Continuing efforts are focused on adapting this technique to photonic integrated circuit-based fabrication and chip-scale Raman spectroscopy for trace chemical vapor detection.This presentation will highlight the design of these next-generation sorbents as a tool to facilitate MIR- and Raman-based sensing of threat chemicals. It will focus on analyzing sorbent-analyte spectral interactions and discuss how to exploit these features to develop MIR- and Raman-based sensors.

Application of Affinity-Capture Self-Interaction Nanoparticle Spectroscopy in Predicting Protein Stability, Especially for Co-Formulated Antibodies.

From traditional monoclonal antibodies to more and more complex mAb-based formulations, biopharmaceutical faces one challenge after another. To avoid these issues, identification of therapeutic proteins in the initial discovery process that has high stability and low self-interaction would simplify the development of safe and effective antibody therapeutics. Affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) is a new prediction method capable of identifying mAbs with different self-association propensity. In this study, 10 formulated monoclonal antibody (mAb) therapeutics include different mAb isotypes and co-formulated antibodies were measured by AC-SINS and some biophysical methods to predict protein stability. The prediction results of all 10 mAbs were compared to their stability data (Δ%monomer and Δ%HMWs) at accelerated (25°C and 40°C) and long-term storage conditions (4°C) as measured by size exclusion chromatography. AC-SINS method has a good predictive correlation with each mAbs and co-formulated antibodies. There were no physicochemical, intermolecular, or biological interactions that occurred between the two components of co-formulated antibodies which confirmed by Analytical ultracentrifugation (AUC). Here we discuss the correlation between each method and protein stability, and also use AC-SINS assay to predict the stability of co-formulated antibodies for the first time. This may be an effective way to predict the stability of these complex mAb-based formulations such as co-formulated mAbs.

Fluorinated Bisphenol Sorbent Materials for Spectroscopic Chemical Threat Sensing and Photonics Applications

Alcoholic and phenolic hydrogen-bond (HB) acidic absorbents activated by fluorine chemistries have been previously developed at the U.S. Naval Research Laboratory and elsewhere to augment sorbent HB acidity, reduce sorbent HB basicity and target complementary HB basicity of a wide range of hazardous target chemicals. A significant limitation of HB sorbents developed to date has been the propensity for sorbent self-association. This intermolecular bonding between sorbent molecules hinders sorbate access to active HB acid sites in the sorbent, limiting its overall efficacy. Sorbent self-association is evidenced by broadened hydroxyl peaks in the mid-infrared (MIR) region, which can obscure important absorption frequencies that appear upon absorption of an analyte into the sorbent. In this current work our aim is to develop improved HB acidic sorbent compounds which minimize undesired sorbent self-association in order to apply these sorbents to infrared- and Raman-based sensing devices for chemical threat detection.A series of new HB acidic sorbents have been synthesized and the subsequent sorbent-sorbate interactions have been characterized by a number of methods. MIR spectroscopy has been used to help elucidate sorbent-analyte vapor interactions. The newly synthesized sorbents have been challenged with various analyte vapors, including toxic industrial chemicals, chemical warfare agent simulants, and background interferents. A particular focus of these characterization efforts has been to observe the spectral changes that occur in the hydroxyl region of the MIR upon exposure of a sorbent material to an analyte vapor. Analyte binding of an HB base occurs principally at the sorbent hydroxyl site. The resulting redshift of the hydroxyl stretching frequency is characteristic of the basicity of the analyte.These sorbent materials are specifically designed to be selective toward hazardous chemicals through complementary hydrogen-bonding interactions between sorbent and analyte molecules. Generally, common interferents, such as hydrocarbons, have little or no hydrogen-bond basicity, while hazardous chemicals of interest have moderate to high basicity. More strongly HB basic analytes trigger larger redshifts of the hydroxyl absorption frequency. Benchtop FTIR characterization has confirmed that these newly designed sorbent materials are responsive to threat chemicals of interest at low concentrations and largely unresponsive to interferent chemicals, even at relatively high concentrations.Based on the strong affinity of these sorbents to threat chemicals of interest and the significant spectral changes that occur in the MIR upon formation of the hydrogen-bonded complex, these sorbent materials make useful candidates for MIR sensing applications. A frequency shift of the hydroxyl stretch indicates a sorbate has formed a HB with the sorbent. The magnitude of the frequency shift correlates with the basicity of the analyte, which is indicative of the class of compound to which the newly bound chemical belongs. In a sensing application, this feature can provide an alert that a hazardous chemical is present, even if it is an unknown threat. While the hydroxyl region allows for class specificity of unknown compounds, the fingerprint region complexity may facilitate specific analyte recognition. At present, this work has been focused on analysis of the hydroxyl region and distinction of different classes of compounds, but future efforts will turn to the fingerprint region to provide an avenue for specific chemical identification.Raman spectroscopy has also been used to characterize these sorbent materials. Specifically, a technique known as waveguide-enhanced Raman spectroscopy (WERS) has been used, which features the use of highly evanescent, low-loss waveguides with the sorbent material as a top cladding.2 WERS can be achieved using an incredibly small footprint with a sorbent-functionalized nanophotonic waveguide that is approximately a few centimeters long. Using WERS, the differential Raman spectra of the sorbent material interacting with different chemical warfare agent simulants has been measured at parts-per-billion detection levels. The spectra exhibit extrapolated three-sigma detection limits as low as 3 ppb. Continuing efforts are focused on adapting this technique to photonic integrated circuit-based fabrication and chip-scale Raman spectroscopy for trace chemical vapor detection.This presentation will highlight the design of these next-generation sorbents as a tool to facilitate MIR- and Raman-based sensing of threat chemicals. It will focus on analyzing sorbent-analyte spectral interactions and discuss how to exploit these features to develop MIR- and Raman-based sensors.

Do the solvent properties affect the propensity for self-association of α-cyclodextrin? Insights from NMR self-diffusion

Whether cyclodextrins (CDs) self-aggregate or not in solution is an interesting and timely scientific question. Yet, we are far from a scientific consensus as regards the nature of the phenomenon. Those who are in favor of the formation of CD aggregates justify the phenomenon by the presence of H-bonds, occurring between the hydroxyl groups located at both rims of cyclodextrins. To gain further insight with regard to the importance of these intermolecular CD-CD interactions, a systematic study of 1H NMR self-diffusion of α-cyclodextrin in binary mixtures of deuterated DMSO and water, at different mole fractions, has been carried out. The choice of these solvents was made based on their different degree of polarity and the fact that they are miscible at any molar ratio. The experimental α-CD diffusion coefficients scale with the solution viscosity. Moreover, hydrodynamic radii of α-CD are independent of the solvent composition. No evidence of α-cyclodextrin aggregation at any DMSO mole fraction is found. Finally, experimental diffusion data are compared with those obtained from atomic level hydrodynamic calculations.In addition, diffusion coefficients of heavy water and DMSO in binary mixed solvents are also measured and analyzed. The obtained diffusion coefficients for DMSOd6 and D2O show a minimum at the eutectic composition and depend on the solution viscosity. The experimental results are compared with published molecular dynamic simulations.

Effect of C-terminal His-tag and purification routine on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of the bacteriophage T5

In this work, we studied the effect of the C-terminally attached poly-histidine tag (His-tag), as well as the peculiarities of the protein purification procedure by the immobilized metal affinity chromatography (IMAC) on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of bacteriophage T5 (EndoT5), whose zinc binding site and catalytic aspartate are located near the C-terminus. By itself, His-tag did not have a significant effect on either activity or folding of the polypeptide chain, nor on the binding of zinc and calcium ions to the protein. However, the His-tagged EndoT5 samples had low shelf-life, with storage of these samples resulting in an increased propensity for protein self-association and decreased enzymatic activity of EndoT5. Furthermore, disastrous effects on the activity of the enzyme were exerted by the presence of imidazole and nickel ions accompanying metal chelate chromatography. The activity of the protein can be restored by thorough washing off of these low molecular impurities via the prolonged dialysis of the His-tagged EndoT5 samples at the specifically elaborated conditions.

Developability Assessment of Engineered Monoclonal Antibody Variants with a Complex Self-Association Behavior Using Complementary Analytical and in Silico Tools.

Monoclonal antibodies (mAbs) are complex molecular structures. They are often prone to development challenges particularly at high concentrations due to undesired solution properties such as reversible self-association, high viscosity, and liquid-liquid phase separation. In addition to formulation optimization, applying protein engineering can provide an alternative mitigation strategy. Protein engineering during the discovery phase can provide great benefits to optimize molecular properties, resulting in improved developability profiles. Here, we present a case study utilizing complementary analytical and predictive in silico methods. We have systematically identified and reengineered problematic residues responsible for the self-association of a model mAb, driven by a complex combination of hydrophobic and electrostatic interactions. Noteworthy findings include a more dominant contribution of hydrophobic interactions to self-association and potential feasibility of mutations in the CDR regions to mitigate self-association. The engineered mutation panel enabled us to assess potential correlations among commonly utilized developability screening assays, including affinity capture self-interaction nanospectroscopy (AC-SINS), dynamic light scattering (DLS), and apparent solubility by PEG-precipitation. In addition, we evaluated the correlations between experimental measurements and computational predictions. CamSol, an in silico computational tool that accounts for complex molecular interactions and neighboring hotspots, was found to be an effective screening tool. Our work led to reengineered mAb variants, better suited for high-concentration liquid formulation development. The engineered mAbs exhibited enhanced in vitro and simulated in vivo solubility and reduced self-association propensity, while maintaining binding affinity and thermal stability.

The Nature of Excimer Formation in Crystalline Pyrene Nanoparticles

Pyrene is an exemplary conjugated organic chromophore with a strong propensity for self-association through an excited-state process known as excimer formation. Pyrene “excimers” and molecular “exc...

A peptoid supramolecular host for benzo[a]pyrene in water

Studies that identify a new supramolecular host for the highly toxic and prevalent carcinogen benzo[a]pyrene (BP) are reported here. By monitoring changes to absorption and fluorescence spectra, the interaction between BP and two peptoids (N-substituted glycine oligomers) of different length and self-association propensities were compared. Peptoid length and solvent choice influence spectral features. Observed trends support that peptoid self-association in aqueous solution generates a hydrophobic core to which BP can associate.

DCM associated LMNA mutations cause distortions in lamina structure and assembly

BackgroundA and B-type lamins are integral scaffolding components of the nuclear lamina which impart rigidity and shape to all metazoan nuclei. Over 450 mutations in A-type lamins are associated with 16 human diseases including dilated cardiomyopathy (DCM). Here, we show that DCM mutants perturb the self-association of lamin A (LA) and it's binding with lamin B1 (LB1). MethodsWe used confocal and superresolution microscopy (NSIM) to study the effect of LA mutants on the nuclear lamina. We further used circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry (ITC) to probe the structural modulations, self-association and heteropolymeric association of mutant LA. ResultsTransfection of mutants in cultured cell lines result in the formation of nuclear aggregates of varied size and distribution. Endogenous LB1 is sequestered into these aggregates. This is consistent with the ten-fold increase in association constant of the mutant proteins compared to the wild type. These mutants exhibit differential heterotypic interaction with LB1, along with significant secondary and tertiary structural alterations of the interacting proteins. Thermodynamic studies demonstrate that the mutants bind to LB1 with different stoichiometry, affinity and energetics. ConclusionsIn this report we show that increased self-association propensity of mutant LA modulates the LA-LB1 interaction and precludes the formation of an otherwise uniform laminar network. General significanceOur results might highlight the role of homotypic and heterotypic interactions of LA in the pathogenesis of DCM and hence laminopathies in the broader sense.

A Biopharmaceutical Industry Perspective on the Control of Visible Particles in Biotechnology-Derived Injectable Drug Products.

In the 2011 monoclonal antibody monograph revision, European Pharmacopoeia experts acknowledged that protein products may also contain proteinaceous particles at release or that protein particles may form during storage. Indeed, industry experience has demonstrated that therapeutic proteins such as monoclonal antibodies can exhibit a propensity for self-association leading to the formation of aggregates that range in size from nanometres (oligomers) to microns (subvisible and visible particles). As a result, the requirement for drug product appearance for monoclonal antibodies was changed from "without visible particles" to "without visible particles unless otherwise authorised or justified". In our view, "practically free from particles" should be considered a suitable acceptance criterion for injectable biotechnology and small-molecule products, as long as appropriately defined. Furthermore, we argue that visual inspection is a suitable quality control release test and that "practically free from particles" is a suitable specification when adequately described.

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

Uncovering and Reprogramming G Protein Coupled Receptor Signaling

G-protein-coupled receptors (GPCRs) constitute one of the largest classes of signaling membrane receptors and drug targets. GPCRs display a large diversity of constitutive activities and responses to extracellular stimuli defining their complex physiologies. Yet, how receptor sequences and structures encode these allosteric properties remains poorly understood and difficult to study in absence of high-resolution structural information for many GPCRs. To address this problem, my laboratory has developed computational techniques to model GPCRs, predict ligand-bound GPCR conformations from a large diversity of structural homologs and design membrane receptors with novel functions. Using these methods, we have rationally engineered structurally-uncharacterized GPCRs with novel ligand binding selectivities, allosteric regulation properties and self-association propensities. I will also discuss how, during the course of these studies, we uncovered novel molecular determinants of membrane protein functions.

Discovery of highly soluble antibodies prior to purification using affinity-capture self-interaction nanoparticle spectroscopy.

Self-association of monoclonal antibodies (mAbs) at high concentrations can result in developability challenges such as poor solubility, aggregation, opalescence and high viscosity. There is a significant unmet need for methods that can evaluate self-association propensities of concentrated mAbs at the earliest stages in antibody discovery to avoid downstream issues. We have previously developed a method (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) that is capable of detecting weak antibody self-interactions using unusually dilute mAb solutions (tens of µg/ml). Here we optimize and implement this assay for characterization of unpurified and highly dilute mAbs directly in cell culture media. This assay was applied to screen 87 mAbs obtained via immunization. Our measurements reveal a wide range of self-associative propensities for mAbs that bind to the same antigen and which differ mainly in their complementarity-determining regions. The least associative mAbs identified by AC-SINS were confirmed to be highly soluble when purified and concentrated by three to five orders of magnitude. This approach represents a key advance in screening mAb variants using unpurified antibody samples, and it holds significant potential to both improve initial candidate selection as well as to guide protein engineering efforts to improve the properties of specific mAb candidates.

Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations

GsMTx4, a gating modifier peptide acting on cationic mechanosensitive channels, has a positive charge (+5e) due to six Lys residues. The peptide does not have a stereospecific binding site on the channel but acts from the boundary lipids within a Debye length of the pore probably by changing local stress. To gain insight into how these Lys residues interact with membranes, we performed molecular dynamics simulations of Lys to Glu mutants in parallel with our experimental work. In silico, K15E had higher affinity for 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine bilayers than wild-type (WT) peptide or any other mutant tested, and showed deeper penetration than WT, a finding consistent with the experimental data. Experimentally, the inhibitory activities of K15E and K25E were most compromised, whereas K8E and K28E inhibitory activities remained similar to WT peptide. Binding of WT in an interfacial mode did not influence membrane thickness. With interfacial binding, the direction of the dipole moments of K15E and K25E was predicted to differ from WT, whereas those of K8E and K28E oriented similarly to that of WT. These results support a model in which binding of GsMTx4 to the membrane acts like an immersible wedge that serves as a membrane expansion buffer reducing local stress and thus inhibiting channel activity. In simulations, membrane-bound WT attracted other WT peptides to form aggregates. This may account for the positive cooperativity observed in the ion channel experiments. The Lys residues seem to fine-tune the depth of membrane binding, the tilt angle, and the dipole moments.

Optimized UV Detection of High-Concentration Antibody Formulations using High-Throughput SE-HPLC

High-concentration antibody solutions (>100 mg/mL) present significant challenges for formulation and process development, including formulation attributes such as increased solution viscosity, and the propensity for self-association. An additional challenge comes from the adaptation of analytical methods designed for low-concentration formulations to the high-concentration regime. The oligomeric state is a good example: it is a quality attribute monitored during pharmaceutical development and is one that can be affected by dilution; a typical first step in the analysis of high-concentration solutions. The objective of this work was to develop a size-exclusion HPLC (SE-HPLC) method that would allow the injection of high-concentration antibody formulations without the need for dilution prior to injection and their analysis in a high-throughput manner that does not create a bottleneck for the execution of complex formulation development studies. It was found that changing the UV detection wavelength from 215 to 235 nm simplified sample preparation by allowing for an approximately fivefold increase in injection load while maintaining the signal within the linear range of detection. In addition, the chromatographic peak properties (i.e., peak symmetry, resolution, and sensitivity) were determined to be consistent when compared with analytical methods developed for formulations with lower antibody concentrations.