Protein quaternary structures in solution are a mixture of multiple forms.

Over half the proteins in the E. coli cytoplasm form homo or hetero-oligomeric structures. Experimentally determined structures are often considered in determining a protein's oligomeric state, but static structures miss the dynamic equilibrium between different quaternary forms. The problem is exacerbated in homo-oligomers, where the oligomeric states are challenging to characterize. Here, we re-evaluated the oligomeric state of 17 different bacterial proteins across a broad range of protein concentrations and solutions by native mass spectrometry (MS), mass photometry (MP), size exclusion chromatography (SEC), and small-angle X-ray scattering (SAXS), finding that most exhibit several oligomeric states. Surprisingly, some proteins did not show mass-action driven equilibrium between the oligomeric states. For approximately half the proteins, the predicted oligomeric forms described in publicly available databases underestimated the complexity of protein quaternary structures in solution. Conversely, AlphaFold multimer provided an accurate description of the potential multimeric states for most proteins, suggesting that it could help resolve uncertainties on the solution state of many proteins.

Diffusion of small molecule drugs is affected by surface interactions and crowder proteins.

Crowded environments are known to affect the diffusion of macromolecules, but their effects on the diffusion of small molecules are largely uncharacterized. We investigate how three protein crowders, bovine serum albumin (BSA), hen egg-white lysozyme, and myoglobin, influence the diffusion rates and interactions of four small molecules: fluorescein, and three drugs, doxorubicin, glycogen synthase kinase-3 inhibitor SB216763, and quinacrine. Using Line-FRAP measurements, Brownian dynamics simulations, and molecular docking, we find that the diffusion rates of the small molecules are highly affected by self-aggregation, interactions with the proteins, and surface adsorption. The diffusion of fluorescein is decreased because of its interactions with the protein crowders and their surface adsorption. Protein crowders increase the diffusion rates of doxorubicin and SB216763 by reducing surface interactions and self-aggregation, respectively. Quinacrine diffusion was not affected by protein crowders. The mechanistic insights gained here may assist in optimization of compounds for higher mobility in complex macromolecular environments.

A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets.

Here, we enhanced the popular yeast display method by multiple rounds of DNA and protein engineering. We introduced surface exposure-tailored reporters, eUnaG2 and DnbALFA, creating a new platform of C and N terminal fusion vectors. The optimization of eUnaG2 resulted in five times brighter fluorescence and 10 °C increased thermostability than UnaG. The optimized DnbALFA has 10-fold the level of expression of the starting protein. Following this, different plasmids were developed to create a complex platform allowing a broad range of protein expression organizations and labeling strategies. Our platform showed up to five times better separation between nonexpressing and expressing cells compared with traditional pCTcon2 and c-myc labeling, allowing for fewer rounds of selection and achieving higher binding affinities. Testing 16 different proteins, the enhanced system showed consistently stronger expression signals over c-myc labeling. In addition to gains in simplicity, speed, and cost-effectiveness, new applications were introduced to monitor protein surface exposure and protein retention in the secretion pathway that enabled successful protein engineering of hard-to-express proteins. As an example, we show how we optimized the WD40 domain of the ATG16L1 protein for yeast surface and soluble bacterial expression, starting from a nonexpressing protein. As a second example, we show how using the here-presented enhanced yeast display method we rapidly selected high-affinity binders toward two protein targets, demonstrating the simplicity of generating new protein-protein interactions. While the methodological changes are incremental, it results in a qualitative enhancement in the applicability of yeast display for many applications.

Line-FRAP, A Versatile Method to Measure Diffusion Rates In Vitro and In Vivo.

The crowded cellular milieu affect molecular diffusion through hard (occluded space) and soft (weak, non-specific) interactions. Multiple methods have been developed to measure diffusion coefficients at physiological protein concentrations within cells, each with its limitations. Here, we show that Line-FRAP, combined with rigours data analysis, is able to determine diffusion coefficients in a variety of environments, from in vitro to in vivo. The use of Line mode greatly improves time resolution of FRAP data acquisition, from 20-100 Hz in the classical mode to 800 Hz in the line mode. This improves data analysis, as intensity and radius of the bleach at the first post-bleach frame is critical. We evaluated the method on different proteins labelled chemically or fused to YFP in a wide range of environments. The diffusion coefficients measured in HeLa and in E. coli were ~2.5-fold and 15-fold slower than in buffer, and were comparable to previously published data. Increasing the osmotic pressure on E. coli further decreases diffusion, to the point at which proteins virtually stop moving. The method presented here, which requires a confocal microscope equipped with dual scanners, can be applied to study a large range of molecules with different sizes, and provides robust results in a wide range of environments and protein concentrations for fast diffusing molecules.

Quantifying enzyme activity in living cells.

For over a century, enzymatic activity has been studied in vitro, assuming similar activity in the crowded cellular milieu. Here, we determined in real time the catalytic activity of TEM1-ß-lactamase inside living cells and compared the values to those obtained in vitro We found the apparent in vivo catalytic efficiency, kcat/Km , to be lower than in vitro, with significant cell-to-cell variability. Surprisingly, the results show that inside the cell the apparent catalytic efficiency decreases, and Km increases with increasing enzyme concentration. To rationalize these findings, we measured enzyme and substrate diffusion rates in the cell and found the latter to be slower than expected. Simulations showed that for attenuated diffusion the substrate flux becomes rate-limiting, explaining why reaction rates in vivo can be independent on enzyme concentrations. The octanol/water partition of the substrate is 4.5, which is in the range of Food and Drug Administration-approved drugs. This suggests substrate-limited reaction rates to be common. These findings indicate that in vitro data cannot be simply extrapolated to the crowded in vivo environment.

Polymeric nanostructures with pH-labile core for controlled drug release.

Efficient and stimuli-triggered controlled delivery of therapeutics is one of the important issues in modern advanced therapy. In the present work, a versatile route for the synthesis of core cross-linked polymeric nanostructures (CLPN) through thiol-acrylate Michael addition reaction via the formation of ß-thiopropionate has been described. The acid groups of the poly(acrylic acid) block of poly(ethylene glycol)-b-poly(N-isopropylacrylamide)-b-poly(acrylic acid) triblock copolymer were reacted with 2-hydroxyethyl acrylate (HEA) to yield the corresponding acrylate-functionalized copolymer (P1). Following this, P1 was reacted with a thiol functionalized cross-linker (CL) resulting in the formation of core cross-linked polymeric nanoparticles through acrylate-thiol Michael reaction. The ability of these nanoparticles to encapsulate drug molecules inside their core and their effective release following a pH-triggered controlled degradation of the core were demonstrated. The temperature sensitive release behaviour of the system was also studied. The non-toxic nature of the precursor polymers and the core cross-linked polymeric nanoparticles was also established, that further substantiated their potential as carriers for controlled release of drugs.

Interaction between calf thymus DNA and cationic bottle-brush copolymers: equilibrium and stopped-flow kinetic studies.

Interaction studies between a set of poly(ethylene glycol) (PEG) based cationic bottle-brush block copolymers (BBCPs) and calf thymus DNA (ctDNA) were carried out using steady state fluorescence spectroscopy, UV melting experiments and dynamic light scattering measurements. Results suggested that these cationic block copolymers could effectively bind with negatively charged DNA. Although electrostatic interaction is believed to be the predominant contributing factor in the overall binding process, hydrophobic interactions between the PEG chains and the DNA base pairs affected the binding process to some extent. Cationic block copolymers with higher PEG content were found to bind more efficiently with DNA. DLS studies revealed the details of the compaction process of elongated DNA chains into a globular structure in the presence of cationic block copolymers. Further, the kinetics of the DNA-cationic BBCP binding process was monitored via the stopped-flow fluorescence technique. In general, a two-step mechanistic pathway was observed in the case of all the cationic BBCP-DNA binding processes and the relative rate constants (k1'and k2') were found to increase with the copolymer concentration. The first step corresponded to a fast electrostatic binding between the cationic BBCP and the anionic ctDNA, while the slow second step indicated a conformational change of the DNA polyplex that led to DNA compaction. In addition to the polymer-DNA charge ratios, the PEG content in the cationic BBCPs was found to have a significant effect on the kinetics of the ctDNA-BBCP polyplex formation.

Polyplex formation between PEGylated linear cationic block copolymers and DNA: equilibrium and kinetic studies.

The basic requirement for understanding the nonviral gene delivery pathway is a thorough biophysical characterization of DNA polyplexes. In this work, we have studied the interactions between calf-thymus DNA (ctDNA)and a new series of linear cationic block copolymers (BCPs). The BCPs were synthesized via controlled radical polymerization using [3-(methacryloylamino)propyl] -trimethylammonium chloride (MAPTAC) and poly(ethyleneglycol) methyl ether (PEGMe) as comonomers. UV−visible spectroscopy, ethidium bromide dye exclusion, and gel electrophoresis study revealed that these cationic BCPs were capable of efficiently binding with DNA. Steady-state fluorescence, UV melting, gel electrophoresis, and circular dichroism results suggested increased binding for BCPs containing higher PEG. Hydrophobic interactions between the PEG and the DNA base pairs became significant at close proximity of the two macromolecules, thereby influencing the binding trend. DLS studies showed a decrease in the size of DNA molecules at lower charge ratio (the ratio of "+" charge of the polymer to "−" charge of DNA) due to compaction, whereas the size increased at higher charge ratio due to aggregation among the polyplexes. Additionally, we have conducted kinetic studies of the binding process using the stop-flow fluorescence method. All the results of BCP−DNA binding studies suggested a two-step reaction mechanism--a rapid electrostatic binding between the cationic blocks and DNA, followed by a conformational change of the polyplexes in the subsequent step that led to DNA condensation. The relative rate constant(k'(1)) of the first step was much higher compared to that of the second step (k'(2)), and both were found to increase with an increase in BCP concentration. The charge ratios as well as the PEG content in the BCPs had a marked effect on the kinetics of the DNA−BCP polyplex formation. Introduction of a desired PEG chain length in the synthesized cationic blocks renders them potentially useful as nonviral gene delivery agents.

Thermoregulated formation and disintegration of cationic block copolymer vesicles: fluorescence resonance energy transfer study.

Formation and disintegration of self-assembled nanostructures in response to external stimuli are important phenomena that have been widely explored for a variety of biomedical applications. In this contribution, we report the thermally triggered assembly of block copolymer molecules in aqueous solution to form vesicles (polymersomes) and their disassembly on reduction of temperature. A new thermoresponsive diblock copolymer of poly(N-isopropylacrylamide) poly((3-methacrylamidopropyl)trimethylammonium chloride) (PNIPA-b-PMAPTAC) was synthesized by reversible addition-fragmentation chain transfer technique. The solution properties and self-assembling behavior of the block copolymer molecules were studied by turbidimetry, temperature-dependent proton nuclear magnetic resonance, fluorescence spectroscopy, dynamic light scattering, and transmission electron microscopy. Fluorescence resonance energy transfer studies between coumarin-153 (C-153, donor) and rhodamine 6G (R6G, acceptor) have been performed by steady-state and picosecond-resolved fluorescence spectroscopy to probe the structural and dynamic heterogeneity of the vesicles. The occurrence of efficient energy transfer was evident from the shortening of donor lifetime in the presence of the acceptor. The capability of the vesicles to encapsulate both hydrophobic and hydrophilic molecules and release them in response to decrease in temperature makes them potentially useful as drug delivery vehicles.

Stopped-flow kinetic studies of poly(amidoamine) dendrimer-calf thymus DNA to form dendriplexes.

Poly(amidoamine) (PAMAM) dendrimers are known to be highly efficient nonviral carriers in gene delivery. Dendrimer-mediated transfection is known to be a function of the dendrimer to DNA charge ratio as well as the size of the dendrimer. In the present study, the binding kinetics of four PAMAM dendrimers (G1, G2, G3, and G4) with calf thymus DNA (CT-DNA) has been studied using stopped-flow fluorescence spectroscopy. The effect of dendrimer-to-DNA charge ratio and dendrimer generation on the binding kinetics was investigated. In most cases, the results of dendrimer-CT-DNA binding can be explained by a two-step reaction mechanism: a rapid electrostatic binding between the dendrimer and DNA, followed by a conformational change of the dendrimer-DNA complex that ultimately leads to DNA condensation. It was observed that the charge ratio on the dendrimer and the DNA phosphate groups, as well as the dendrimer generation (size), has a marked effect on the kinetics of binding between the DNA and the dendrimers. The rate constant (k'1) of the first step was much higher compared to that of the second step (k'2), and both were found to increase with an increase in dendrimer concentration. Among the four generations of dendrimers, G4 exhibited significantly faster binding kinetics compared to the three smaller generation dendrimers.

Operational issues and network effects in vaccine markets.

One of the most important concerns for managing public health is the prevention of infectious diseases. Although vaccines provide the most effective means for preventing infectious diseases, there are two main reasons why it is often difficult to reach a socially optimal level of vaccine coverage: (i) the emergence of operational issues (such as yield uncertainty) on the supply side, and (ii) the existence of negative network effects on the consumption side. In particular, uncertainties about production yield and vaccine imperfections often make manufacturing some vaccines a risky process and may lead the manufacturer to produce below the socially optimal level. At the same time, negative network effects provide incentives to potential consumers to free ride off the immunity of the vaccinated population. In this research, we consider how a central policy-maker can induce a socially optimal vaccine coverage through the use of incentives to both consumers and the vaccine manufacturer. We consider a monopoly market for an imperfect vaccine; we show that a fixed two-part subsidy is unable to coordinate the market, but derive a two-part menu of subsidies that leads to a socially efficient level of coverage.