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Polyethylene glycol (PEG) conjugation provides a protective modification that enhances the pharmacokinetics and solubility of proteins for therapeutic use. A knowledge of the structural ensemble of these PEGylated proteins is necessary to understand the molecular details that contribute to their hydrodynamic and colligative properties. Because of the large size and dynamic flexibility of pharmaceutically important PEGylated proteins, the determination of structure is challenging. In addition, the hydration of these conjugates that contain large polymers is difficult to determine with traditional methods that identify only first shell hydration water, which does not account for the complete hydrodynamic volume of a macromolecule. Here, we demonstrate that structural ensembles, generated by coarse-grained simulations, can be analyzed with HullRad and used to predict sedimentation coefficients and concentration-dependent hydrodynamic and diffusion nonideality coefficients of PEGylated proteins. A knowledge of these concentration-dependent properties enhances the ability to design and analyze new modified protein therapeutics. HullRad accomplishes this analysis by effectively accounting for the complete hydration of a macromolecule, including that of flexible polymers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Covalent labeling of therapeutic drugs and proteins with polyethylene glycol (PEGylation) is an important modification for improving stability, solubility, and half-life. PEGylation alters protein solution behavior through its impact on thermodynamic nonideality by increasing the excluded volume, and on hydrodynamic nonideality by increasing the frictional drag. To understand PEGylation's impact, we investigated the thermodynamic and hydrodynamic properties of a model system consisting of PEGylated human serum albumin derivatives using analytical ultracentrifugation (AUC) and dynamic light scattering (DLS). We constructed PEGylated human serum albumin derivatives of single, linear 5K, 10K, 20K, and 40K PEG chains and a single branched-chain PEG of 40K (2 × 20K). Sedimentation velocity (SV) experiments were analyzed using SEDANAL direct boundary fitting to extract ideal sedimentation coefficients s o , hydrodynamic nonideality k s , and thermodynamic nonideality 2BM 1 SV terms. These quantities allow the determination of the Stokes radius R s , the frictional ratio f/f o , and the swollen or entrained volume V s /v, which measure size, shape, and solvent interaction. We performed sedimentation equilibrium experiments to obtain independent measurements of thermodynamic nonideality 2BM 1 SE . From DLS measurements, we determined the interaction parameter, k D , the concentration dependence of the apparent diffusion coefficient, D, and from extrapolation of D to c = 0 a second estimate of R s . R s values derived from SV and DLS measurements and ensemble model calculations (see complementary study) are then used to show that k s  + k D  = theoretical 2B 22 M 1 . In contrast, experimental BM 1 values from SV and sedimentation equilibrium data collectively allow for similar analysis for protein-PEG conjugates and show that k s  + k D  = 1.02-1.07 ∗ BM 1 , rather than the widely used k s  + k D  = 2BM 1 developed for hard spheres. The random coil behavior of PEG dominates the colloidal properties of PEG-protein conjugates and exceeds the sum of a random coil and hard-sphere volume due to excess entrained water., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The red seaweed Asparagopsis taxiformis has a potent antimethanogenic effect, which has been proven both in vitro and in vivo. Vegetable oil immersions of this seaweed (hereafter Bromoil) help stabilise the bromoform (CHBr 3 ) responsible for its antimethanogenic effect. We evaluate the effects of increasing the levels of CHBr 3 in lamb diets on growth performance, methane (CH 4 ) production, animal health and meat quality. Twenty-four Merino Branco ram lambs were fed a ground complete compound feed, supplemented with 50 mL/kg DM of sunflower oil with different CHBr 3 content. The treatments were defined by the CHBr 3 doses in the oil: 0 mg (control - B0), 15 mg (B15), 30 mg (B30) and 45 mg (B45) of CHBr 3 per kg of feed DM. The feed was prepared daily by mixing Bromoil with the compound feed. At the end of the experiment, the lambs were sacrificed, the ruminal content was collected for in vitro fermentation to evaluate CH 4 production and organic matter (OM) degradability, and the rumen mucosa was sampled for histological examination. Meat samples were collected for chemical composition and CHBr 3 analysis. The half-life of CHBr 3 in the air-exposed feed was 3.98 h making it very difficult to establish the practiced level of CHBr 3 supplementation. Lambs-fed treatments B30 and B45 decreased DM intake by up to 28%. Average daily gain was also reduced due to CHBr 3 supplementation, with B45 showing results 40% lower than B0. DM feed conversion ratio was similar for all treatments. The degradability of OM, the volume of total gas and of gas without CH 4 were unaffected by the experimental treatments, evaluated by the in vitro method. However, the volume of CH 4 decreased by up to 75% for treatments above 30 mg/kg DM, while the yield of CH 4 /g OM degraded was reduced by up to 78% with treatments above 30 mg/kg DM. Meat chemical composition was not affected by Bromoil supplementation and no traces of CHBr 3 were found in meat samples. During this experiment, the animals presented normal health and behaviour. However, postslaughter examination of the rumen showed distinct lesions on the ventral region of the rumen mucosa of animals supplemented with Bromoil. These lesions were more severe in the animals receiving treatments B30 and B45. This research determined that although concentrations of CHBr 3 in the diet above 30 mg/kg DM helped to reduce CH 4 emissions, it negatively affected the performance and rumen wall., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Atrial fibrillation (AF), a prevalent cardiac arrhythmia, is associated with increased morbidity and mortality worldwide. Stroke, the leading cause of serious disability in the United States, is among the important complications of this arrhythmia. Recent studies have demonstrated that certain clinical variables can be useful in the prediction of AF development in the future. The electrocardiogram (ECG) is a simple and cost-effective technology that is widely available in various healthcare settings. An emerging body of evidence has suggested that ECG tracings preceding the development of AF can be useful in predicting this arrhythmia in the future. Various variables on ECG especially different P wave parameters have been investigated in the prediction of new-onset AF and found to be useful. Several risk models were also introduced using these variables along with the patient's clinical data. However, current guidelines do not provide a clear consensus regarding implementing these prediction models in clinical practice for identifying patients at risk of AF. Also, the role of intensive screening via ECG or implantable devices based on this scoring system is unclear. The purpose of this review is to summarize AF and various related terminologies and explain the pathophysiology and electrocardiographic features of this tachyarrhythmia. We also discuss the predictive electrocardiographic features of AF, review some of the existing risk models and scoring system, and shed light on the role of monitoring device for screening purposes., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Takotsubo cardiomyopathy (TTC) is a severe, acute, reversible, and self-limited cardiac dysfunction. It usually affects postmenopausal women and is mostly triggered by physical or emotional stressors. Following the COVID-19 pandemic, millions of doses of different types of COVID-19 vaccines are being administered globally. There have been reports of different cardiac complications after receiving COVID-19 vaccines. To our knowledge, there have been 16 reported cases of COVID-19 vaccination-associated TTC. In this study, we first provide a brief overview of TTC and then an overview of selected reported TTC cases following COVID-19 vaccinations. It is crucial to highlight that the occurrence of TTC after vaccination does not establish a direct cause-and-effect relationship between immunization and TTC. Further investigations are necessary to examine any potential association between COVID-19 vaccines and the incidence of TTC. Additionally, the benefits of receiving COVID-19 vaccines significantly outweigh the potential risks of developing adverse events., Competing Interests: Conflict of interest: Authors have no conflict of interest to disclose.

Hydration of biological macromolecules is important for their stability and function. Historically, attempts have been made to describe the degree of macromolecular hydration using a single parameter over a narrow range of values. Here, we describe a method to calculate two types of hydration: surface shell water and entrained water. A consideration of these two types of hydration helps to explain the "hydration problem" in hydrodynamics. The combination of these two types of hydration allows accurate calculation of hydrodynamic volume and related macromolecular properties such as sedimentation and diffusion coefficients, intrinsic viscosities, and the concentration-dependent non-ideality identified with sedimentation velocity experiments., (© 2023. European Biophysical Societies' Association.)

There is a long tradition in the Biophysics community of using simulations as a means to understand macromolecular behavior in various physicochemical methods. This allows a rigorous means to interpret observations in terms of fundamental principles, including chemical equilibrium, reaction kinetics, transport processes and thermodynamics. Here we simulate data for the Gilbert Theory for self-association, a fundamental analytical ultracentrifuge (AUC) technique to understand the shape of sedimentation velocity reaction boundaries that involve reversible monomer-Nmer interactions. Simulating monomer-dimer through monomer-hexamer systems as a function of concentration about the equilibrium constant allows a visual means to differentiate reaction stoichiometry by determining end points and inflection positions. Including intermediates (eg A 1 -A 2 -A 3 -A 4 -A 5 -A 6 ) in the simulations reveals the smoothing of the reaction boundary and the removal of sharp inflections between monomers and polymers. The addition of cooperativity restores sharp boundaries or peaks to the observation and allows more discrimination in the selection of possible fitting models. Thermodynamic nonideality adds additional features when applied across wide ranges of concentration that might be appropriate for high-concentration therapeutic monoclonal antibody (mAb) solutions. This presentation serves as a tutorial for using modern AUC analysis software like SEDANAL for selecting potential fitting models., (© 2023. European Biophysical Societies' Association.)

The biotech industry has great interest in investigating therapeutic proteins in high concentration environments like human serum. The fluorescence detection system (Aviv-FDS) allows the performance of analytical ultracentrifuge (AUC) sedimentation velocity (SV) experiments in tracer or BOLTS protocols. Here, we compare six pooled human serum samples by AUC SV techniques and demonstrate the potential of this technology for characterizing therapeutic antibodies in serum. Control FDS SV experiments on serum alone reveal a bilirubin-HSA complex whose sedimentation is slowed by solution nonideality and exhibits a Johnston-Ogston (JO) effect due to the presence of high concentrations of IgG. Absorbance SV experiments on diluted serum samples verify the HSA-IgG composition as well as a significant IgM pentamer boundary at 19 s. Alexa-488 labeled Simponi (Golimumab) is used as a tracer to investigate the behavior of a therapeutic monoclonal antibody (mAb) in serum, and the sedimentation behavior of total IgG in serum. Serum dilution experiments allow extrapolation to zero concentration to extract s o , while global direct boundary fitting with SEDANAL verifies the utility of a matrix of self- and cross-term phenomenological nonideality coefficients (k s and BM 1 ) and the source of the JO effect. The best fits include weak reversible association (~ 4 × 10 3  M -1 ) between Simponi and total human IgG. Secondary mAbs to human IgG and IgM verify the formation of a 10.2 s 1:1 complex with human IgG and a 19 s complex with human IgM pentamers. These results demonstrate that FDS AUC allows a range of approaches for investigating therapeutic antibodies in human serum., (© 2023. European Biophysical Societies' Association.)

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have special physiological functions in both brain and retinal tissues that are related to the modulation of inflammatory processes and direct effects on neuronal membrane fluidity, impacting mental and visual health. Among them, the long-chain (LC) n-3 PUFAs, as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are of special importance. Scarce data are available about the fatty acid (FA) composition of the ruminant brain in response to dietary intervention. However, we decided to examine the brain and retina FA composition of lambs supplemented with an EPA-rich microalga feed for 21 days, as it is known that despite the extensive biohydrogenation of dietary PUFAs in the rumen, ruminants can selectively accumulate some n-3 LC-PUFAs in their brain and retinal tissues. Twenty-eight male lambs were fed a control diet, or the same diet further supplemented with Nannochloropsis sp. microalga. Their brains and retina were collected for FA characterization. Overall, the brain FA profile remained unchanged, with little alteration in omega-3 docosapentaenoic acid (DPA) enhancement in both the hippocampus and prefrontal cortex. Retinal tissues were particularly responsive to the dietary intervention, with a 4.5-fold enhancement of EPA in the freeze-dried-fed lambs compared with the control lambs. We conclude that retinal tissues are sensitive to short-term n-3 PUFA supplementation in lambs.

Protein phase separation is thought to be a primary driving force for the formation of membrane-less organelles, which control a wide range of biological functions from stress response to ribosome biogenesis. Among phase-separating (PS) proteins, many have intrinsically disordered regions (IDRs) that are needed for phase separation to occur. Accurate identification of IDRs that drive phase separation is important for testing the underlying mechanisms of phase separation, identifying biological processes that rely on phase separation, and designing sequences that modulate phase separation. To identify IDRs that drive phase separation, we first curated datasets of folded, ID, and PS ID sequences. We then used these sequence sets to examine how broadly existing amino acid property scales can be used to distinguish between the three classes of protein regions. We found that there are robust property differences between the classes and, consequently, that numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. This result indicates that multiple, redundant mechanisms contribute to the formation of phase-separated droplets from IDRs. The top-performing scales were used to further optimize our previously developed predictor of PS IDRs, ParSe. We then modified ParSe to account for interactions between amino acids and obtained reasonable predictive power for mutations that have been designed to test the role of amino acid interactions in driving protein phase separation. Collectively, our findings provide further insight into the classification of IDRs and the elements involved in protein phase separation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Urban habitats offer spatially and temporally predictable anthropogenic food sources for opportunistic species, such as several species of gulls that are known to exploit urban areas and take advantage of accessible and diverse food sources, reducing foraging time and energy expenditure. However, human-derived food may have a poorer nutritional quality than the typical natural food resources and foraging in urban habitats may increase birds' susceptibility of ingesting anthropogenic debris materials, with unknown physiological consequences for urban dwellers. Here we compare the fatty acids (FA) composition of two opportunistic gull species (the yellow-legged gull, Larus michahellis, and the lesser black-backed gull, Larus fuscus) from areas with different levels of urbanization, to assess differences in birds' diet quality among foraging habitats, and we investigate the effects of ingesting anthropogenic materials, a toxicological stressor, on gulls' FA composition. Using GC-MS, 23 FAs were identified in the adipose tissue of both gull species. Significant differences in gulls' FA composition were detected among the three urbanization levels, mainly due to physiologically important highly unsaturated FAs that had lower percentages in gulls from the most urbanized habitats, consistent with a diet based on anthropogenic food resources. The deficiency in omega (ω)-3 FAs and the higher ω-6:ω-3 FAs ratio in gulls from the most urbanized location may indicate a diet-induced susceptibility to inflammation. No significant differences in overall FA composition were detected between gull species. While we were unable to detect any effect of ingested anthropogenic materials on gulls' FA composition, these data constitute a valuable contribution to the limited FA literature in gulls. We encourage studies to explore the long-term physiological effects of the lower nutritional quality diet for urban dwellers, and to detect the sub-lethal impacts of the ingestion of anthropogenic materials., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The complex cellular milieu can spontaneously demix, or phase separate, in a process controlled in part by intrinsically disordered (ID) proteins. A protein's propensity to phase separate is thought to be driven by a preference for protein-protein over protein-solvent interactions. The hydrodynamic size of monomeric proteins, as quantified by the polymer scaling exponent (v), is driven by a similar balance. We hypothesized that mean v, as predicted by protein sequence, would be smaller for proteins with a strong propensity to phase separate. To test this hypothesis, we analyzed protein databases containing subsets of proteins that are folded, disordered, or disordered and known to spontaneously phase separate. We find that the phase-separating disordered proteins, on average, had lower calculated values of v compared with their non-phase-separating counterparts. Moreover, these proteins had a higher sequence-predicted propensity for β-turns. Using a simple, surface area-based model, we propose a physical mechanism for this difference: transient β-turn structures reduce the desolvation penalty of forming a protein-rich phase and increase exposure of atoms involved in π/sp 2 valence electron interactions. By this mechanism, β-turns could act as energetically favored nucleation points, which may explain the increased propensity for turns in ID regions (IDRs) utilized biologically for phase separation. Phase-separating IDRs, non-phase-separating IDRs, and folded regions could be distinguished by combining v and β-turn propensity. Finally, we propose a new algorithm, ParSe (partition sequence), for predicting phase-separating protein regions, and which is able to accurately identify folded, disordered, and phase-separating protein regions based on the primary sequence., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Allosteric proteins with multiple subunits and ligand-binding sites are central in regulating biological signals. The cAMP receptor protein from Mycobacterium tuberculosis (CRP MTB ) is a global regulator of transcription composed of two identical subunits, each one harboring structurally conserved cAMP- and DNA-binding sites. The mechanisms by which these four binding sites are allosterically coupled in CRP MTB remain unclear. Here, we investigate the binding mechanism between CRP MTB and cAMP, and the linkage between cAMP and DNA interactions. Using calorimetric and fluorescence-based assays, we find that cAMP binding is entropically driven and displays negative cooperativity. Fluorescence anisotropy experiments show that apo-CRP MTB forms high-order CRP MTB -DNA oligomers through interactions with nonspecific DNA sequences or preformed CRP MTB -DNA complexes. Moreover, we find that cAMP prevents and reverses the formation of CRP MTB -DNA oligomers, reduces the affinity of CRP MTB for nonspecific DNA sequences, and stabilizes a 1-to-1 CRP MTB -DNA complex, but does not increase the affinity for DNA like in the canonical CRP from Escherichia coli (CRP Ecoli ). DNA-binding assays as a function of cAMP concentration indicate that one cAMP molecule per homodimer dissociates high-order CRP MTB -DNA oligomers into 1-to-1 complexes. These cAMP-mediated allosteric effects are lost in the double-mutant L47P/E178K found in CRP from Mycobacterium bovis Bacille Calmette-Guérin (CRP BCG ). The functional behavior, thermodynamic stability, and dimerization constant of CRP BCG are not due to additive effects of L47P and E178K, indicating long-range interactions between these two sites. Altogether, we provide a previously undescribed archetype of cAMP-mediated allosteric regulation that differs from CRP Ecoli , illustrating that structural homology does not imply allosteric homology., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The Aviv fluorescence detection system (Aviv-FDS) has allowed the performance of sedimentation velocity experiments on therapeutic antibodies in highly concentrated environments like formulation buffers and serum. Methods were implemented in the software package SEDANAL for the analysis of nonideal, weakly associating AUC data acquired on therapeutic antibodies and proteins (Wright et al. Eur Biophys J 47:709-722, 2018, Anal Biochem 550:72-83, 2018). This involved fitting both hydrodynamic, k s , and thermodynamic, BM 1 , nonideality where concentration dependence is expressed as s = s o /(1 + k s c) and D = D o (1 + 2BM 1 c)/(1 + k s c) and s o and D o are values extrapolated to c = 0 (mg/ml). To gain insight into the consequences of these phenomenological parameters, we performed simulations with SEDANAL of a monoclonal antibody as a function of k s (0-100 ml/g) and BM 1 (0-100 ml/g). This provides a visual understanding of the separate and joint impact of k s and BM 1 on the shape of high-concentration sedimentation velocity boundaries and the challenge of their unique determination by finite element methods. In addition, mAbs undergo weak self- and hetero-association (Yang et al. Prot Sci 27:1334-1348, 2018) and thus we have simulated examples of nonideal weak association over a wide range of concentrations (1-120 mg/ml). Here we demonstrate these data are best analyzed by direct boundary global fitting to models that account for k s , BM 1 and weak association. Because a typical clinical dose of mAb is 50-200 mg/ml, these results have relevance for biophysical understanding of concentrated therapeutic proteins.

Previously, we found that elastin-like polypeptide (ELP), when dried above the lower critical solution temperature on top of a hydrophilic fused silica disk, exhibited a dynamic coalescence behavior. The ELP initially wet the silica, but over the next 12 h, dewett the surface and formed aggregates of precise sizes and shapes. Using Fourier-transform infrared (FT-IR) spectroscopy, the present study explores the role of secondary structures present in ELP during this progressive desiccation and their effect on aggregate size. The amide I peak (1600-1700 cm -1 ) in the ELP's FT-IR spectrum was deconvoluted using the second derivative method into eight subpeaks (1616, 1624, 1635, 1647, 1657, 1666, 1680, 1695 cm -1 ). These peaks were identified to represent extended strands, β-turns, 3(10)-helix, polyproline I, and polyproline II using previous studies on ELP and molecules similar in peptide composition. Positive correlations were established between the various subpeaks, water content, and aggregate size to understand the contributions of the secondary structures in particle formation. The positive correlations suggest that type II β-turns, independent of the water content, contributed to the growth of the aggregates at earlier time points (1-3.5 h). At later time points (6-12 h), the aggregate growth was attributed to the formation of 3(10)-helices that relied on a decrease in water content. Understanding these relationships gives greater control in creating precisely sized aggregates and surface coatings with varying roughness., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

The lower critical solution temperature (LCST) of the thermo-responsive engineered elastin-like polypeptide (ELP) biopolymer is being exploited for the thermal targeted delivery of doxorubicin (Dox) to solid tumors. We examine the impact of Dox labeling on the thermodynamic and hydrodynamic behavior of an ELP drug carrier and how Dox influences the liquid-liquid phase separation (LLPS). Turbidity, dynamic light scattering (DLS), and differential scanning calorimetry measurements show that ELP undergoes a cooperative liquid-liquid phase separation from a soluble to insoluble coacervated state that is enhanced by Dox labeling. Circular dichroism measurements show that below the LCST ELP consists of both random coils and temperature-dependent β-turn structures. Labeling with Dox further enhances β-turn formation. DLS measurements reveal a significant increase in the hydrodynamic radius of ELP below the LCST consistent with weak self-association. Dox-labeled SynB1-ELP1 (Dox-ELP) has a significant increase in the hydrodynamic radius by DLS measurements that is consistent with stable oligomers and, at high Dox-ELP concentrations, micelle structures. Enhanced association by Dox-ELP is confirmed by sedimentation velocity analytical ultracentrifugation measurements. Both ELP self-association and the ELP inverse phase transition are entropically driven with positive changes in enthalpy and entropy. We show by turbidity and DLS that the ELP phase transition is monophasic, whereas mixtures of ELP and Dox-ELP are biphasic, with Dox-labeled ELP phase changing first and unlabeled ELP partitioning into the coacervate as the temperature is raised. DLS reveals a complex growth in droplet sizes consistent with coalescence and fusion of liquid droplets. Differential scanning calorimetry measurements show a -11 kcal/mol change in enthalpy for Dox-ELP coacervation relative to the unlabeled ELP, consistent with droplet formation being stabilized by favorable enthalpic interactions. We propose that the ELP phase change is initiated by ELP self-association, enhanced by increased Dox-ELP oligomer and micelle formation and stabilized by favorable enthalpic interactions in the liquid droplets., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In this study, scanning electron microscopy (SEM) was used to observe the interaction between de-solvated SynB1-elastin-like polypeptide (SynB1-ELP) and silica at a temperature above ELP's lower critical solution temperature (LCST). ELP was seen to initially wet the surface of the silica before rearranging to form narrowly distributed spherical particles. After formation, the ELP particles dynamically rearranged to increase and subsequently decrease in size until 24 h at which time they collapsed. SEM and Energy Dispersive X-ray Spectroscopy revealed that the formation of a thin layer of salt from the PBS solution preceded the initial wetting of ELP on silica, which was shown to play a role in the continuous rearrangement of ELP. FT-IR revealed that the salt, in combination with the hydrophilic silica, trapped water that provided a repulsive surface to the hydrophobic ELP and forced the ELP to continuously minimize its surface area until the water evaporated. This behavior shows that ELP's thermo-responsive nature coupled with its hydrophobicity can be used to create ELP particles and surfaces that can reorganize with minimal water present., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The goal of this work is to develop a preclinical method for quantitative hydrodynamic and thermodynamic analysis of therapeutic proteins in crowded environments like human serum. The method utilizes tracer amounts of fluorescently labeled monoclonal antibodies and the Aviv AU-FDS optical system. We have performed sedimentation velocity experiments as a function of mAb, human serum albumin and human IgG concentration to extract self- and cross-term hydrodynamic nonideality effects. SV measurements are consistently complicated by weak mAb-mAb and mAb-IgG interactions (Wright et al. in Anal Biochem 550:72-83, 2018). In an attempt to explore different approaches we have investigated measurements of diffusion coefficients by traditional synthetic boundary experiments. Here we present a new technique incorporated into SEDANAL that can globally analyze the full time course of synthetic boundary experiments. This approach also utilizes F-mAb against a high concentration of unlabeled carrier protein (HSA or IgG). In principle both diffusion and sedimentation coefficient information can be extracted including hydrodynamic and thermodynamic nonideality. The method can be performed at a traditional low speed (5-7K rpm) or at high speeds. The high speed method can also be used to measure D and s for small molecules like fluorescein (often contaminants of F-HSA and F-mAb). The advantage of synthetic boundary over the standard sedimentation velocity method is that it allows for higher precision determination of diffusion coefficients. The concentration dependence of D can be corrected for hydrodynamic nonideality effects by plotting D * (1 + k ij c j ) vs total carrier concentration. The slope of the fitted data allows an alternate approach to determine self- and cross-term thermodynamic nonideality. This method can also explore cross-term diffusion coefficient effects. These results are compared to dynamic light scattering approaches which are limited to k D determinations for solutions of pure protein.

We have developed a new spectro-gonio radiometer, SHADOWS, to study in the laboratory the bidirectional reflectance distribution function of dark and precious samples. The instrument operates over a wide spectral range from the visible to the near-infrared (350-5000 nm) and is installed in a cold room to operate at a temperature as low as -20° C . The high flux monochromatic beam is focused on the sample, resulting in an illumination spot of about 5.2 mm in diameter. The reflected light is measured by two detectors with high sensitivity (down to 0.005% in reflectance) and absolute accuracy of 1%. The illumination and observations angles, including azimuth, can be varied over wide ranges. This paper presents the scientific and technical constraints of the spectro-gonio radiometer, its design and additional capabilities, as well as the performances and limitations of the instrument.

Weak protein-protein interactions may be important to binding cooperativity. A panel of seven fluorescently labeled tracer monoclonal IgG antibodies, differing in variable (V) and constant (C) region sequences, were sedimented in increasing concentrations of unlabeled IgGs of identical, similar, and different backgrounds. Weak IgG::IgG attractive interactions were detected and characterized by global analysis of the hydrodynamic nonideality coefficient, k s . The effects of salt concentration and temperature on k s suggest the interactions are predominantly enthalpic in origin. The interactions were found to be variable in strength, affected by both the variable and constant regions, but indiscriminate with respect to IgG subclass. Furthermore, weak attractive interactions were observed for all the mAbs with freshly purified human poly-IgG. The universality of the weak interactions suggest that they may contribute to effector function cooperativity in the normal immune response, and we postulate that the generality of the interactions allows for a broader range of epitope spacing for complement activation. These studies demonstrate the utility of analytical ultracentrifuge fluorescence detection in measuring weak protein-protein interactions. It also shows the strength of global analysis of sedimentation velocity data by SEDANAL to extract hydrodynamic nonideality k s to characterize weak macromolecular interactions., (© 2018 The Protein Society.)

The preclinical characterization of biopharmaceuticals seeks to determine the stability, state of aggregation, and interaction of the antibody/drug with other macromolecules in serum. Analytical ultracentrifugation is the best experimental method to understand these factors. Sedimentation velocity experiments using the AU-FDS system were performed in order to quantitatively characterize the nonideality of fluorescently labeled therapeutic antibodies in high concentrations of human serum proteins. The two most ubiquitous serum proteins are human serum albumin, HSA, and γ-globulins, predominantly IgG. Tracer experiments were done pairwise as a function of HSA, IgG, and therapeutic antibody concentration. The sedimentation coefficient for each fluorescently labeled component as a function of the concentration of the unlabeled component yields the hydrodynamic nonideality (k s ). This generates a 3x3 matrix of k s values that describe the nonideality of each pairwise interaction. The k s matrix is validated by fitting both 2:1 mixtures of HSA (1-40 mg/ml) and IgG (0.5-20 mg/ml) as serum mimics, and human serum dilutions (10-100%). The data are well described by SEDANAL global fitting with the k s nonideality matrix. The k s values for antibodies are smaller than expected and appear to be masked by weak association. Global fitting to a k s and K 2 model significantly improves the fits., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Elastin-like proteins (ELPs) are known to undergo liquid-liquid phase separation reversibly above a concentration-dependent transition temperature. Previous studies suggested that, as temperature increases, ELPs experience an increased propensity for type II β-turns. However, how the ELPs behave below the phase transition temperature itself is still elusive. Here, we investigate the importance of β-turn formation during the early stages of ELP self-association. We examined the behavior of two ELPs, a 150-repeat construct that had been investigated previously (ELP[V 5 G 3 A 2 -150] as well as a new 40-repeat construct (ELP40) suitable for nuclear magnetic resonance measurements. Structural analysis of ELP40 reveals a disordered conformation, and chemical shifts throughout the sequence are insensitive to changes in temperature over 20°C. However, a low population of β-turn conformation cannot be ruled out based on chemical shifts alone. To examine the structural consequences of β-turns in ELPs, a series of structural ensembles of ELP[V 5 G 3 A 2 -150] were generated, incorporating differing amounts of β-turn bias throughout the chain. To mimic the early stages of the phase change, two monomers were paired, assuming preferential interaction at β-turn regions. This approach was justified by the observation that buried hydrophobic turns are commonly observed to interact in the Protein Data Bank. After dimerization, the ensemble-averaged hydrodynamic properties were calculated for each degree of β-turn bias, and the results were compared with analytical ultracentrifugation experiments at various temperatures. We find that the temperature dependence of the sedimentation coefficient (s 20,w o ) can be reproduced by increasing the β-turn content in the structural ensemble. This analysis allows us to estimate the presence of β-turns and weak associations under experimental conditions. Because disordered proteins frequently exhibit weak biases in secondary structure propensity, these experimentally-driven ensemble calculations may complement existing methods for modeling disordered proteins generally., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: In the regular wildlife monitoring action carried out in the summer of the past few years at the Berlenga Island, wild rabbits (Oryctolagus cuniculus) have been repeatedly found dead. However, the origin of those deaths was never investigated. Our aim was to investigate the cause of death of 11 rabbits collected between April and May 2016., Results: While screening samples from rabbit carcasses for the major viral rabbit pathogens, five tested positive to RHDV2 but all were negative for RHDV and myxoma virus (MYXV). For six RHDV2-negative specimens, emaciation and parasitism were considered the most probable cause of death. Lesions identified in the RHDV2-positive rabbits included non-suppurative diffuse hepatic necrosis and pulmonary lesions varying from congestion and oedema of the lungs to interstitial pneumonia. Sequencing analysis of the vp60 gene obtained from two specimens showed identical vp60 sequences. Comparison with other known RHDV2 strains from public databases through BLAST analysis revealed a closer similarity with strains from Alentejo collected during 2013. Maximum Likelihood and Bayesian phylogenetic analysis showed that the 2016 strains from the archipelago have a higher resemblance with a group of strains mostly collected in the South of Portugal between 2013 and 2014., Conclusion: The results suggest that RHDV2 may have been introduced on the Berlenga Island a few years ago, having evolved separately from mainland strains due to insularity.

Background: Dirofilaria immitis is a mosquito-borne pathogen that is spreading worldwide, and the associated infection (i.e. dirofilariosis) is becoming a threat to animals and humans living in endemic areas. Little is known about the occurrence and risk of infection of D. immitis in pinnipeds. Here we report dirofilariosis by D. immitis in several pinniped species kept in captivity in Portugal., Methods: Animals were housed in an oceanographic park located in Algarve, southern Portugal, a geographical area endemic for canine dirofilariosis. To assess the occurrence of D. immitis, blood was collected from the park's resident pinniped population, which consisted of 16 animals (5 common seals Phoca vitulina, 2 grey seals Halichoerus grypus, 3 California sea lions Zalophus californianus and 6 South African fur seals Arctocephalus pusillus pusillus). Dirofilaria immitis nematodes were detected by real-time PCR and by the presence of circulating antigens. In addition, modified Knott's technique was performed to detect circulating microfilariae. Necropsies and histopathological examination of two animals which died during the study were also conducted., Results: Out of the 16 pinnipeds housed at the park, seven (43.8%) were positive for D. immitis by real-time PCR (3 P. vitulina, 2 Z. californianus and 2 A. p. pusillus), two of which (P. vitulina) were also positive for the nematode's antigen. Additionally, D. immitis microfilariae were detected in one A. p. pusillus. Furthermore, several D. immitis specimens were retrieved from the right ventricle and pulmonary arteries at the necropsy of one P. vitulina and one A. p. pusillus., Conclusions: This study provides new epidemiological data on D. immitis infection in pinnipeds diagnosed through clinical, molecular and pathological findings. Additionally, the South African fur seal is herein reported as a new host for this zoonotic filarioid. The situation herein described could also occur in other parks located in areas where canine dirofilariosis is endemic. Active surveillance and preventive measures of dirofilariosis in pinnipeds on a local and global scale are therefore vital to improve the early diagnosis and control of dirofilariosis.

In this study, we hypothesized that the incorporation of docosahexaenoic acid (DHA) in tissues will be higher when it is ingested as triacylglycerols (TAG) structured at the sn-2 position, which enhances efficacy and health benefits of dietary DHA n-3 supplementation. Ten-week-old Golden Syrian male hamsters were randomly allocated into 4 dietary groups with 10 animals in each: linseed oil (LSO; control group), fish oil (FO), fish oil ethyl esters (FO-EE), and structured DHA at the sn-2 position of TAG (DHA-SL). After 12 weeks, there were no variations in the hamsters' body composition parameters across dietary groups. The DHA-SL diet had the lowest values of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total lipids, and aspartate aminotransferase activity, whereas the inverse was observed for the FO diet. Glucose was increased in the LSO diet without affecting insulin and insulin resistance markers. Whereas n-3 polyunsaturated fatty acid was increased in the brain of hamsters fed the DHA-SL diet, higher levels of n-6 polyunsaturated fatty acid were observed in the liver and erythrocytes of the LSO. The highest omega-3 index was obtained with the DHA-SL diet. The principal component analyses discriminated DHA from other metabolites and set apart 4 clusters matching the 4 diets. Similarly, liver, erythrocytes, and brain were separated from each other, pointing toward an individual signature on fatty acid deposition. The structured sn-2 position DHA-containing TAG ameliorated blood lipids and fatty acid incorporation, in particular eicosapentaenoic acid and DHA in liver, erythrocytes, and brain, relative to commercially FOs, thus improving the health benefits of DHA due to its higher bioavailability., (Copyright © 2016 Elsevier Inc. All rights reserved.)

An approach consisting in combining in situ and laboratory experiments is often favoured for investigating the mechanisms involved in the weathering of the materials of the cultural heritage. However, the realistic simulation in the laboratory of the environmental conditions ruling the interactions of atmospheric compounds with materials is a very complex task. The aim of this work is to characterise CIME, a new chamber specially built to simulate the interactions between materials of the cultural heritage and the environment. The originality of this instrument is that beside the usual climatic parameters (temperature, relative humidity, solar radiation) and gaseous pollutants, it also allows the controlled injection of different types of particulate matter such as terrigenous, marine and anthropogenic. Therefore, varied realistic atmospheric environments (marine or urban) can be easily simulated within CIME. In addition to the technical description of CIME, this paper shows the first results obtained by the impact of gaseous pollutants on non-durable glass, bronze and limestone. The first experiments for the deposition of different particles (calcite, clays, soot and halite) are also presented.

Here we give an overview of the history of sedimentation velocity analysis focusing on seminal and fundamental contributions that derived from early ultracentrifugation studies. We introduce the concepts of nonequilibrium thermodynamics and outline the derivation of the Svedberg and the Lamm equations and the requirements for including both hydrodynamic and thermodynamic nonideality. We introduce the phenomenological equations for coupled flows as developed from the principles of nonequilibrium or irreversible thermodynamics and derive a form of the Lamm equation that incorporates cross-diffusion coefficients and coupled gradient terms. We give an historical overview of solutions to the Lamm equation including Fujita-MacCosham solutions and Claverie finite-element numerical solutions and discuss the software that have implemented these solutions. We discuss the three major optical systems (absorbance, interference, and fluorescence) and recently developed multiwavelength systems. We also suggest a number of experimental practices and guidelines for optimizing the determination of s and D and discuss the appropriate centerpiece components and their utility. This chapter complements other recent reviews submitted by the authors (Correia, Lyons, Sherwood, & Stafford, 2015; Stafford, 2015) and should be considered an effort to revive the importance of irreversible thermodynamics in the understanding and analysis of sedimentation velocity ultracentrifugation data., (© 2015 Elsevier Inc. All rights reserved.)

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C(+) basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C(+) hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Elastin-like polypeptides (ELPs) are large, nonpolar polypeptides under investigation as components of a novel drug delivery system. ELPs are soluble at low temperatures, but they desolvate and aggregate above a transition temperature (TT). This aggregation is being utilized for targeting systemically delivered ELP-drug conjugates to heated tumors. We previously examined the structural, thermodynamic, and hydrodynamic properties of ELP[V5G3A2-150] to understand its behavior as a therapeutic agent. In this study, we investigate the effect that adding basic cell-penetrating peptides (CPPs) to ELP[V5G3A2-150] has on the polypeptide's solubility, structure, and aggregation properties. CPPs are known to enhance the uptake of ELP into cultured cells in vitro and into tumor tissue in vivo. Interestingly, the asymmetric addition of basic residues decreased the solubility of ELP[V5G3A2-150], although below the TT we still observed a low level of self-association that increased with temperature. The ΔH of the aggregation process correlates with solubility, suggesting that the basic CPPs stabilize the aggregated state. This is potentially beneficial as the decreased solubility will increase the fraction aggregated and enhance drug delivery efficacy at a heated tumor. Otherwise, the basic CPPs did not significantly alter the biophysical properties of ELP. All constructs were monomeric at low temperatures but self-associate with increasing temperature through an indefinite isodesmic association. This self-association was coupled to a structural transition to type II β-turns. All constructs reversibly aggregated in an endothermic reaction, consistent with a reaction driven by the release of water.

The coupling of a gas reactor, named APSIS, with a vacuum-ultraviolet (VUV) beamline at the SOLEIL synchrotron radiation facility, for a photochemistry study of gas mixtures, is reported. The reactor may be irradiated windowless with gas pressures up to hundreds of millibar, and thus allows the effect of energetic photons below 100 nm wavelength to be studied on possibly dense media. This set-up is perfectly suited to atmospheric photochemistry investigations, as illustrated by a preliminary report of a simulation of the upper atmospheric photochemistry of Titan, the largest satellite of Saturn. Titan's atmosphere is mainly composed of molecular nitrogen and methane. Solar VUV irradiation with wavelengths no longer than 100 nm on the top of the atmosphere enables the dissociation and ionization of nitrogen, involving a nitrogen chemistry specific to nitrogen-rich upper atmospheres.

Sedimentation velocity analytical ultracentrifugation is a classical biophysical technique that is commonly used to analyze the size, shape, and interactions of biological macromolecules in solution. Fluorescence detection provides enhanced sensitivity and selectivity relative to the standard absorption and refractrometric detectors, but data acquisition is more complex and can be subject to interference from several photophysical effects. Here, we describe methods to configure sedimentation velocity measurements using fluorescence detection and evaluate the performance of the fluorescence optical system. The fluorescence detector output is linear over a concentration range of at least 1 to 500nM fluorescein and Alexa Fluor 488. At high concentrations, deviations from linearity can be attributed to the inner filter effect. A duplex DNA labeled with Alexa Fluor 488 was used as a standard to compare sedimentation coefficients obtained using fluorescence and absorbance detectors. Within error, the sedimentation coefficients agree. Thus, the fluorescence detector is capable of providing precise and accurate sedimentation velocity results that are consistent with measurements performed using conventional absorption optics, provided the data are collected at appropriate sample concentrations and the optics are configured correctly., (Copyright © 2013 Elsevier Inc. All rights reserved.)

EB1 is a highly conserved microtubule (MT) plus end tracking protein (+TIP) involved in regulating MT dynamics, but the mechanisms of its effects on MT polymerization remain undefined. Resolving this question requires understanding how EB1 interacts with MTs. Previous electron microscopy of the S. pombe EB1 homolog Mal3p suggested that Mal3p binds specifically to the MT seam, implying that EB1 family members promote MT polymerization by stabilizing the seam. However, more recent electron microscopy indicates that Mal3p binds everywhere except the seam. Neither set of experiments investigated the behavior of human EB1, or provided an explanation for why these studies arrived at different answers. To resolve these questions, we have used a combination of MT-binding assays and theoretical modeling with MTBindingSim. Our results indicate that human EB1 binds to the lattice, consistent with the recent Mal3p results, and show that Mal3p-binding assays that were previously interpreted as evidence for preferential seam binding are equally consistent with weak lattice binding. In addition, we used analytical ultracentrifugation to investigate the possibility that the EB1 monomer-dimer equilibrium might contribute to EB1 binding behavior, and determined that the EB1 dimerization dissociation constant is approximately 90 nM. We and others find that the cellular concentration of EB1 is on the order of 200 nM, suggesting that a portion of EB1 may be monomeric at physiological concentrations. These observations lead us to suggest that regulation of EB1 dimerization might play a role in controlling EB1 function., (Copyright © 2013 Wiley Periodicals, Inc.)

The therapeutic potential of elastin-like polypeptide (ELP) conjugated to therapeutic compounds is currently being investigated as an approach to target drugs to solid tumors. ELPs are hydrophobic polymers that are soluble at low temperatures and cooperatively aggregate above a transition temperature (TT), allowing for thermal targeting of covalently attached drugs. They have been shown to cooperatively transition from a disordered structure to a repeating type II β-turn structure, forming a β-spiral above the TT. Here we present biophysical measurements of the structural, thermodynamic, and hydrodynamic properties of a specific ELP being investigated for drug delivery, ELP[V5G3A2-150]. We examine the biophysical properties below and above the TT to understand and predict the therapeutic potential of ELP-drug conjugates. We observed that below the TT, ELP[V5G3A2-150] is soluble, with an extended conformation consisting of both random coil and heterogeneous β structures. Sedimentation velocity experiments indicate that ELP[V5G3A2-150] undergoes weak self-association with increasing temperature, and above the TT the hydrophobic effect drives aggregation entropically. These experiments also reveal a previously unreported temperature-dependent critical concentration (Cc) that resembles a solubility constant. Labeling ELP[V5G3A2-150] with fluorescein lowers the TT by 3.5°C at 20 μM, whereas ELP[V5G3A2-150] dissolution in physiological media (fetal bovine serum) increases the TT by ∼2.2°C., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5's bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiled-coil 'BASS' (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments.

Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ aggregates, low molecular weight species ranging between ~2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12-18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.

During the finishing phase of bovines, large amounts of subcutaneous and visceral fats are deposited leading to production inefficiencies with major impact on meat quality. A better understanding of the cellularity features of the main fat depots could provide strategies for adipose tissue manipulation. This study assessed the effect of feeding diets with distinct forage to concentrate ratios on the cellularity of two fat depots of beef cattle and their implications on the fatty acid profile. Thus, two phylogenetically distant Portuguese bovine breeds, Alentejana and Barrosã, were selected. The results did not show differences in subcutaneous fat deposition nor in visceral fat depots partitioning. Plasma adipokines concentration failed to show a consistent relationship with fatness, as leptin remained constant in all experimental groups, whereas interleukin-6 was influenced by breed. Fat depot seems to determine the area and number of adipocytes, with larger adipocytes and a lower number of cells in subcutaneous fat than in mesenteric fat. Neither breed nor diet influenced adipocytes area and number. The contents of total fatty acids, partial sums of fatty acids and conjugated linoleic acid isomeric profile were affected by breed and fat depot. The incorporation of saturated fatty acids (SFA), trans fatty acids, polyunsaturated fatty acids (PUFA) and branched chain fatty acids (BCFA) was higher in mesenteric fat depot, whereas subcutaneous fat depot had greater percentages of monounsaturated fatty acids (MUFA). In addition, SFA and MUFA proportions seem to be breed-related. In spite of the less relevant role of diet, the percentages of PUFA and BCFA were influenced by this factor. Under these experimental conditions, the effect of fat depot on cellularity and fatty acid composition prevails over breed or diet, as reinforced by the principal component analysis.

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The Escherichia coli RNA binding protein Hfq is involved in many aspects of post-transcriptional gene expression. Tight binding of Hfq to polyadenylate sequences at the 3' end of mRNAs influences exonucleolytic degradation, while Hfq binding to small noncoding RNAs (sRNA) and their targeted mRNAs facilitate their hybridization which in turn effects translation. Hfq binding to an A-rich tract in the 5' leader region of the rpoS mRNA and to the sRNA DsrA have been shown to be important for DsrA enhanced translation initiation of this mRNA. The complexes of Hfq-A(18) and Hfq-DsrA provide models for understanding how Hfq interacts with these two RNA sequence/structure motifs. Different methods have reported different values for the stoichiometry of Hfq-A(18) and Hfq-DsrA. In this work, mass spectrometry and analytical ultracentrifugation provide direct evidence that the strong binding mode of the Hfq hexamer (Hfq(6)) for A(18) and domain II of DsrA (DsrA(DII)) involve 1:1 complexes. This stoichiometry was also supported by fluorescence anisotropy and a competition gel mobility shift experiment using wild-type and truncated Hfq. More limited studies of Hfq binding to DsrA as well as to the sRNAs RprA, OxyS, and an 18-nt segment of OxyS were also consistent with 1:1 stoichiometry. Mass spectrometry of cross-linked samples of Hfq(6), A(18), and DsrA(DII) exhibit intensity corresponding to a ternary 1:1:1 complex; however, the small intensity of this peak and fluorescence anisotropy experiments did not provide evidence that this ternary complex is stable in solution.

The RNA-binding protein Hfq has been studied extensively for its function as a modulator of gene expression at the post-transcriptional level. While most Hfq studies have focused on the protein's interaction with sRNAs and mRNAs, Hfq binding to DNA has been observed but is less explored. During the isolation of Hfq from Escherichiacoli, we found genomic DNA fragments associated with the protein after multiple steps of purification. Sequences of 41 amplified segments from the DNA fragments associated with Hfq were determined. A large fraction of the DNA segments were predicted to have significant helical axis curvature and were from genes associated with membrane proteins, characteristics unexpected for non-specific binding. Analysis by analytical ultracentrifugation indicated that rA(18) binding to Hfq disrupts Hfq-DNA interactions. The latter observation suggests Hfq binding to DNA involves its distal surface. This was supported by a gel mobility shift assay that showed single amino acid mutations on the distal surface of Hfq inhibited Hfq binding to duplex DNA, while six of seven mutations on the proximal surface and outer circumference of the hexamer did not prevent Hfq binding. Two mutated Hfq which have portions of their C-terminal domain removed also failed to bind to DNA. The apparent K(d) for binding wild type Hfq to several duplex DNA was estimated from a gel mobility shift assay to be ~400nM., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Real-time polymerase chain reaction (PCR) has been used for quantification of intracellular mRNA levels in cell culture and tissue samples. It is an important tool for studying antimitotic drug effects on tubulin isotype and microtubule-interacting protein levels and for measuring differences in normal and tumor tissue samples that could have predictive or prognostic applications. Both quantitative and comparative methods are valuable approaches; however, the selection of either approach requires an understanding of their benefits and challenges. In this chapter, we provide detailed protocols for real-time PCR experiments, discuss issues to consider in selecting real-time PCR methodologies, and give examples utilizing either quantitative or comparative approaches., (Copyright 2010 Elsevier Inc. All rights reserved.)

This chapter describes the use of analytical ultracentrifugation in a Beckman XLA to study the self-association properties of tubulin and the interaction of tubulin with antimitotic drugs. Procedures for sample preparation, operation of the ultracentrifuge, and collection of data conform to standard modern methods. Analysis of sedimentation velocity data initially includes generation of g(s) sedimentation coefficient distributions with DCDT(+2) and determination of weight average sedimentation coefficients S(w). S(w) versus concentration data are then fit to isodesmic or indefinite assembly models to extract K(iso) values, the association constant for each successive assembly step. Alternatively the raw data can also be analyzed by direct boundary analysis methods using the program Sedanal. Direct boundary analysis also extracts the K(iso) value by fitting to the shape of the sedimentation boundary as a function of total concentration. While the fitting of weight average data as a function of protein or drug concentration to indefinite assembly models has been shown to be equivalent to direct boundary fitting of multiple data sets with Sedanal, direct boundary fitting is preferred because it robustly identifies the presence of irreversible aggregation or mechanisms that are more complex., (Copyright 2010 Elsevier Inc. All rights reserved.)

Halichondrin B is an antimitotic drug that inhibits microtubule assembly. To understand the molecular details of its interaction with tubulin, we investigated the binding of two halichondrin B analogues, eribulin (previously, ER-086526, E7389) and ER-076349, to tubulin by quantitative analytical ultracentrifugation. Eribulin is currently undergoing phase III clinical trials for cancer; ER-076349 is a closely related analogue with C.35 hydroxyl instead of C.35 primary amine [Towle, M. J., et al. (2001) Cancer Res. 61, 1013]. Below the critical concentration for microtubule assembly and in the presence of GDP, tubulin undergoes weak self-association into short curved oligomers. Eribulin inhibits this oligomer formation 4-6-fold, while ER-076349 slightly stimulates oligomer formation by 2-fold. This is in contrast to vinblastine which strongly stimulates large spiral polymers by 1000-fold under these same conditions. Vinblastine-induced spiral formation is strongly inhibited by both eribulin and ER-076349. Colchicine binding to the intradimer interface has no significant effect on small oligomer formation or the inhibitory activity of eribulin on this process. These results suggest that halichondrin B analogues bind to the interdimer interface or to the beta-subunit alone, disrupt polymer stability, and compete with vinblastine-induced spiral formation. Stathmin is known to form a tight 1:2 complex with tubulin. Eribulin strongly inhibits formation of the 1:2 stathmin-tubulin complex (>3.3 kcal/mol), while ER-076349 weakens formation of the 1:2 complex by approximately 1.9 kcal/mol. These results suggest that eribulin is a global inhibitor of tubulin polymer formation, disrupting tubulin-tubulin contacts at the interdimer interface. ER-076349 also perturbs tubulin-tubulin contacts, but in a more polymer specific manner, reflecting adaptability of the interdimer interface to drug and polymer polymorphism. These results suggest halichondrin B analogues exhibit unique tubulin-based activities that may underlie the clinical utility of these compounds.