Hydrodynamic and thermodynamic analysis of PEGylated human serum albumin.

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).

The molecular basis for hydrodynamic properties of PEGylated human serum albumin.

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.

Revisiting macromolecular hydration with HullRadSAS.

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.

Intrinsically disordered regions that drive phase separation form a robustly distinct protein class.

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.

Beta turn propensity and a model polymer scaling exponent identify intrinsically disordered phase-separating proteins.

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.

cAMP is an allosteric modulator of DNA-binding specificity in the cAMP receptor protein from Mycobacterium tuberculosis.

Allosteric proteins with multiple subunits and ligand-binding sites are central in regulating biological signals. The cAMP receptor protein from Mycobacterium tuberculosis (CRP) 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 remain unclear.

FT-IR Spectroscopic Analysis of the Secondary Structures Present during the Desiccation Induced Aggregation of Elastin-Like Polypeptide on Silica.

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.

Effects of Doxorubicin on the Liquid-Liquid Phase Change Properties of Elastin-Like Polypeptides.

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.

Visualization of the temperature dependent rearrangement of SynB1 elastin-like polypeptide on silica using scanning electron microscopy.

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.

AUC measurements of diffusion coefficients of monoclonal antibodies in the presence of human serum proteins.

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.

Characterization of therapeutic antibodies in the presence of human serum proteins by AU-FDS analytical ultracentrifugation.

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.

Modeling the Early Stages of Phase Separation in Disordered Elastin-like Proteins.

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.

Weak IgG self- and hetero-association characterized by fluorescence analytical ultracentrifugation.

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 .

Sedimentation Velocity: A Classical Perspective.

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.

Folding and hydrodynamics of a DNA i-motif from the c-MYC promoter determined by fluorescent cytidine analogs.

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.

Effect of basic cell-penetrating peptides on the structural, thermodynamic, and hydrodynamic properties of a novel drug delivery vector, ELP[V5G3A2-150].

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.

Biochemical evidence that human EB1 does not bind preferentially to the microtubule seam.

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.

Structural and hydrodynamic analysis of a novel drug delivery vector: ELP[V5G3A2-150].

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.

Are fluorescence-detected sedimentation velocity data reliable?

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 bipolar assembly domain of the mitotic motor kinesin-5.

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.

Specific soluble oligomers of amyloid-β peptide undergo replication and form non-fibrillar aggregates in interfacial environments.

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.

Introduction: twenty five years of the Gibbs Conference on Biothermodynamics.

In 2011, the Gibbs Conference on Biothermodynamics will celebrate its 25th anniversary. Since the inaugural meeting in 1987, it has brought together laboratories that lived, breathed and argued about the molecular logic of macromolecular machines. The participants have a deep commitment to understanding the nature of physico-chemical forces that govern regulation of biological systems, and share a passion for applying linkage theory.

The use of analytical sedimentation velocity to extract thermodynamic linkage.

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.

The stoichiometry of the Escherichia coli Hfq protein bound to RNA.

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.