Understanding the Phase Behavior of a Multistimuli-Responsive Elastin-like Polymer: Insights from Dynamic Light Scattering Analysis.

Elastin-like polymers are a class of stimuli-responsive protein polymers that hold immense promise in applications such as drug delivery, hydrogels, and biosensors. Yet, understanding the intricate interplay of factors influencing their stimuli-responsive behavior remains a challenging frontier. Using temperature-controlled dynamic light scattering and zeta potential measurements, we investigate the interactions between buffer, pH, salt, water, and protein using an elastin-like polymer containing ionizable lysine residues. We observed the elevation of transition temperature in the presence of the common buffering agent HEPES at low concentrations, suggesting a "salting-in" effect of HEPES as a cosolute through weak association with the protein. Our findings motivate a more comprehensive investigation of the influence of buffer and other cosolute molecules on elastin-like polymer behavior.

Application of Voltage in Dynamic Light Scattering Particle Size Analysis.

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. Modern instrumentation permits measurement of particle size as a function of time and/or temperature, but currently there is no simple method for performing DLS particle size distribution measurements in the presence of applied voltage. The ability to perform such measurements would be useful in the development of electroactive, stimuli-responsive polymers for applications such as sensing, soft robotics, and energy storage. Here, a technique using applied voltage coupled with DLS and a temperature ramp to observe changes in aggregation and particle size in thermoresponsive polymers with and without electroactive monomers is presented. The changes in aggregation behavior observed in these experiments were only possible through the combined application of voltage and temperature control. To obtain these results, a potentiostat was connected to a modified cuvette in order to apply voltage to a solution. Changes in polymer particle size were monitored using DLS in the presence of constant voltage. Simultaneously, current data were produced, which could be compared with particle size data, to understand the relationship between current and particle behavior. The polymer poly(N-isopropylacrylamide) (pNIPAM) served as a test polymer for this technique, as pNIPAM's response to temperature is well-studied. Changes in the lower-critical solution temperature (LCST) aggregation behavior of pNIPAM and poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate), an electrochemically active block-copolymer, in the presence of applied voltage are observed. Understanding the mechanisms behind such changes will be important when trying to achieve reversible polymer structures in the presence of applied voltage.

Insights on the Lower Critical Solution Temperature Behavior of pNIPAM in an Applied Electric Field.

Poly(N-isopropylacrylamide), or pNIPAM, is a free-radical polymer that is commonly studied for uses in surface coatings, tissue engineering, energy storage, biosensing, and more, due to its temperature responsiveness. pNIPAM is known to solubilize at temperatures below its lower critical solution temperature (LCST) and agglomerate above its LCST. This behavior has been shown to be reproducible and reversible. We confirmed this reversibility and the value of the LCST by performing dynamic light scattering (DLS) with a temperature sweep (increase and decrease). However, performing the same experiment under an applied voltage from copper electrodes, we observed a decrease in the LCST of pNIPAM and irreversible aggregation. Here we present preliminary data comparing the LCST behavior of pNIPAM in the presence of applied voltage using copper, aluminum, and carbon electrodes. We present data in support of the hypothesis that a phenomenon is occurring specifically with the use of copper electrodes that is altering pNIPAM LCST behavior.

Electrochemical characterization of the stimuli-response of surface-immobilized elastin-like polymers.

Elastin-like polymers (ELPs) are frequently used in a variety of bioengineering applications because of their stimuli-responsive properties. Above their transition temperature, ELPs will adopt different structures that promote intra- and intermolecular hydrophobic contacts to minimize unfavorable interactions with an aqueous environment. We electrochemically characterize the stimuli-responsive behavior of surface-immobilized ELPs corresponding to two proposed states: extended and collapsed. In the extended state the ELPs are more solvated. In the collapsed state, triggered by introducing an environmental stimulus, non-polar intramolecular contacts within ELPs are favored, resulting in quantifiable morphological changes on the surface characterized using electrochemical impedance spectroscopy (EIS). Charge transfer resistance, a component of impedance, was shown to increase after exposing an ELP modified electrode to a high salt concentration environment (3.0 M NaCl). An increase in charge transfer resistance indicates an increase in the insulating layer on the electrode surface consistent with the proposed mechanism of collapse, as the ELPs have undergone morphological changes to hinder the kinetics of the redox couple exchange. Further characterization of the surface-immobilized ELPs showed a reproducible surface modification, as well as reversibility and tunability of the stimuli-response.

Flow imaging microscopy as a novel tool for high-throughput evaluation of elastin-like polymer coacervates.

Biological and bioinspired polymer microparticles have broad biomedical and industrial applications, including drug delivery, tissue engineering, surface modification, environmental remediation, imaging, and sensing. Full realization of the potential of biopolymer microparticles will require methods for rigorous characterization of particle sizes, morphologies, and dynamics, so that researchers may correlate particle characteristics with synthesis methods and desired functions. Toward this end, we evaluated biopolymer microparticles using flow imaging microscopy. This technology is widely used in the biopharmaceutical industry but is not yet well-known among the materials community. Our polymer, a genetically engineered elastin-like polypeptide (ELP), self-assembles into micron-scale coacervates. We performed flow imaging of ELP coacervates using two different instruments, one with a lower size limit of approximately 2 microns, the other with a lower size limit of approximately 300 nanometers. We validated flow imaging results by comparison with dynamic light scattering and atomic force microscopy analyses. We explored the effects of various solvent conditions on ELP coacervate size, morphology, and behavior, such as the dispersion of single particles versus aggregates. We found that flow imaging is a superior tool for rapid and thorough particle analysis of ELP coacervates in solution. We anticipate that researchers studying many types of microscale protein or polymer assemblies will be interested in flow imaging as a tool for quantitative, solution-based characterization.

Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent.

Protein-ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. In this work, we focus on the conjugation of amphiphilic variants of elastin-like polypeptide (ELP), short elastin (sEL), to poorly water-soluble compounds like OPPVs ( p-phenylenevinylene oligomers), triarylamines, and polypyridine-metal complexes. These conjugations are problematic when carried out in aqueous phase because hydrophobic ligands tend to avoid exposure to water, which in turn causes the ligand to self-aggregate and/or interact noncovalently with hydrophobic regions of the amphiphile. Ultimately, this behavior leads to low conjugation efficiency and contamination with strong noncovalent "conjugates". After exploring the solubility of sEL in various organic solvents, we have established an efficient conjugation methodology for obtaining covalent conjugates virtually free of contaminating noncovalent complexes. When conjugating carboxylated ligands to the amphiphile amines, we demonstrate that even when only one amine (the N-terminus) is present, its derivatization is 98% efficient. When conjugating amine moieties to the amphiphile carboxyls (a problematic configuration), protein multimerization is avoided, 98-100% of the protein is conjugated, and the unreacted ligand is recovered in pure form. Our syntheses occur in "one pot", and our purification procedure is a simple workup utilizing a combination of water and organic solvent extractions. This conjugation methodology might provide a solution to problems arising from solubility mismatch of protein and ligand, and it is likely to be widely applied for modification of recombinant amphiphiles used for drug delivery (PEG-antibodies, polymer-enzymes, food proteins), cell adhesion (collagen, hydrophobins), synthesis of nanostructures (peptides), and engineering of biocompatible optoelectronics (biological polymers), to cite a few.

A metallo-biopolymer conjugate of elastin-like polypeptide: photoluminescence enhancement in the coacervate microenvironment.

An optically active metallo-polymer assembly is demonstrated via conjugation of a genetically engineered elastin-like polypeptide (ELP) and a ruthenium(II) polypyridyl complex. By taking advantage of the phase transition of ELPs in water, photophysical properties of the resultant conjugate are investigated for both phases, below and above the critical transition temperature. Upon coacervation, the luminescence of the metallo-ELP is greatly enhanced as a consequence of local effects on the metal-ligand luminophore. These findings open a possibility to harness the temperature control of stimuli-responsive properties of biopolymers.

Stimuli-Responsive Genetically Engineered Polymer Hydrogel Demonstrates Emergent Optical Responses.

Biopolymer-based optical hydrogels represent an emerging class of materials with potential applications in biocompatible integrated optoelectronic devices, bioimaging applications, and stretchable/flexible photonics. We have synthesized stimuli-responsive three-dimensional hydrogels from genetically engineered elastin-like polymers (ELPs) and have loaded these hydrogels with an amine-containing p-phenylenevinylene oligomer (OPPV) derivative featuring highly tunable, environmentally sensitive optical properties. The composite ELP/OPPV hydrogels exhibit both pH- and temperature-dependent fluorescence emission, from which we have characterized a unique optical behavior that emerged from OPPV within the hydrogel environment. By systematic comparison with free OPPV in solution, our results suggest that this distinct behavior is due to local electronic effects arising from interactions between the hydrophobic ELP microenvironment and the nonprotonated OPPV species at pH 7 or higher.

Multisite phosphorylation networks as signal processors for Cdk1.

The order and timing of cell-cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDKs). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, distribution of serines and threonines as phosphoacceptors and positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phosphoadaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within networks of phosphorylation sites in different targets provides a wide range of possibilities for differential amplification of Cdk1 signals, thus providing a mechanism to generate a wide range of thresholds in the cell cycle.

Cks confers specificity to phosphorylation-dependent CDK signaling pathways.

Cks is an evolutionarily conserved protein that regulates cyclin-dependent kinase (CDK) activity. Clarifying the underlying mechanisms and cellular contexts of Cks function is critical because Cks is essential for proper cell growth, and its overexpression has been linked to cancer. We observe that budding-yeast Cks associates with select phosphorylated sequences in cell cycle-regulatory proteins. We characterize the molecular interactions responsible for this specificity and demonstrate that Cks enhances CDK activity in response to specific priming phosphosites. Identification of the binding consensus sequence allows us to identify putative Cks-directed CDK substrates and binding partners. We characterize new Cks-binding sites in the mitotic regulator Wee1 and discover a new role for Cks in regulating CDK activity at mitotic entry. Together, our results portray Cks as a multifunctional phosphoadaptor that serves as a specificity factor for CDK activity.

Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase.

Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.

The structure of a monomeric mutant Cks protein reveals multiple functions for a conserved hinge-region proline.

Cks (cyclin-dependent kinase subunit) proteins are essential eukaryotic cell cycle regulatory proteins that physically associate with cyclin-dependent kinases (Cdks) to modulate their activity. Cks proteins have also been studied for their ability to form domain-swapped dimers by exchanging ß-strands. Domain swapping is mediated by a conserved ß-hinge region containing two proline residues. Previous structural studies indicate that Cks in its dimer form is unable to bind Cdk, suggesting that the monomer-dimer equilibrium of Cks may have an effect on Cks-mediated Cdk regulation. We present the crystal structure of a proline-to-alanine mutant Saccharomyces cerevisiae Cks protein (Cks1 P93A) that preferentially adopts the monomer conformation but surprisingly fails to bind Cdk. Comparison of the Cks1 P93A structure to that of other Cks proteins reveals that Pro93 is critical for stabilizing a multiple ß-turn structure in the hinge region that properly positions an essential Cdk-binding residue. Additionally, we find that these ß-turn formations, conserved in Cks homologs, have implications for the mechanism and preferentiality of strand exchange. Together, our observations suggest that the conservation of Cks hinge-region prolines reflects their functions in forming a Cdk binding interface and that the ability of these prolines to control partitioning between monomer and dimer is a consequence of the ß-turn networks within the hinge.