Elastic-contractile model proteins: Physical chemistry, protein function and drug design and delivery.

This review presents the structure and physico-chemical properties of ECMPs, elastic-contractile model proteins using sparse design modifications of elastic (GVGVP)(n); it describes the capacity of ECMP to perform the energy conversions that sustain living organisms; it arrives at the hydration thermodynamics of ECMP in terms of the change in Gibbs free energy of hydrophobic association, ΔG(HA), and the apolar-polar repulsive free energy of hydration, ΔG(ap); it applies ΔG(HA), ΔG(ap), and the nature of elasticity to describe the function of basic diverse proteins, namely - the F₁-motor of ATP synthase, Complex III of mitochondria, the KscA potassium-channel, and the molecular chaperonin, GroEL/ES; it applies ΔG(HA) and ΔG(ap) to describe the function of ABC exporter proteins that confer multi-drug resistance (MDR) on micro-organisms and human carcinomas and suggests drug modifications with which to overcome MDR. Using ECMP, means are demonstrated, for quantifying drug hydrophobicity with which to combat MDR and for preparing ECMP drug delivery nanoparticles, ECMPddnp, decorated with synthetic antigen-binding fragments, Fab1 and Fab2, with which to target specific up-regulated receptors, characteristic of human carcinoma cells, for binding and localized drug release.

Function and frustration of multi-drug ABC exporter protein and design of model proteins for drug delivery using protein hydration thermodynamics.

The mechanism is presented whereby simultaneous hydrolysis of two molecules of ATP in the ATP-binding cassette (ABC) exporter protein, Sav 1866, opens a transmembrane channel to pump drug out of the cell and confers drug resistance, e.g., gives rise to methicillin resistant Staphylococcus aureus, MRSA.

Effect of NaCl on the exothermic and endothermic components of the inverse temperature transition of a model elastin-like polymer.

TMDSC data have been employed to observe the effect of NaCl on the inverse temperature transition of the model elastin-like polymer (GVGVP)251. NaCl causes a decrease in Tt and an increase in DeltaH. The increase in enthalpy appears both in the enthalpy related with the folding of the polymer and in the contribution associated with disruption of the structured water of hydrophobic hydration.

Controlled release of phosphorothioates by protein-based polymers.

Protein-based polymers are water soluble at lower temperatures but undergo a phase transition with increasing temperature. The polymers' hydrophobicity controls the transition temperature and the free energy of its charged groups through an apolar-polar repulsive free energy of hydration, which drives the binding of charged drugs. Binding and release of phosphorothioates were obtained with polymers containing 1 lysine alone or coupled with 2 to 5 phenylalanines per 30 residues.

Human amniotic cell sheet harvest using a novel temperature-responsive culture surface coated with protein-based polymer.

Human amniotic epithelial (hAE) and mesenchymal (hAM) cells are believed to have the potential to differentiate into various functional cells, such as neurons, hepatocytes, cardiomyocytes, and pancreatic beta cells. However, cell transplantation has been performed by injection of cell suspensions, and thus it is difficult to control shape, size, location, and functions of differentiated cells. To overcome these problems, we developed a novel temperature-responsive culture surface coated with elastic protein-based polymer.

Function of the F1-motor (F1-ATPase) of ATP synthase by apolar-polar repulsion through internal interfacial water.

Within the structurally-confined internal aqueous cavity of the F1-motor of ATP synthase, function results from free energy changes that shift the balance between interfacial charge hydration and interfacial hydrophobic hydration. TRANSITION STATE DESCRIPTION: At the beta-P end of ADP x Mg occurs an inorganic phosphate, P(i). This P(i) resides at the base of a water-filled cleft that functions like an aperture to focus, into an aqueous chamber, a competition for hydration (an apolar-polar repulsion) between charged phosphate and hydrophobic surface of the gamma-rotor.

Crystal structure of cyclic (APGVGV)2, an analog of elastin, and a suggested mechanism for elongation/contraction of the molecule.

Tropoelastin is a complex polymeric protein composed primarily of repeating segments of Val-Pro-Gly-Gly, Val-Pro-Gly-Val-Gly, and Ala-Pro-Gly-Val-Gly-Val that occurs in connective tissue and arteries. It has rubber-like extensible properties. A synthetic cyclic dodecapeptide, with a double repeat of the hexapeptide sequence, has been shown to undergo a reversible inverse temperature transition; that is, crystals grow at 60 degrees C and dissolve in the mother liquor upon cooling.

Bioelastic membranes for topical application of a thromboxane synthetase inhibitor for protection of skin from pressure injury: a preliminary study.

A previous study showed that topical exposure to bioelastic-thromboxane synthetase inhibitor-matrix resulted in local tissue concentrations of thromboxane synthetase inhibitor sufficient for thromboxane synthetase inhibition. The objective of this research was to use an animal model to determine if a dressing having controlled release of thromboxane synthetase inhibitor (dazmegrel) could be used to prevent tissue breakdown over pressure points, i.e.

In vitro skin penetration of dazmegrel delivered with a bioelastic matrix.

The penetration of dazmegrel, a selective thromboxane synthetase inhibitor, through excised human and greyhound skin was measured. A bioelastic matrix was used for topical delivery. Results demonstrated that dazmegrel readily penetrated the skin.

Skin concentrations of thromboxane synthetase inhibitor after topical application with bioelastic membrane.

Elevated thromboxane levels are associated with a number of disease states, including dermal pressure ulcers. When dazmegrel was orally administered to greyhound dogs wearing leg casts, it resulted in a sparring effect on the skin areas of potential pressure ulcer development. The objective of this research was to determine if bioelastic matrices could provide controlled release of thromboxane A2 synthetase inhibitor (dazmegrel) at tissue concentrations sufficient for inhibition of thromboxane synthesis.

Prevention of postlaminectomy epidural fibrosis using bioelastic materials.

Study Design: The use of elastic protein-based polymers for the prevention of epidural fibrosis following lumbar spine laminectomy was investigated in a rabbit model.

Objectives: To determine the safety and efficacy of two bioelastic polymers in matrix and gel forms as interpositional materials in preventing postlaminectomy epidural fibrosis.

Summary Of Background Data: Postlaminectomy epidural fibrosis complicates revision spine surgery and is implicated in cases of "failed back syndrome.

Mechanics of elastin: molecular mechanism of biological elasticity and its relationship to contraction.

Description of the mechanics of elastin requires the understanding of two interlinked but distinct physical processes; the development of entropic elastic force and the occurrence of hydrophobic association. Elementary statistical-mechanical analysis of AFM single-chain force-extension data of elastin model molecules identifies damping of internal chain dynamics on extension as a fundamental source of entropic elastic force and eliminates the requirement of random chain networks. For elastin and its models, this simple analysis is substantiated experimentally by the observation of mechanical resonances in the dielectric relaxation and acoustic absorption spectra, and theoretically by the dependence of entropy on frequency of torsion-angle oscillations, and by classical molecular-mechanics and dynamics calculations of relaxed and extended states of the beta-spiral description of the elastin repeat, (GVGVP)n.

Elastin: a representative ideal protein elastomer.

During the last half century, identification of an ideal (predominantly entropic) protein elastomer was generally thought to require that the ideal protein elastomer be a random chain network. Here, we report two new sets of data and review previous data. The first set of new data utilizes atomic force microscopy to report single-chain force-extension curves for (GVGVP)(251) and (GVGIP)(260), and provides evidence for single-chain ideal elasticity.

Flow cytometry as a useful tool for process development: rapid evaluation of expression systems.

Flow cytometry is an established tool in fundamental studies of single-cell microbial physiology. Here we show that it can also provide valuable information for process development. Using recombinant Escherichia coli strains, which express the protein-based polymer (GVGIP)(260)GVGVP, the utility of flow cytometry in monitoring and optimization of fermentations is demonstrated.

Elastomeric polypentapeptides cross-linked into matrixes and fibers.

Microbially prepared polypentapeptides were cross-linked by two chemical methods. In one chemical approach, (GVGIP)(260) where G = glycine, V = valine, I = isoleucine, and P = proline with no functional groups in its side chains, was cross-linked using dicumyl peroxide, and reaction conditions were systematically examined. Successful cross-linking was obtained even under severe conditions for proteins, i.

Wheat seed proteins exhibit a complex mechanism of protein elasticity.

Elastomeric proteins are found in a number of animal tissues (elastin, abductin and resilin), where they have evolved to fulfil a range of biological functions. All exhibit rubber-like elasticity, undergoing deformation without rupture, storing the energy involved in deformation, and then recovering to their initial state when the stress is removed. The second part of the process is passive, entropy decreasing when the proteins are deformed, with the higher entropy of the relaxed state providing the driving force for recoil.

Phase transition and elasticity of protein-based hydrogels.

Reported are specific materials characterizations of three protein-based polymers comprised of repeating pentapeptide sequences, namely (GVGVP)251, (GVGIP)260 and (GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP)n](GVGVP) where G = glycine, V = valine, P = proline, I = isoleucine, and E = glutamic acid, which had been previously prepared and gamma-irradiation cross-linked into elastic matrices. These polymers exhibit a hydrophobic folding and assembling transition on raising the temperature above a critical temperature, designated by Tt. Their equilibrium swelling ratio, uniaxial tensile and dynamic shear behavior were studied.

Interaction of processes on different length scales in a bioelastomer capable of performing energy conversion.

This work concerns the aggregation properties of (Gly-Val-Gly-Val-Pro)(251) rec, a polypentapeptide reflecting a highly conserved repetitive unit of the bioelastomer, elastin. On raising the temperature of aqueous solutions above 25 degrees C, this polypeptide was already known to undergo concurrent conformational changes (hydrophobic folding), phase separation, and self-assembly with formation of aggregated three-stranded filaments composed of dynamic polypeptide helices, called beta-spirals. Aggregates obtained from the solution can be shaped into bands that acquire entropic elastic properties upon gamma-irradiation and can perform a variety of energy conversions.

Elastic molecular machines in metabolism and soft-tissue restoration.

Elastic protein-based machines (bioelastic materials) can be designed to perform diverse biological energy conversions. Coupled with the remarkable energy-conversion capacity of cells, this makes possible a tissue-restoration approach to tissue engineering. When properly attached to the extracellular matrix, cells sense the forces to which they are subjected and respond by producing an extracellular matrix that will withstand those forces.

Engineering protein-based machines to emulate key steps of metabolism (biological energy conversion).

Metabolism is the conversion of available energy sources to those energy forms required for sustaining and propagating living organisms; this is simply biological energy conversion. Proteins are the machines of metabolism; they are the engines of motility and the other machines that interconvert energy forms not involving motion. Accordingly, metabolic engineering becomes the use of natural protein-based machines for the good of society.

Elastic protein-based polymers in soft tissue augmentation and generation.

Five elastic protein-based polymers, designed as variations of polymer I, (GVGVP)251, elicited different responses when injected as subcutaneous implants in the guinea pig, a preclinical test used to evaluate materials for soft tissue augmentation and specifically for correction of urinary incontinence. All six polymers, prepared using recombinant DNA technology, expressed at good levels using transformed E. coli fermentation.

Elastic protein-based materials in tissue reconstruction.

In natural tissues, cells form multiple attachment sites to their extracellular matrix. By means of those attachments, cells deform as the tissue deforms in response to the natural mechanical stresses and strains that the tissue must sustain during function. These mechanical forces are the energy input that instruct the cells to produce the extracellular matrix sufficient to sustain those forces.

Expression of a synthetic protein-based polymer (elastomer) gene in Aspergillus nidulans.

A gene for a synthetic protein-based polymer, G-(VPGVG)119-VPGV, coding for the EG-120mer (elastomer), was cloned into a fungal expression vector to allow constitutive expression of the polymer controlled by the gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter sequence of Aspergillus nidulans. Stable transformants of A. nidulans showed plasmid integration with varying copy number when analyzed by Southern-blot hybridization.