Superdomains in the protein structure hierarchy: The case of PTP-C2.

Superdomain is uniquely defined in this work as a conserved combination of different globular domains in different proteins. The amino acid sequences of 25 structurally and functionally diverse proteins from fungi, plants, and animals have been analyzed in a test of the superdomain hypothesis. Each of the proteins contains a protein tyrosine phosphatase (PTP) domain followed by a C2 domain. Four novel conserved sequence motifs have been identified, one in the PTP domain and three in the C2 domain. All contribute to the PTP-C2 domain interface in PTEN, a tumor suppressor, and all are more conserved than the PTP signature motif, HCX3 (K/R)XR, in the 25 sequences. We show that PTP-C2 was formed prior to the fungi, plant, and animal kingdom divergence. A superdomain as defined here does not fit the usual protein structure classification system. The demonstrated existence of one superdomain suggests the existence of others.

Molecular physiology of the tensin brotherhood of integrin adaptor proteins.

Numerous proteins have been identified as constituents of the adhesome, the totality of molecular components in the supramolecular assemblies known as focal adhesions, fibrillar adhesions and other kinds of adhesive contact. The transmembrane receptor proteins called integrins are pivotal adhesome members, providing a physical link between the extracellular matrix (ECM) and the actin cytoskeleton. Tensins are ever more widely investigated intracellular adhesome constituents. Involved in cell attachment and migration, cytoskeleton reorganization, signal transduction and other processes relevant to cancer research, tensins have recently been linked to functional properties of deleted in liver cancer 1 (DLC1) and a mitogen-activated protein kinases (MAPK), to cell migration in breast cancer, and to metastasis suppression in the kidney. Tensins are close relatives of phosphatase homolog/tensin homolog (PTEN), an extensively studied tumor suppressor. Such findings are recasting the earlier vision of tensin (TNS) as an actin-filament (F-actin) capping protein in a different light. This critical review aims to summarize current knowledge on tensins and thus to highlight key points concerning the expression, structure, function, and evolution of the various members of the TNS brotherhood. Insight is sought by comparisons with homologous proteins. Some historical points are added for perspective.

Protein- and peptide-based electrospun nanofibers in medical biomaterials.

Electrospun fibers are being studied and developed because they hold considerable promise for realizing some advantages of nanostructured materials. The fibers can be made of biocompatible and biodegradable polymers. Electrospinning has therefore attracted interest in biotechnology and medicine, and there has been rapid growth in this area in recent years. This review presents an introduction to polymer nanofiber electrospinning, focusing on the use of natural proteins and synthetic peptides. We summarize key physical properties of protein-based and peptide-based nanofiber mats, survey biomedical applications of these materials, identify key challenges, and outline future prospects for development of the technology for tissue engineering, drug delivery, wound healing, and biosensors. FROM THE CLINICAL EDITOR: This review focuses on polymer nanofiber electrospinning using natural proteins and synthetic peptides. The authors describe key properties and applications of these materials, and outline future prospects for tissue engineering, drug delivery, wound healing, and biosensors based on these nanomats and nanofibers.

A synthetic polypeptide electrospun biomaterial.

Fiber mats of a synthetic anionic copolypeptide of l-glutamic acid and l-tyrosine (PLEY) have been produced by electrospinning, and physical, chemical, and biological properties of the fibers have been characterized in vitro. Fibers were obtained from polymer dissolved in water at concentrations of 20-60% (w/v) but not below this range. Applied voltage and spinneret-collector distance were also found to influence polymer spinnability. Oriented fibers were obtained by changing the geometry of the collector. Fiber diameter was measured by scanning electron microscopy (SEM). A common chemical reagent was used to cross-link polymers postspinning. Fiber solubility in aqueous solution varied as a function of cross-linking time. Cationic polypeptides labeled with a fluorescent dye became noncovalently associated with cross-linked fibers, enabling visualization by fluorescence microscopy. Spectroscopy provided information on polymer chain conformation in solution and in fibers. Degradation of cross-linked fibers by different proteases has been demonstrated. Fibroblasts were cultured on cross-linked fiber mats to test basic cytocompatibility. Synthetic polypeptide fiber mats may be useful in applications in medicine, biotechnology, and other areas.

Insoluble synthetic polypeptide mats from aqueous solution by electrospinning.

Water-insoluble nanofiber mats of synthetic polypeptides of defined composition have been prepared by a process involving electrospinning from aqueous solution. L-ornithine is a physiological amino acid. Fibers of poly(L-ornithine) (PLO) were produced at feedstock concentrations above 20% w/v. Applied voltage and needle-to-collector distance were crucial for nanofiber formation. Attractive fibers were obtained at 35-40% w/v. Fiber diameter and mat morphology have been characterized by electron microscopy. Polymer cross-linking with glutaraldehyde (GTA) vapor rendered fiber mats water-insoluble. The study has yielded two advances on previous work in the area: avoidance of an animal source of peptides and avoidance of inorganic solvent.

Present and future prospects for polypeptide multilayer nanofilms in biotechnology and medicine.

Polypeptide multilayer nanofilms are a promising nanotechnology for commercial product development because the processes used to prepare them are simple, flexible, reliable, automatable, and scalable. Moreover, these materials can display a remarkable diversity of physical, chemical, and biological properties. Furthermore, the constituents of these nanofilms, in most cases the nanofilms themselves, and the fabrication process are environmentally benign. Nanofilm structure and function can be tailored to address two Grand Challenges of the US National Nanotechnology Initiative.

Reversibility of structural changes of polypeptides in multilayer nanofilms.

Structural properties of different polypeptide multilayer nanofilms fabricated at neutral pH have been analyzed by UV spectroscopy, circular dichroism spectroscopy (CD), and Fourier-transform infrared spectroscopy (FTIR). The various peptides studied exhibit a strong tendency to adopt a beta sheet conformation in the films. Changes in film structure on dehydration are completely reversed on rewetting. The time scale of reversibility is, however, substantially shorter for the polymer backbone than the side chains, as in protein folding.

Controlled loading and release of a model drug from polypeptide multilayer nanofilms.

A major concern of medicine today is the sustained release of therapeutic compounds. Delivery vehicles for such compounds must be biocompatible. Ideally, loading a drug into the delivery vehicle will be a simple process, and vehicle properties will allow control over the drug release profile under desired conditions. Here, polypeptide multilayer nanofilms have been prepared by electrostatic layer-by-layer self-assembly to study the post-fabrication loading and release of a model therapeutic, methylene blue (MB). Drug loading and release have been characterized by optical spectroscopy for different peptide designs at different pH values, and film surface morphology has been characterized by atomic force microscopy (AFM). Differences in peptide structure have been found to influence MB loading and release under otherwise fixed conditions. Release is also influenced by pH, salt concentration, and number of "capping" layers. Although more research will be needed to exhaust the potential of polypeptide multilayer films, present results would suggest that the technology holds considerable promise for applications in medicine.

Polypeptide multilayer nanofilm artificial red blood cells.

Reliable encapsulation of hemoglobin (Hb) within polypeptide multilayer nanofilms has been achieved by a template-based approach, and protein functionality has been demonstrated postencapsulation. The method is general in scope and could be useful for many other encapsulants. Met-Hb was adsorbed onto 5 microm-diameter CaCO3 microparticles, and the Hb-coated particles were encapsulated within a multilayer nanofilm of poly(L-glutamic acid) (PLGA) and poly(L-lysine) (PLL) by layer-by-layer assembly. The CaCO3 templates were then dissolved within the PLGA/PLL nanofilms by addition of ethylenediaminetetraacetic acid. Encapsulation of Hb was proved by fluorescence microscopy, the pH-dependence of retention of Hb was determined by visible wavelength absorbance, and conversion of the encapsulated met-Hb to deoxy-Hb and oxy-Hb was demonstrated by spectroscopic analysis of the Soret absorption peak under various conditions. It thus has been shown that control of Hb oxygenation within polypeptide multilayer nanofilm artificial cells is possible, and that Hb thus encapsulated can bind, release, and subsequently rebind molecular oxygen. This work therefore represents an advance in the development of polypeptide multilayer film artificial red blood cells.

Context dependence of the assembly, structure, and stability of polypeptide multilayer nanofilms.

Polyelectrolyte multilayer nanofilms and nanocomposites have shown considerable promise for the rational development of multifunctional materials with wide-ranging properties. Polypeptides are a distinctive and largely unexplored class of polyelectrolytes in this context. Methods now exist for the synthesis of peptides with control at the level of the amino acid sequence, and for the preparation of these polymers in massive quantities. Here, we analyze the roles of six designed 32mer peptides in the fabrication, structure, and stability of multilayer nanofilms prepared by layer-by-layer self-assembly. The data show that amino acid sequence and the specific combination of anionic and cationic peptides together have a marked impact on nanofilm growth behavior, secondary structure content, and density in experimental studies. The same factors determine physical properties of the corresponding interpolypeptide complexes in molecular dynamics simulations.

Antimicrobial polypeptide multilayer nanocoatings.

A multilayer coating (or film) of nanometer-thick layers can be made by sequential adsorption of oppositely charged polyelectrolytes on a solid support. The method is known as layer-by-layer assembly (LBL). No special apparatus is required for LBL and nanofilms can be prepared under mild, physiological conditions. A multilayer nanofilm in which at least one of the constituent species is a polypeptide is a polypeptide multilayer nanofilm. The present work was aimed at assessing whether polypeptide multilayer nanofilms with specific antimicrobial properties could be prepared by incorporation of a known antimicrobial agent in the film structure, in this case the edible protein hen egg white lysozyme (HEWL). The chicken enzyme is widely employed as a human food preservative. An advantage of LBL in this context is that the nanofilm is fabricated directly on the surface of interest, eliminating the need to incorporate the antimicrobial in other packaging materials. Here, nanofilms were made of poly(L-glutamic acid) (PLGA), which is highly negatively charged in the mildly acidic pH range, and HEWL, which has a high net positive charge at acidic pH. We show that PLGA/HEWL nanofilms inhibit growth of the model microbe Microccocus luteus in the surrounding liquid medium. The amount of HEWL released from PLGA/HEWL films depends on the number of HEWL layers and therefore on the total quantity of HEWL in the films. This initial study provides a sketch of the scope for further development of LBL in the area of antimicrobial polypeptide multilayer films. Potential applications of such films include strategies for food preservation and coatings for implant devices.

Quantal self-assembly of polymer layers in polypeptide multilayer nanofilms.

Simple molecular models predict key aspects of the "microscopic" assembly behavior of various peptide systems in the fabrication of multilayer films. Such films show substantial differences in density for different peptide systems. The data suggest that exponential film growth is possible in the absence of polymer diffusion and that "macroscopic" assembly behavior is more a function of peptide-peptide interactions than peptide sequence alone.

A molecular dynamics study of the physical basis of stability of polypeptide multilayer nanofilms.

Electrostatic layer-by-layer assembly (LBL) is a versatile method of fabricating ultrathin multilayer films, coatings, and microcapsules from materials in solution, notably, oppositely charged polyelectrolytes in water. Polypeptides, a special type of polyelectrolyte, have recently shown promise for a range of applications in biotechnology and medicine, for example, artificial cells, drug delivery systems, cell/tissue scaffolds, artificial viruses, and implantable device coatings. Poly(L-lysine) (PLL) and poly(L-glutamic acid) (PLGA) at neutral pH are model oppositely charged polypeptides. Experimental studies have shown that PLL/PLGA multilayer films contain a substantial amount of beta-sheets. Here, we present findings of a molecular dynamics (MD) study of the physical basis of interaction between PLL and PLGA in multilayer film models. Simulations have been carried out to study structural and dynamical properties of PLL/PLGA aggregates in beta-sheet conformation. The results suggest that hydrophobic interactions, in addition to electrostatics interactions, play a significant role in PLL/PLGA multilayers. The preferred orientation of peptides in the beta-sheet structures is antiparallel within sheets and parallel between sheets. Intersheet hydrogen-bond formation is more likely the result of peptide association than the cause. The approach provides a general means to understand better how various types of noncovalent interactions contribute to the structure and stability of polypeptide multilayer films.

Straightforward and effective protein encapsulation in polypeptide-based artificial cells.

A simple and straightforward approach to encapsulating an enzyme and preserving its function in polypeptide-based artificial cells is demonstrated. A model enzyme, glucose oxidase (GOx), was encapsulated by repeated stepwise adsorption of poly(L-lysine) and poly(L-glutamic acid) onto GOx-coated CaCO3 templates. These polypeptides are known from previous research to exhibit nanometer-scale organization in multilayer films. Templates were dissolved by ethylenediaminetetraacetic acid (EDTA) at neutral pH. Addition of polyethylene glycol (PEG) to the polypeptide assembly solutions greatly increased enzyme retention on the templates, resulting in high-capacity, high-activity loading of the enzyme into artificial cells. Assay of enzyme activity showed that over 80 mg-mL(-1) GOx was retained in artificial cells after polypeptide multilayer film formation and template dissolution in the presence of PEG, but only one-fifth as much was retained in the absence of PEG. Encapsulation is a means of improving the availability of therapeutic macromolecules in biomedicine. This work therefore represents a means of developing polypeptide-based artificial cells for use as therapeutic biomacromolecule delivery vehicles.

Physics of polypeptide multilayer films.

Polypeptide multilayer films are promising for the development of coatings for implant devices, biosensors, and artificial cells. This paper discusses aspects of the physics of these films. Three sub-topics in the physics of peptide adsorption in multilayer film assembly covered here are peptide structure at the film/solid support interface, adsorbed layer thickness, and dynamics of peptide adsorption. A synopsis of work in these areas is preceded by an introduction to the subject and a review of some aspects of polymer theory.

Structural stability of polypeptide nanofilms under extreme conditions.

Self-assembly of designed peptides is a promising area of biomaterials research and development. Here, polypeptide nanofilms have been prepared by electrostatic layer-by-layer self-assembly (LBL) of cysteine (Cys)-containing 32mers designed to be oppositely charged at neutral pH, and structural stability of the films has been probed by subjecting them to various extreme physical and chemical conditions. The results suggest that although electrostatic attraction plays a key role in strengthening polypeptide films, stability is inversely related to absolute net charge of the supramolecular complex. This behavior is similar to the typical behavior of small globular proteins. Film structure is very stable in organic solvent and, when dehydrated, at extreme temperatures. Such stability is in marked contrast to the behavior of proteins, which tend to denature under comparable conditions. Similar to proteins, peptide nanofilms cross-linked by disulfide (S-S) bonds are considerably stronger than films stabilized by electrostatic, van der Waals, or hydrophobic interactions alone. This effect is particularly evident at extremes of pH and at elevated temperature when the film is hydrated. These results, the great variety of possible peptide structures, the inherent biocompatibility of l-amino acids, and current applications of thin films in commercial products together suggest that polypeptide films are promising for the development of new or enhanced products in food technology, drug delivery and medical device coatings, and biomaterials.

Fine tuning of physical properties of designed polypeptide multilayer films by control of pH.

Adjustment of pH can alter the ensemble of three-dimensional structures of a polypeptide in solution by changing the distribution of charge and Coulombic interactions. The role of pH in layer-by-layer self-assembly (LbL) of designed 32mer peptides containing the amino acid cysteine has been investigated using a combination of physical methods. Results show that pH can have a substantial influence on the mass of adsorbed peptide, surface roughness, and film density over a range of 1.5 pH units. Peptide film thickness depends on the number of layers, as with "conventional" polyelectrolytes. Film density and morphology, however, vary more with pH than does thickness, translating into a change in density on the order of 70% over the pH range 7.4-8.9. Results of this work provide insight on the physical basis of LbL and suggest that peptides are a promising class of polyelectrolytes for the creation of designer thin films for applications in biotechnology and other areas.

High-capacity functional protein encapsulation in nanoengineered polypeptide microcapsules.

Addition of polyethylene glycol to aqueous assembly solutions of oppositely charged polypeptides enables high-capacity "loading" of functional protein in biocompatible microcapsules by template-supported layer-by-layer nanoassembly.

Protein-inspired multilayer nanofilms: science, technology and medicine.

The field of polypeptide multilayer nanofilm research flourishes where study of protein structure and function shares a border with development of polyelectrolyte multilayers. The soil is fertile for creative input and promises a harvest of interesting results: the structure of a film can be predetermined on a layer-by-layer (LBL) basis, a huge variety of polypeptide sequences can be realized in large quantities by modern methods of synthesis, and the fabrication process is environmentally benign. In electrostatic LBL assembly, multilayer film assembly is driven primarily by coulombic interactions, but hydrophobic interactions and hydrogen bonds also contribute to film formation and stability, the amount depending on polypeptide design. Most peptides suitable for LBL assembly form films with a large percentage of beta-sheet at neutral pH; it would appear that beta-sheet is favored over alpha-helix in this context by the contribution to entropy of the number of ways of forming a beta-sheet from a single polypeptide chain. Film thickness and roughness depend rather substantially on amino acid composition. Promising applications of the polypeptide multilayer film platform technology include coatings for medical implant devices, scaffolds for tissue engineering, coatings for targeted drug delivery, artificial cells for oxygen therapeutics, and artificial viruses for immunization. In each case peptide structure is tailored to the application. Here we summarize recent results of experimental studies and computational work from our laboratory, showing how the study of protein structure has inspired the design of polypeptide films and pointing out new opportunities for technology development. This work also provides a brief introduction to polypeptide structure and multilayer films.