Tailoring Synthetic Polypeptide Design for Directed Fibril Superstructure Formation and Enhanced Hydrogel Properties.

The biological significance of self-assembled protein filament networks and their unique mechanical properties have sparked interest in the development of synthetic filament networks that mimic these attributes. Building on the recent advancement of autoaccelerated ring-opening polymerization of amino acid N-carboxyanhydrides (NCAs), this study strategically explores a series of random copolymers comprising multiple amino acids, aiming to elucidate the core principles governing gelation pathways of these purpose-designed copolypeptides. Utilizing glutamate (Glu) as the primary component of copolypeptides, two targeted pathways were pursued: first, achieving a fast fibrillation rate with lower interaction potential using serine (Ser) as a comonomer, facilitating the creation of homogeneous fibril networks; and second, creating more rigid networks of fibril clusters by incorporating alanine (Ala) and valine (Val) as comonomers. The selection of amino acids played a pivotal role in steering both the morphology of fibril superstructures and their assembly kinetics, subsequently determining their potential to form sample-spanning networks. Importantly, the viscoelastic properties of the resulting supramolecular hydrogels can be tailored according to the specific copolypeptide composition through modulations in filament densities and lengths. The findings enhance our understanding of directed self-assembly in high molecular weight synthetic copolypeptides, offering valuable insights for the development of synthetic fibrous networks and biomimetic supramolecular materials with custom-designed properties.

Modeling and Designing Particle-Regulated Amyloid-like Assembly of Synthetic Polypeptides in Aqueous Solution.

In cells, actin and tubulin polymerization is regulated by nucleation factors, which promote the nucleation and subsequent growth of protein filaments in a controlled manner. Mimicking this natural mechanism to control the supramolecular polymerization of macromolecular monomers by artificially created nucleation factors remains a largely unmet challenge. Biological nucleation factors act as molecular scaffolds to boost the local concentrations of protein monomers and facilitate the required conformational changes to accelerate the nucleation and subsequent polymerization. An accelerated assembly of synthetic poly(l-glutamic acid) into amyloid fibrils catalyzed by cationic silica nanoparticle clusters (NPCs) as artificial nucleation factors is demonstrated here and modeled as supramolecular polymerization with a surface-induced heterogeneous nucleation pathway. Kinetic studies of fibril growth coupled with mechanistic analysis demonstrate that the artificial nucleators predictably accelerate the supramolecular polymerization process by orders of magnitude (e.g., shortening the assembly time by more than 10 times) when compared to the uncatalyzed reaction, under otherwise identical conditions. Amyloid-like fibrillation was supported by a variety of standard characterization methods. Nucleation followed a Michaelis-Menten-like scheme for the cationic silica NPCs, while the corresponding anionic or neutral nanoparticles had no effect on fibrillation. This approach shows the effectiveness of charge-charge interactions and surface functionalities in facilitating the conformational change of macromolecular monomers and controlling the rates of nucleation for fibril growth. Molecular design approaches like these inspire the development of novel materials via biomimetic supramolecular polymerizations.

Controlling the Graphene-Bio Interface: Dispersions in Animal Sera for Enhanced Stability and Reduced Toxicity.

Liquid phase exfoliation of graphite in six different animal sera and evaluation of its toxicity are reported here. Previously, we reported the exfoliation of graphene using proteins, and here we extend this approach to complex animal fluids. A kitchen blender with a high-turbulence flow gave high quality and maximum exfoliation efficiency in all sera tested, when compared to the values found with shear and ultrasonication methods. Raman spectra and electron microscopy confirmed the formation of three- or four-layer, submicrometer size graphene, independent of the serum used. Graphene prepared in serum was directly transferred to cell culture media without post-treatments. Contrary to many reports, a nanotoxicity study of this graphene fully dispersed to human embryonic kidney cells, human lung cancer cells, and nematodes (Caenorhabditis elegans) showed no acute toxicity for up to 7 days at various doses (50-500 µg/mL), but prolonged exposure at higher doses (300-500 µg/mL, 10-15 days) showed cytotoxicity to cells (∼95% death) and reproductive toxicity to C. elegans (5-10% reduction in brood size). The origin of toxicity was found to be due to the highly fragmented smaller graphene sheets (<200 nm), while the larger sheets were nontoxic (50-300 µg/mL dose). In contrast, graphene produced with sodium cholate as the mediator has been found to be cytotoxic to these cells at these dosages. We demonstrated the toxicity of liquid phase exfoliated graphene is attributed to highly fragmented fractions or nonbiocompatible exfoliating agents. Thus, low-toxicity graphene/serum suspensions are produced by a facile method in biological media, and this approach may accelerate the much-anticipated development of graphene for biological applications.

Fluorescent, bioactive protein nanoparticles (prodots) for rapid, improved cellular uptake.

A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15-50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1-3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers.

Ultrasensitive carbohydrate-peptide SPR imaging microarray for diagnosing IgE mediated peanut allergy.

Severity of peanut allergies is linked to allergen-specific immunoglobulin E (IgE) antibodies in blood, but diagnostics from assays using glycoprotein allergen mixtures may be inaccurate. Measuring IgEs specific to individual peptide and carbohydrate epitopes of allergenic proteins is promising. We report here the first immunoarray for IgEs utilizing both peptide and carbohydrate epitopes. A surface plasmon resonance imaging (SPRi) microarray was equipped with peptide and ß-xylosyl glycoside (BXG) epitopes from major peanut allergen glycoprotein Arachis hypogaea h2 (Ara-h2). A monoclonal anti-IgE antibody was included as positive control. IgEs were precaptured onto magnetic beads loaded with polyclonal anti-IgE antibodies to enhance sensitivity and minimize non-specific binding. As little as 0.1 attomole (0.5 pg mL(-1)) IgE was detected from dilute serum in 45 min. IgEs binding to Ara-h2 peptide and BXG were quantified in 10 µL of patient serum and correlated with standard ImmunoCAP values.

Highly efficient binding of paramagnetic beads bioconjugated with 100,000 or more antibodies to protein-coated surfaces.

We report here the first kinetic characterization of 1 µm diameter superparamagnetic particles (MP) decorated with over 100,000 antibodies binding to protein antigens attached to flat surfaces. Surface plasmon resonance (SPR) was used to show that these antibody-derivatized MPs (MP-Ab(2)) exhibit irreversible binding with 100-fold increased association rates compared to free antibodies. The estimated upper limit for the dissociation constant of MP-Ab(2) from the SPR sensor surface is 5 fM, compared to 3-8 nM for the free antibodies. These results are explained by up to 2000 interactions of MP-Ab(2) with protein-decorated surfaces. Findings are consistent with highly efficient capture of protein antigens in solution by the MP-Ab(2) and explain in part the utility of these beads for ultrasensitive protein detection into the fM and aM range. Aggregation of these particles on the SPR chip, probably due to residual magnetic microdomains in the particles, also contributes to ultrasensitive detection and may also help drive the irreversible binding.

Measurement of biomarker proteins for point-of-care early detection and monitoring of cancer.

This critical review evaluates progress toward viable point-of-care protein biomarker measurements for cancer detection and diagnostics. The ability to measure panels of specific, selective cancer biomarker proteins in physicians' surgeries and clinics has the potential to revolutionize cancer detection, monitoring, and therapy. The dream envisions reliable, cheap, automated, technically undemanding devices that can analyze a patient's serum or saliva in a clinical setting, allowing on-the-spot diagnosis. Existing commercial products for protein assays are reliable in laboratory settings, but have limitations for point-of-care applications. A number of ultrasensitive immunosensors and some arrays have been developed, many based on nanotechnology. Multilabel detection coupled with high capture molecule density in immunosensors and arrays seems to be capable of detecting a wide range of protein concentrations with sensitivity ranging into the sub pg mL(-1) level. Multilabel arrays can be designed to detect both high and ultralow abundance proteins in the same sample. However, only a few of the newer ultrasensitive methods have been evaluated with real patient samples, which is key to establishing clinical sensitivity and selectivity.

Folding control and unfolding free energy of yeast iso-1-cytochrome c bound to layered zirconium phosphate materials monitored by surface plasmon resonance.

The free energy change (Delta G degrees ) for the unfolding of immobilized yeast iso-1-cytochrome c (Cyt c) at nanoassemblies was measured by surface plasmon resonance (SPR) spectroscopy. Data show that SPR is sensitive to protein conformational changes, and protein solid interface exerts a major influence on bound protein stability. First, Cyt c was self-assembled on the Au film via the single thiol of Cys-102. Then, crystalline sheets of layered alpha-Zr(O(3)POH)(2).H(2)O (alpha-ZrP) or Zr(O(3)PCH(2)CH(2)COOH)(2).xH(2)O (alpha-ZrCEP) were adsorbed to construct alpha-ZrP/Cyt c/Au or alpha-ZrCEP/Cyt c/Au nanoassemblies. The construction of each layer was monitored by SPR, in real time, and the assemblies were further characterized by atomic force microscopy and electrochemical studies. Thermodynamic stability of the protein nanoassembly was assessed by urea-induced unfolding. Surprisingly, unfolding is reversible in all cases studied here. Stability of Cyt c in alpha-ZrP/Cyt c/Au increased by approximately 4.3 kJ/mol when compared to the unfolding free energy of Cyt c/Au assembly. In contrast, the protein stability decreased by approximately 1.5 kJ/mol for alpha-ZrCEP/Cyt c/Au layer. Thus, OH-decorated surfaces stabilized the protein whereas COOH-decorated surfaces destabilized it. These data quantitate the role of specific functional groups of the inorganic layers in controlling bound protein stability.

Photocleavage of lysozyme by cobalt(III) complexes.

Photochemical reagents that cleave proteins at specific sites (photoproteases) are useful for studying protein structure and protein-ligand interactions. PolyammineCo(III) complexes are tested here as photochemical probes to cleave proteins. Irradiation of a mixture of lysozyme, a model protein, and polyammineCo(III) complexes resulted in the facile cleavage of the peptide backbone. Photocleavage yielded two fragments of molecular weights 10.6 and 3.7 kDa, and these masses sum to the molecular mass of lysozyme (14.3 kDa). No cleavage was detected in the absence of the metal complex, in the dark, or upon irradiation at wavelengths of >420 nm. The photocleavage yield increased with irradiation time and with the concentrations of the metal complex and the protein. N-terminal sequencing of the 10.6 kDa fragment indicated residues that are identical to the N-terminus of lysozyme, and sequencing of the 3.7 kDa fragment indicated Val-Ala-Trp-Arg, an internal sequence of lysozyme. From the known primary sequence of lysozyme and the sequencing data, the cleavage site was assigned to Trp108-Val109. Molecular modeling indicates that the observed cleavage site is within few angstroms from the proposed metal binding site at Glu35-Asp52. This is the first report of the successful photocleavage of proteins, with high selectivity, by transition metal complexes. This novel observation can facilitate the rational design of transition metal complexes for the photochemical footprinting of metal binding sites on proteins.

Contributions of a long side chain to the binding affinity of an anthracene derivative to DNA.

Systematic studies on the DNA binding of a new anthracene derivative, carrying a 1,8-octyldiamine side chain, were carried out. Calorimetric, spectroscopic, and helix melting studies show that the side chain, consisting of eight methylene groups, enhances the binding constant by a factor of approximately 35 when compared to the binding of a probe lacking the long side chain. Furthermore, the enthalpy of binding of the long-chain derivative to calf thymus DNA (Delta H = 4.1 +/- 0.1 kcal/mol) is far greater than the sum of the enthalpy changes associated with the binding of the probe lacking the long side chain, and the enthalpy for the binding of 1,8-octyldiamine.2HCl. Strong synergistic effects, therefore, are seen with the long-chain derivative. Spectroscopic data indicate bathochromism, strong hypochromism, and quenching of anthryl fluorescence when the above ligand binds to calf thymus DNA. Fluorescence energy transfer studies and circular dichroism data strongly suggest intercalation of the anthryl ring system. The binding stabilizes the double helix, and the helix melting temperature is increased from 78 degrees C to >90 degrees C. The binding to DNA is reversible, depended on the ionic strength, and the major binding mode was suppressed at high ionic strengths and a new mode begins to dominate binding. Substitution of the anthracene ring with 1,8-octyldiamine chain provided a simple method to enhance the binding constant by nearly a factor of 35.