Bioengineered dual fluorescent carbon nano dots from Indian long pepper leaves for multifaceted environmental and health utilities.

In this article, we present the synthesis of Piper longum leaves-derived ethanolic carbon dots (PLECDs) using the most simplistic environmentally friendly solvothermal carbonization method. The PLECDs fluoresced pink color with maximum emission at 670 nm at 397 nm excitation. Additionally, the dried PLECDs dissolved in water showed green fluorescence with higher emission at 452 nm at 370 nm excitation. The UV spectra showed peaks in the UV region (271.25 nm and 320.79 nm) and a noticeable tail in the visible region, signifying the efficient synthesis of nano-sized carbon particles and the Mie scattering effect. Various functional groups (-OH, -N-H, -C-H, -C = C, -C-N, and -C-O) were identified using Fourier transform infrared spectroscopy (FTIR). Its nanocrystalline property was revealed by the sharp peaks in the X-ray diffraction (XRD). High-resolution transmission electron microscopy (HRTEM) photomicrograph displayed a roughly spherical structure with a mean size of 2.835 nm. The energy dispersive X-ray (EDAX) and X-ray photoelectron spectroscopy (XPS) revealed the elemental abundance of C, O, and N. The high-performance thin-layer chromatography (HPTLC) fingerprint of PLECDs showed an altered pattern than its precursor (Piper longum leaves ethanolic extract or PLLEE). The PLECDs sensed Cu2+ selectively with a limit of detection (LOD) and limit of quantification (LOQ) of 0.063 µM and 0.193 µM, respectively. It showed excellent cytotoxicity toward MDA-MB-231 (human breast cancer), SiHa (human cervical carcinoma), and B16F10 (murine melanoma) cell lines with excellent in vitro bioimaging outcomes. It also has free radical scavenging activity. The PLECDs also showed outstanding bacterial biocompatibility, pH-dependent fluorescence stability, photostability, physicochemical stability, and thermal stability.

Tinospora cordifolia Leaves Derived Carbon dots for Cancer Cell Bioimaging, Free radical Scavenging, and Fe3+ Sensing Applications.

Herein, we report the fabrication of Tinospora cordifolia leaves-derived carbon dots (TCLCDs) from aqueous extract of leaves as carbon source via simple, environmentally friendly, hydrothermal carbonization (HTC) technique. The synthesized TCLCDs were characterized for their physicochemical properties and further explored for in-vitro cancer cell bioimaging, radical scavenging, and metal ion sensing. The synthesized TCLCDs showed excitation-dependent emission property with maximum emission at 435 nm under the excitation of 350 nm. The High-Resolution Transmission Electron Microscopy (HRTEM) results revealed a roughly spherical shape with an average diameter of 5.47 nm. The diffused ring pattern of Selected Area Electron Diffraction (SAED) and halo diffraction pattern of X-ray diffraction (XRD) disclosed their amorphous nature. The Energy Dispersive X-ray (EDX) showed the existence of C, N, and O. The Fourier-transform infrared spectroscopy (FTIR) revealed the presence of -OH, -NH, -CN, and -CH groups. The TCLCDs showed excellent cellular biocompatibility with dose-dependent bioimaging results in melanoma (B16F10) and cervical cancer (SiHa) cell lines. Also, they exhibited excellent scavenging of free radicals with an IC50 value of 0.524 mg/mL & selective Fe3+ ion sensing with a detection limit of 0.414 µM. Further, they exerted excellent bacterial biocompatibility, photostability, and thermal stability. The overall results reflected their potential for in-vitro cancer cell bioimaging, free radical scavenging, and selective Fe3+ ion sensing.

Carbon dots from an immunomodulatory plant for cancer cell imaging, free radical scavenging and metal sensing applications.

Aim: This work aimed to develop Tinospora cordifolia stem-derived carbon dots (TCSCD) for cancer cell imaging, free radical scavenging and metal sensing applications. Method: The TCSCDs were synthesized by a simple, one-step, and ecofriendly hydrothermal carbonization method and characterized for their optical properties, morphology, hydrodynamic size, surface functionality, crystallinity, stability, bacterial biocompatibility, in vitro cellular imaging, free radical scavenging and metal sensing ability. Results: The TCSCDs exhibited excellent biocompatibility with dose-dependent bioimaging results in melanoma (B16F10) and cervical cancer (SiHa) cell lines. They exerted good free radical scavenging, Fe3+ sensing, bacterial biocompatibility, photostability, colloidal dispersion stability and thermal stability. Conclusion: The results reflect the potential of TCSCDs for biomedical and pharmaceutical applications.

Multi-Functional Carbon Dots from an Ayurvedic Medicinal Plant for Cancer Cell Bioimaging Applications.

The combination of an Ayurvedic wisdom and nanotechnology may help us to resolve the complex healthcare challenges. A facile and economical one-pot hydrothermal synthesis method has been adopted for preparing a blue fluorescent carbon dots (CDs) with a quantum yield of 15.10% from an Ayurvedic medicinal plant Andrographis paniculata (AP). The Andrographis paniculata derived CDs (AAPCDs) were then characterized using different techniques. Through High Performance Thin Layer Chromatography (HPTLC) profiling of the AP extract and the CDs, it was found that some of the phytoconstituents are retained as such while others may have been converted into their derivatives during the process of formation of CDs. The CDs are designed to possess cellular imaging of human breast carcinoma cells (MCF-7), apart from free radicals sensing and scavenging capabilities. AAPCDs showed minimal cytotoxicity in Multi Drug Resistant clinically isolated strains of gram positive and gram negative bacteria which may be employed for microbiology oriented experiments. These results suggest potential of multi-functional AAPCDs as nano-probes for various pharmaceutical, biomedical and bioengineering applications.

Circular Dichroism Spectroscopy of Collagen Fibrillogenesis: A New Use for an Old Technique.

Type-I collagen assembles in a stepwise, hierarchic fashion from the folding of the triple helix to the assembly of fibrils into fibers. The mature assembled fibers are crucial for tissue structure and mechanics, cell interactions, and other functions in vivo. Although triple helix folding can be followed with the use of optical methods such as circular dichroism (CD) spectroscopy, fibrillogenesis is typically measured by alternative methods such as turbidity, rheology, and microscopy. Together, these approaches allow for investigation of the mechanical properties and architectures of collagen-based scaffolds and excised tissues. Herein, we demonstrate that CD spectroscopy, a technique that is used primarily to evaluate the secondary structure of proteins, can also be employed to monitor collagen fibrillogenesis. Type-I collagen suspensions demonstrated a strong, negative ellipticity band between 204 and 210 nm under conditions consistent with fibrillogenesis. Deconvolution of CD spectra before, during, and after fibrillogenesis identified a unique fibril spectrum distinct from triple helix and random coil conformations. The ability to monitor multiple states of collagen simultaneously in one experiment using one modality provides a powerful platform for studying this complex assembly process and the effects of other factors, such as collagenases, on fibrillogenesis and degradation.

Dissecting Electrostatic Contributions to Folding and Self-Assembly Using Designed Multicomponent Peptide Systems.

We investigate formation of nano- to microscale peptide fibers and sheets where assembly requires association of two distinct collagen mimetic peptides (CMPs). The multicomponent nature of these designs allows the decoupling of amino acid contributions to peptide folding versus higher-order assembly. While both arginine and lysine containing CMP sequences can favor triple-helix folding, only arginine promotes rapid supramolecular assembly in each of the three two-component systems examined. Unlike lysine, the polyvalent guanidyl group of arginine is capable of both intra- and intermolecular contacts, promoting assembly. This is consistent with the supramolecular diversity of CMP morphologies observed throughout the literature. It also connects CMP self-assembly with a broad range of biomolecular interaction phenomena, providing general principles for modeling and design.

Metal Stabilization of Collagen and de Novo Designed Mimetic Peptides.

We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacency of the carboxy-termini of the three chains makes this region an attractive target for introducing metal binding sites. We engineered His3 sites based on structural modeling constraints into a series of designed homotrimeric and heterotrimeric peptides, assessing the capacity of metal binding to improve stability and in the case of heterotrimers, affect specificity of assembly. Notable enhancements in stability for both homo- and heteromeric systems were observed upon addition of zinc(II) and several other metal ions only when all three histidine ligands were present. Metal binding affinities were consistent with the expected Irving-Williams series for imidazole. Unlike other metals tested, copper(II) also bound to peptides lacking histidine ligands. Acetylation of the peptide N-termini prevented copper binding, indicating proline backbone amide metal-coordination at this site. Copper similarly stabilized animal extracted Type I collagen in a metal-specific fashion, highlighting the potential importance of metal homeostasis within the extracellular matrix.

Computational design of metalloproteins.

A number of design strategies exist for the development of novel metalloproteins. These strategies often exploit the inherent symmetry of metal coordination and local topology. Computational design of metal binding sites in flexible regions of proteins is challenging as the number of conformational degrees of freedom is significantly increased. Additionally, without pre-organization of the primary shell ligands by the protein fold, metal binding sites can rearrange according to the coordination constraints of the metal center. Examples of metal incorporation into existing folds, full fold design exploiting symmetry, and fold design in asymmetric scaffolds are presented.

Methacrylation induces rapid, temperature-dependent, reversible self-assembly of type-I collagen.

Type-I collagen self-assembles into a fibrillar gel at physiological temperature and pH to provide a cell-adhesive, supportive, structural network. As such, it is an attractive, popular scaffold for in vitro evaluations of cellular behavior and for tissue engineering applications. In this study, type-I collagen is modified to introduce methacrylate groups on the free amines of the lysine residues to create collagen methacrylamide (CMA). CMA retains the properties of collagen such as self-assembly, biodegradability, and natural bioactivity but is also photoactive and can be rapidly cross-linked or functionalized with acrylated molecules when irradiated with ultraviolet light in the presence of a photoinitiator. CMA also demonstrates unique temperature-dependent behavior. For natural type-I collagen, the overall structure of the fiber network remains largely static over time scales of a few hours upon heating and cooling at temperatures below its denaturation point. CMA, however, is rapidly thermoreversible and will oscillate between a liquid macromer suspension and a semisolid fibrillar hydrogel when the temperature is modulated between 10 and 37 °C. Using a series of mechanical, scattering, and spectroscopic methods, we demonstrate that structural reversibility is manifest across multiple scales from the protein topology of the triple helix up through the rheological properties of the CMA hydrogel. Electron microscopy imaging of CMA after various stages of heating and cooling shows that the canonical collagen-like D-periodic banding ultrastructure of the fibers is preserved. A rapidly thermoreversible collagen-based hydrogel is expected to have wide utility in tissue engineering and drug delivery applications as a biofunctional, biocompatible material. Thermal reversibility also makes CMA a powerful model for studying the complex process of hierarchical collagen self-assembly.

Design of net-charged abc-type collagen heterotrimers.

Net-negatively-charged heterospecific A:B:C collagen peptide heterotrimers were designed using an automated computational approach. The design algorithm considers both target stability and the energy gap between the target states and misfolded competing states. Structural characterization indicates the net-negative charge balance on the new designs enhances the specificity of the target state at the expense of its stability.

Self-assembly of left- and right-handed molecular screws.

Stereoselectivity is a hallmark of biomolecular processes from catalysis to self-assembly, which predominantly occur between homochiral species. However, both homochiral and heterochiral complexes of synthetic polypeptides have been observed where stereoselectivity hinges on details of intermolecular interactions. This raises the question whether general rules governing stereoselectivity exist. A geometric ridges-in-grooves model of interacting helices indicates that heterochiral associations should generally be favored in this class of structures. We tested this principle using a simplified molecular screw, a collagen peptide triple-helix composed of either l- or d-proline with a cyclic aliphatic side chain. Calculated stabilities of like- and opposite-handed triple-helical pairings indicated a preference for heterospecific associations. Mixing left- and right-handed helices drastically lowered solubility, resulting in micrometer-scale sheet-like assemblies that were one peptide-length thick as characterized with atomic force microscopy. X-ray scattering measurements of interhelical spacing in these sheets support a tight ridges-in-grooves packing of left- and right-handed triple helices.

Control of Collagen Stability and Heterotrimer Specificity through Repulsive Electrostatic Interactions.

Charge-pair interactions between acidic and basic residues on the surface of collagen can promote stability as well as control specificity of molecular recognition. Heterotrimeric collagen peptides have been engineered de novo using either rational or computational methods, which in both cases optimize networks of favorable charge-pair interactions in the target structure. Less understood is the role of electrostatic repulsion between groups of like charge in destabilizing structure or directing molecular recognition. To study this, we apply a "charge crowding" approach, where repulsive interactions between multiple aspartate side chains are found to destabilize the homotrimer states in triple helical peptide system and can be utilized to promote the formation of heterotrimers. Neutralizing surface charge by increasing salt concentration or decreasing pH can enhance homotrimer stability, confirming the role of charge crowding on the destabilization of homotrimers via electrostatic repulsion. Charge crowding may be used in conjunction with other approaches to create specific collagen heterotrimers.

A peptide study of the relationship between the collagen triple-helix and amyloid.

Type XXV collagen, or collagen-like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro-Hyp-Gly)10 , an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)(n) domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple-helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple-helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple-helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly-Xaa-Yaa sequence and required the triple-helix conformation. The inhibitory effect of the collagen triple-helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation.

Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association.

The standard collagen triple-helix requires a perfect (Gly-Xaa-Yaa)(n) sequence, yet all nonfibrillar collagens contain interruptions in this tripeptide repeating pattern. Defining the structural consequences of disruptions in the sequence pattern may shed light on the biological role of sequence interruptions, which have been suggested to play a role in molecular flexibility, collagen degradation, and ligand binding. Previous studies on model peptides with 1- and 4-residue interruptions showed a localized perturbation within the triple-helix, and this work is extended to introduce natural collagen interruptions up to nine residue in length within a fixed (Gly-Pro-Hyp)(n) peptide context. All peptides in this set show decreases in triple-helix content and stability, with greater conformational perturbations for the interruptions longer than five residue. The most stable and least perturbed structure is seen for the 5-residue interruption peptide, whose sequence corresponds to a Gly to Ala missense mutation, such as those leading to collagen genetic diseases. The triple-helix peptides containing 8- and 9-residue interruptions exhibit a strong propensity for self-association to fibrous structures. In addition, a small peptide modeling only the 9-residue sequence within the interruption aggregates to form amyloid-like fibrils with antiparallel beta-sheet structure. The 8- and 9-residue interruption sequences studied here are predicted to have significant cross-beta aggregation potential, and a similar propensity is reported for approximately 10% of other naturally occurring interruptions. The presence of amyloidogenic sequences within or between triple-helix domains may play a role in molecular association to normal tissue structures and could participate in observed interactions between collagen and amyloid.

Lysozyme as diffusion tracer for measuring aqueous solution viscosity.

Measuring tracer diffusion provides a convenient approach for monitoring local changes in solution viscosity or for determining viscosity changes in response to multiple solution parameters including pH, temperature, salt concentrations or salt types. One common limitation of tracer diffusion in biologically relevant saline solutions is the loss of colloidal stability and aggregation of the tracer particles with increasing ionic strength. Using dynamic light scattering to measure tracer diffusion, we compared the performance of two different types of tracer particles, polystyrene nanobeads vs. the small protein lysozyme, for viscosity measurements of saline solutions. Polystyrene beads provide reliable values for water viscosity, but begin flocculating at ionic strengths exceeding about 100mM. Using lysozyme, in contrast, we could map out viscosity changes of saline solutions for a variety of different salts, for salt concentrations up to 1M, over a wide range of pH values, and over the temperature range most relevant for biological systems (5-40 degrees C). Due to its inherently high structural and colloidal stability, lysozyme provides a convenient and reliable tracer particle for all these measurements, and its use can be readily extended to other optical approaches towards localized measurements of tracer diffusion such as fluorescence correlation spectroscopy.

Hydration and hydrodynamic interactions of lysozyme: effects of chaotropic versus kosmotropic ions.

Using static and dynamic light scattering we have investigated the effects of either strongly chaotropic, nearly neutral or strongly kosmotropic salt ions on the hydration shell and the mutual hydrodynamic interactions of the protein lysozyme under conditions supportive of protein crystallization. After accounting for the effects of protein interaction and for changes in solution viscosity on protein diffusivity, protein hydrodynamic radii were determined with +/-0.25 A resolution. No changes to the extent of lysozyme hydration were discernible for all salt-types, at any salt concentration and for temperatures between 15-40 degrees C. Combining static with dynamic light scattering, we also investigated salt-induced changes to the hydrodynamic protein interactions. With increased salt concentration, hydrodynamic interactions changed from attractive to repulsive, i.e., in exact opposition to salt-induced changes in direct protein interactions. This anti-correlation was independent of solution temperature or salt identity. Although salt-specific effects on direct protein interactions were prominent, neither protein hydration nor solvent-mediated hydrodynamic interactions displayed any obvious salt-specific effects. We infer that the protein hydration shell is more resistant than bulk water to changes in its local structure by either chaotropic or kosmotropic ions.

Pre-assembled clusters distort crystal nucleation kinetics in supersaturated lysozyme solutions.

Efficient determination of three-dimensional protein structures is critical for unraveling structure-function relationships and for supporting targeted drug design. A major impediment to these efforts is our lack of control over the nucleation and growth of high-quality protein crystals for X-ray structure determinations. While basic research on protein crystal growth mechanisms has provided valuable new insights, studies of crystal nucleation have been plagued by inconsistent and outright contradictory results. Using dynamic light scattering and SDS gel electrophoresis, we have investigated possible causes of these inconsistencies. We find that commercial sources of lyophilized hen-egg white lysozyme (HEWL) used in nucleation studies contain significant populations of large (approximately 100 nm), pre-assembled lysozyme clusters that can readily evade standard assays of sample purity. In supersaturated solutions, these clusters act as heterogeneous nucleation centers that enhance the rate of crystal nucleation and significantly deteriorate the quality of macroscopic crystals.