Biomedical Nanotechnology Lecture 7: Protein & Glyco Nanotechnology PDF

BIOMEDICAL NANOTECHNOLOGY LECTURE 7: PROTEIN & GLYCO NANOTECHNOLOGY Dr.P.GOPINATH DEPARTMENT OF BIOTECHNOLOGY 1  Contents Protein nanotechnology & applications Glyco nanotechnology & applications...

BIOMEDICAL NANOTECHNOLOGY LECTURE 7: PROTEIN & GLYCO NANOTECHNOLOGY Dr.P.GOPINATH DEPARTMENT OF BIOTECHNOLOGY 1  Contents Protein nanotechnology & applications Glyco nanotechnology & applications 2 Protein Nanotechnology 3  Peptide lego Molecular-designed ‘peptide Lego’, at the nanometer scale, resembles the Lego bricks that have both pegs and holes in a precisely determined organization that can be programmed to assemble in well-formed structures. This class of ‘peptide Lego’ can spontaneously assemble into well-formed nanostructures at the molecular level The first member of the peptide lego was discovered from a segment in a left-handed Z-DNA binding protein in yeast and named Zuotin (Zuo means left in Chinese, tin means protein in biology). 4  Peptide lego These peptides form beta-sheet structures in aqueous solution thus they form two distinct surfaces, one hydrophilic, the other hydrophobic, like the pegs and holes in Lego In aqueous solution, the hydrophobic sides shield themselves from water, thus facilitating the peptide to undergo intermolecular self-assembly, similar to what is seen in the case of intramolecular protein folding. The unique structural feature of these ‘‘peptide Lego’’ systems is that they form complementary ionic bonds with regular repeats on the hydrophilic surface. 5 Self-assembly Zhang, S. Fabrication of novel biomaterials through molecular self-assembly. Nature Biotechnol. 2003. 6  Self-assembly Peptide of 16 AA Alternating polar/nonpolar Form stable β-strands and β-sheets Form nanofibers by hydrophobicity Matrices with high H2O content Zhang, S. Fabrication of novel biomaterials through molecular self-assembly. Nature Biotechnol. 2003. 7  Self-assembly Smaller fibers and pore sizes Can include functional motifs Peptide scaffolds are ideal materials for 3-D cell culture The peptide Lego molecules readily undergo self-assembly in aqueous solutions to form well-ordered nanofibers that further associate to form nanofiber scaffolds with well-ordered nanopores averaging 5–200 nm. One of them, RADA16-I, has been widely used and commercialized; it is now called PuraMatrix because of its purity as a molecular designer biological scaffold in contrast to other biologically derived scaffolds from animal collagens and Matrigel which contain unspecified components in addition to known materials. 9 Peptide scaffolds are ideal materials for 3-D cell culture Since these nanofiber scaffolds contain 5–200 nm pores with extremely high water content (99.5% or 5 mg/ml w/v), they were used for the preparation of three- dimensional (3-D) cell culture. These scaffolds closely mimic the porosity and gross structure of extracellular matrices, not only allowing cells to reside and migrate in a 3-D environment, but also allowing molecules, such as growth factors and nutrients, to diffuse in and out very slowly; therefore, these peptide scaffolds are ideal materials for 3-D cell culture, controlled cell differentiation, regenerative medicine and slow drug release applications. 10 Peptide scaffolds are ideal materials for 3-D cell culture Molecular models of several self-assembling peptides, RAD16-I, RAD16-II, EAK16-I and EAK16-II. Each molecule is 5 nm in length with 8 alanines on one side and 4 negatively and 4 positively charged amino acids in an alternating arrangement on the other side. B) SEM image of EAK16-II nanofiber scaffold. Note the nanopores 5–200 nanometers in diameter, the right pore size for biomolecular diffusion. C) AFM image of RADA16-I nanofiber scaffold (also called PuraMatrix). The nanoscale is in sharp contrast to the microfibers of traditional polymer scaffolds, where the fiber diameter is 10–50 microns and the pores range from 10–200 microns. 11  Lipid-like self-assembling peptides Lipid-like self assembling peptides with hydrophobic tails and hydrophilic heads that all undergo self-assembly in water. These peptides have tunable hydrophobic tails with various degrees of hydrophobicity, a hydrophilic head, and either negatively charged aspartic and glutamic acids or positively charged lysine, histidine or arginine. The individual peptides contain 7 to 8 amino acid residues with a hydrophilic head composed of aspartic acid and a tail of hydrophobic amino acids such as alanine, valine or leucine. The length of each peptide is ~2.5 nm, similar to that of natural phospholipids. 12  Lipid-like self-assembling peptides However, the peptide length can also be fine-tuned by adding or removing amino acids one at a time to a desired length. Although individually these lipid-like peptides have completely different composition and sequences, they share a common feature: the hydrophilic heads have 1–2 charged amino acids and the hydrophobic tails have four or more consecutive hydrophobic amino acids. 13 Lipid-like self-assembling peptides A6D (AAAAAAD), V6D (VVVVVVD) peptides have six hydrophobic alanine or valine residues from the N-terminus followed by a negatively charged aspartic acid residue. In contrast, K2V6 (KKVVVVVV) has two positively charged lysines as the hydrophilic head, followed by six valines as the hydrophobic tail. 14  Lipid-like self-assembling peptides Furthermore, we can mimic phospholipid even more closely using phosphoserine as the hydrophilic heads and alanine or valine as hydrophobic tails, pSAAAAAA (pSA6), pSVVVVVV (pSV6). They also exhibited similar self-assembly behaviors to phospholipids, forming well-ordered nanostructures. Lipid-like self-assembling peptides undergo self-assembly in water to form nanotubes and nanovesicles with an average diameter of 30–50 nm 15  Peptide nanotubes Self-assembly: Surfactant-type peptide Form nanotubes and nanovesicles Form interconnected network Similar to carbon nanotube behavior 16  Peptide nanotubes Quick-freeze sample preparation where the sample was instantly flash-frozen at - 190 C produced a 3-D structure with minimal structural disturbance. It revealed a network of open-ended nanotubes observed under transmission electron microscopy. Likewise, A6K cationic peptides also exhibited similar nanotube structures with the opening ends clearly visible. 17 Molecular models of lipid-like peptide nanostructures How could these simple lipid-like peptides form such well structured nanotubes and nanovesicles? It is of great interest that these simple lipid-like peptides readily produce remarkable complex and dynamic structures. If we can fully understand the correlation of their chemical properties and self- assembling behaviors, we will then be able to gain freedom to build materials from the bottom up. These monomer, lipid like peptides were used for molecular modeling. 18  Self-assembly Surface nanocoating peptide (Peptide ink) Three regions: Ligand Anchor Linker Zhang, S. Fabrication of novel biomaterials through molecular self-assembly. Nature Biotechnol. 2003. 19  Peptide ink Peptide or protein inks can be directly printed on surfaces to allow adhesion molecules to interact with cells and adhere to the surface These peptides have three general regions along their lengths: (i) a ligand for specific cell recognition and attachment; (ii) a linker for physical separation from the surface; and (iii) an anchor for covalent attachment to the surface. The ligands might be of the RGD (arginine–glycine–aspartic acid) motif that is known to promote cell adhesion, or other sequences for specific molecular recognition, or specific cell interactions. The linker is usually a string of hydrophobic amino acids such as alanine or valine. The anchor can be a cysteine residue for gold surfaces, asparatic acid linking on amine surfaces, or lysine linking on carboxylic surfaces. 20 Peptide ink Using proteins or peptides as ink we can directly microprint specific features onto the non-adhesive surface of polyethylene glycol to write any arbitrary patterns rapidly without preparing the mask or stamps The process is similar to using an ink pen for writing – here, the printing device is the pen and the biological substances are the inks. This simple and rapid printing technology allowed us to design arbitrary patterns to address questions in neuro-biology that would not have been possible before. 21 Lipid-like peptides stabilize diverse membrane proteins These designer lipid like peptides may now open a new avenue to overcome one of the biggest challenges in structural biology: to obtain high-resolution structures of membrane proteins. Study of membrane proteins will not only enrich and deepen our knowledge of how cells communicate with their surroundings (the response of all living systems to their environments), but also these membrane proteins can be used to fabricate the most advanced molecular devices, such as energy harnessing devices, extremely sensitive sensors, medical detection devices, and other applications we can’t now even imagine. The lipid-like peptides work similarly as other chemical surfactants that encapsulate and protect membrane proteins from undesirable self-aggregation 22  Lipid-like peptides stabilize diverse membrane proteins Chem. Soc. Rev., 2006,35, 1105-1110 23 Protein 'passport' helps nanoparticles get past immune system https://phys.org/news/2013-02-protein-passport-nanoparticles-immune.html 24  Peptides and protein based sensors Peptides and proteins are playing more active roles as useful building blocks in the design of sensors. One of the best-known examples of such a device is the utilization of protein nanopores as nanosensors. In nanopore-based sensors, ionic current blockade occurs as a single molecule is translocated though the channel protein, and this ionic current blockade contains information about the identity, concentration, structure and dynamics of the target molecule.  Protein nanopore based sensor Chem. Soc. Rev., 2010, 39, 3499–3509 Biological protein alpha-hemolysin present a 1 nm diameter pore that makes them ideal candidates for the detection of single- stranded DNA. ss-DNA could be detected label-free in a sequence-specific manner via translocation in nanopores, and this approach shows great promise for the development of new Protein nanopore sensors detect a current economical DNA sequencing devices decay as single stranded DNA molecules are translocated trough the pore. 26 Biomimetic assembly -- multiple nanowire In this biomimetic assembly methodology, multiple nanowires, incorporating various antibodies, were selectively immobilized on different locations on electric substrates where areas were labelled by complementary proteins. When the concentrations of proteins on the substrate were optimized, complex electric device geometries could be fabricated with high yields. 27  Biomimetic assembly -- multiple nanowire Scheme to assemble two different antibody nanotubes, anti-mouse IgG-coated nanotube and anti-human IgG-coated nanotube, into the cross-bar geometry by biomolecular recognition (left). AFM image of the two antibody nanotubes assembled in the cross-bar geometry (right).Scale bar = 200 nm. Chem. Soc. Rev., 2010, 39, 3499–3509 28 Peptide and protein based stimulus responsive materials Stimulus responsive nanomaterials are defined as solid structures that undergo the change with external stimulus such as pH, ionic strength, temperature, electric/magnetic fields, and photon. To trigger this function by peptides, the use of their conformation change is the most popular approach. The basic idea for this approach is that peptides incorporated in nanomaterials assemblies or matrices alter the spacing or the volume by swelling or shrinking induced by the conformation change. For this reason, some people called this peptide as a peptide actuator. 29  Peptide and protein based stimulus responsive materials Schematic representation of ELP fusion protein actuators: (a) When apoCaM binds Ca2+ the attached elastin-like protein (ELP) are assembled into meso– microscale particles (b) Chelation of the bound Ca2+ by EDTA reverses the transition to the apoCaM–ELP monomers. Legend: CaM, cyan; ELPs, orange; Ca2+ , gray. Chem. Soc. Rev., 2010, 39, 3499–3509 30 Glyco-Nanotechnology 31 The glyconanoparticle concept and its potential applications 32 Applications of glyconanoparticles Biochimica et Biophysica Acta 1760 (2006) 636–651 Carbohydrates join DNA, proteins and peptides as biological partner of inorganic nanoparticles. 34 Selective binding of Man-AuNPs to the wild-type E. coli strain ORN178 The mutant ORN208 showed no binding 35  Selective binding of Man-AuNPs to the wild-type E. coli strain ORN178 Dispersed gold Agglomerated gold nanoparticles nanoparticles Schematic representation of the reversible aggregation–dispersion behaviour of lactose gold nanoparticles by sequential addition of RCA120 lectin and galactose with actual concomitant change in colour from pinkish-red to purple to pinkish-red. Biochimica et Biophysica Acta 1760 (2006) 636–651 36  Glyco QDs Glyco-QDs could be applied in cell biology as a routine tool to analyze carbohydrate receptors Biochimica et Biophysica Acta 1760 (2006) 636–651 37  Confocal microscope imaging for staining of sperm with glyco-QDs Acetylglucosamine-encapsulated QDs were concentrated at the sperm heads, while mannose- encapsulated QDs tended to spread over the whole sperm body, due to the different distribution of the GlcNAc and Man receptors on the sperm surface. (A) selective β-N-acetylglucosamine-encapsulated quantum dots labeling on the heads of sea-urchin sperm (B) close-up of β-N-acetylglucosamine-encapsulated quantum dots-labeled sea-urchin sperm, and (C) close-up of mannose encapsulated quantum dots-labeled mouse sperm. Biochimica et Biophysica Acta 1760 (2006) 636–651 38 Glyconanoparticles for studying carbohydrate–carbohydrate interactions. Isothermal titration calorimetry (ITC) is a physical technique used to determine the thermodynamic parameters of interactions in solution Biochimica et Biophysica Acta 1760 (2006) 636–651 The analytical scheme of the Lin's technique for the specific capture of target proteins and the rapid mapping of binding-epitope-containing peptides. 40  Step 1 Nanoparticle encapsulation & Protein mixture addition of protein mixture Carbohydrate ligand Gold nanoparticle 41  Step 2 Protein targeting and digestion Proteolytic enzymes 42  Step 3 Protein digestion Free peptide fragments Bound fragments 43  Step 4 Centrifugation and detection MS Spectra Supernatant Pellet MALDI-TOF-TOF-MS 44 Glycodendrimer inhibition strategy to block bacterial adhesion to host tissues Instead of the usual low affinity of monovalent carbohydrate ligands toward bacterial lectins, multivalent ligand presentation on dendritic scaffolds leads to more potent candidates. Brazilian Journal of Pharmaceutical Sciences,vol. 49, special issue, 2013 45 Possible action mechanism of lactose glyconanoparticles in anti-adhesive therapy Biochimica et Biophysica Acta 1760 (2006) 636–651 46  Summary Protein nanotechnology – Peptide lego – Peptide scaffold – Lipid like peptides – Peptide nanotubes – Peptide ink – Other applications of protein nanotechnology Glyco nanotechnology – Bioimaging – Sensing – Carbohydrate & protein interactions – Anti-adhesive therapy 47