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From carbon nanotechnology to bionanotechnology: Protein and peptide nanofibrils and nanowires
Carbon Nanotechnology Edited by Liming Dai 2006 Published by Elsevier B.V.
Chapter 20 From carbon nanotechnology to bionanotechnology: Protein and peptide nanofibrils and nanowires Oleg I. Kiselev Institute of Influenza Russian Academy of Medical Sciences, Department of Molecular Virology and Genetic Engineering, Russia, Saint Petersburg 1. BIONANOTECHNOLOGY As can be seen from preceding chapters, nanotechnology is no longer a merely social talking point and is beginning to affect the lives of everyone. With the revolutionary discoveries of the C molecule and carbon nanotubes, carbon nanotechnology has become the building block of the entire field of nanotechnology. Whether it is deoxyribonucleic acid (DNA) or synthetic polymers, all are based on carbon backbones. Nature has been doing remarkably well by systematically organizing of matter at nanometer-length scale in biological systems. For example, the well-known proteins collagen and insulin form nanofibrils and the Sup35 yeast protein forms nanowires. Either by using these naturally occurring proteins or creating synthetic derivatives of these molecules, nanotools and nanoelements can be fabricated for molecular recognition, electronic transmission, or for disease diagnosis and treatment. Progress in understanding the molecular basis of human diseases has prompted a new wave of studies in bionanotechnology. Bionanotechnology is a field of science and technology that has resulted from advances in the disciplines of biochemistry, molecular biology, cytology, genetic engineering, and biotechnology. Bionanotechnology takes advantage of the unique properties of biological macromolecules such as peptides, proteins, and prions by using their self-assembling nature for the nano-engineering of molecular templates and supra-molecular structures [1]. The self-assembling of biological macromolecules including nucleic acids such as DNA is based on the recognition of complementary regions. Through complementary binding, these macromolecules naturally link and construct the body*s tissues and organs through fiber-hke structures. Many proteins are amphiphilic and maintain their structure and conformation in both hydrophilic and hydrophobic environments, which makes them attractive candidates for attachment to various materials for biocompatibility. In general, the advantages to use proteins and peptides as building blocks in bionanotechnology include, but not limit to: (1) Proteins are produced in large quantities for many purposes in mild and non-consumptive conditions; (2) Proteins and peptides are more stable and resistant to temperature, irradiation, enzymes etc.; (3) Proteins and
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peptides have many conformations and conformational transitions, which is important for the construction of molecular memory tools based on molecular plasticity; (4) Proteins and peptides undergo folding and many intermediate states; (5) Selfassembling is an intrinsic property of many proteins and peptides. Some of them are capable of forming nanofibrils and nanotubes; (6) Proteins and peptides can be galvanized by metals such as Ag, Au, Zn, Cd, Se, Cu etc.; (7) Polypeptides may have recognition patterns and specific domains for ligand binding; (8) Proteins and peptides can be used for the design of nanowires and manufacturing in very mild conditions; (9) Natural and synthetic peptides are biocompatible for use in the design of retina, neurons, wires, etc.; (10) Using peptide and protein genetic code as an operating system for other types of memory and information operation; (11) Protein-based devices may be able to replace human structures such as the eye lens and retina, skin, bones etc.; and (12) Proteins are capable of forming tight complexes with many other molecules and artificial nanomaterials. Areas of bionanotechnology where applications are beginning to be developed include the assembling of the eye's lens, retina, nerves, as well as chips for the treatment of brain disorders. Devices and approaches that are covered in this chapter include the creation of nanoscale components such as protein nanotubes, nanowires, and nanotools. Synthetic peptides and proteins are discussed as leading materials in bionanotechnology as the nearest materials to be incorporated into practical and commercial applications in medicine. 2. SIMILARITIES BETWEEN FULLERENE C AND VIRUSES Viruses are autonomous genetic machines that reproduce inside cell, and subsequently kill the cells that they infect. Because viruses are very small, difficult to detect, and highly capable of infecting a large number of people or animals, there is a threat for use of viruses as biological warfare agents. Interestingly, C^Q fuUerenes and many viruses share similar morphologies as shown in Fig. 1. Fig. 2 shows how the structure of proteins exists on many levels. For example, different types of Hemagglutinin (HA) exist, each binding to a slightly different type of host molecule. HA is like a key that allows the virus to enter those cells
(a)
(b)
(c)
(d)
Figure 1. Structure of (a) C fullerene, (b) Herpes virus, (c) model of polio virus, and (d) microscopic images of herpes virus.
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Qua ary = active form of protein Can be made of multiple polypeptides
Figure 2. Formation of secondary, tertiary, and quartemary structures by biomolecules.
with a matching lock. Bird flu HA binds to molecules found on birds' intestinal cells. Human flu HA binds to slightly different molecules on human lung cells. Birds are natural hosts to all flue strains while humans are only infected by strains that have adapted to recognize epithelium cell in upper inhalation tract and lungs. Recently humans have been infected by the bird flu H5N1. The HA associated with the virus makes it particularly lethal because people have no natural immunity to it. That is, their antibodies do not recognize this new HA antigen [2]. The protein structure of a flu virus, HA tertiary structure and its physical map are shown in Figs. 3 and 4. Many stages of viral replication are dependent on translocation across the plasma membrane. C fuUerenes are also capable of penetrating the lipid bilayer of the plasma membrane as shown in Fig. 5. In addition, fuUerenes can be used as free radical generators under light irradiation where they initially select and target proteins and membranes. Viral proteins containing the amino acids Cys, Tyr, and Met. are damaged by free radical oxidation, which leads to crosslinking, catalytic inactivation, blocking of conformational transitions, and fusion with cellular membranes. Therefore, many of the similarities between fuUerene C and viruses can be used in biology and medicine in both damaging and beneficial ways.
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®
Figure 3. The HA molecule with the sites used to bind with the host cell membrane indicated by the number one.
HAi (1-328)
HAg (1-221}
Fusion peptide a
HA
m
•s-s1 14
328
1
23 38
137
I I 18S 221
Figure 4. Influenza HA physical map.
3. FULLERENE-BINDING PEPTIDE DOMAIN Fullerenes and carbon natiotubes are the two major constituent members of the carbon family, but the two carbon materials are very different in their properties. The chemistry of fullerene molecules has provided many derivatives for a variety of purposes during the past 5 years. Now, the chemical modification of carbon nanotubes is a growing area in research and technology development. Further developments in nanotechnology will likely rely upon the manufacturing of materials that can have closer interface with biological materials. For example, fullerenes are capable of interacting with proteins [3] and computer modeling of fullerene-peptide complexes showed that the fuUerene molecule specifically interacts with the domain containing duplicated phenylalanine residues [4,5]. Unexpected data were obtained in studies of fullerene complexes with insulin fibers. In Fig. 6, the complexes have fullerene molecules clustered inside bundles of protein fibrils. These bundles
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| | = p h ,[ospholipid
airiino acid chain carbohj'-drate unit cytoplasmic face
Figure 5. Fullerenes in PVP complexes are capable of penetrating into the lipid bilayer of a cell's plasma membrane.
Figure 6. (a) Computer modeling of peptide (Lys-Leu-Val-Phe-Phe), specifically recognize single fullerene molecule and (b) interaction of insulin fibrils with fullerene clusters.
include nucleation sequences of the insulin B-subunits such as the C-terminal domain, containing duplicated phenylalanine residues (Fig. 6b). If carbon nanotubes can be functionalized to bind or recognize similar domains, a multitude of new biocompatible complexes could be formed with great potential applications.
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4. BIOLOGICAL MACROMOLECULES: PROTEINS, PEPTIDES, AND PRIONS Carbon, hydrogen, nitrogen, and oxygen, along with sulfur, phosphorus, iron, or iodine, are basic elements for generating biological macromolecules. When these elements are combined into basic amino groups and one or more acidic carboxyl groups, they can form an amino acid, which is the building block of peptides and proteins. Peptides are compounds that are composed of two or more amino acids joined by peptide bonds. Proteins are polypeptides composed of large groups of amino acids that form the human body's hormones, enzymes, antibodies, tissues, and organs. Some symptoms of defects or deficiencies in proteins are: abnormal tissue development, poor resistance to infection, and delayed healing. Proteins in the body are capable of recognizing millions of molecules, which can lead to beneficial interactions such as triggering an immune response to inactivate and destroy viruses. However, not all natural biological macromolecules are beneficial. Proteinaceous particles called prions are believed to be responsible for transmissible neurodegenerative diseases. They can cause damage to the body because they go undetected as foreign material by the immune system due to their lack of nucleic acid, which is the material responsible for genetic encoding. 5. NATURAL NANOSTRUCTURES: NANOFIBRILS AND NANOWIRES 5.1. Collagen Collagen is a rather heterogeneous protein secreted by cells into the extracellular matrix [6]. It is found throughout the human body where it forms tissues such as bones, tendons, and skin. Its conformation allows it to make long, thin, and mechanically resistant fibrils that become organized as a microfilm as shown in Fig. 7. This structure results in complicated visco-elastic mechanical behavior [6,7].
Figure 7. Transmission electron micrograph of collagen fibrils where the diameter of the collagen fibrils is 10-50 nm.
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Collagen is also a target of many post-translational modifications including ionfilling and cross-linking because it is enriched with proline and glycine amino acid residues in repeated motifs as shown in Fig. 8. These post-translational modifications result in triple-helix fibrils that are 50-200 nm in length as shown in Fig. 9. Collagen is widely used in medicine and cosmetics, but it has not been used in molecular and nano design at this point. However, understanding the structure of this protein may provide information for modeling other biomolecules with similar structures and properties. 5.2. Prions and prion-like proteins Many proteins are able to form fibrils in a wide range of physiological and pathological conditions [7-12]. Some of these proteins have native structures, but have lost their stability in a specific environment whereas others are mutants. All of these proteins are unified in groups of prion-like proteins, which are characterized by their ability to form protein fibrils [7,8]. Prions are infectious proteins having the intrinsic capability to form nanofibrils during their life cycle and biogenesis in infected cells. Fig. 10a shows an example of image of the fibrils that prions form via transmission electron microscopy (TEM) and Fig. 10b shows prion peptides from an amyloidogenic region grown on a solid surface. Prions consist of polypeptide chains that are 254 amino acids in length. The mapping of these molecules has led to the identification of rather short amyloidogenic sequences involved in conformation transformation such as: 06-KTNMKHMAGAAAAGAWGGLG-126
V^^^ NH
• - ^ ;/ /
CH2 CH2
^^"^•gfcv'
•OH
b
Pm X
0=1 " i ^
'1
N-H-
i=0
Pro Y
'^
).i.'OH
W
Gly
°i Figure 8. Primary structures of collagen fragments and inter chain hydrogen bounds. Hydrogen bonding in triple-helical collagen, (a) Ladder of intrastrand ProC^OHGly hydrogen bonds and (b) Electrostatic field of tripeptide Gly-Pro-Hyp segment of collagen molecule.
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Pathway Following exocytosis:
Synthesis in ER
- P r o p e p t i d e s cleaved off b y extracellular e n z y m e s - Triple-helix molecules p o l y m e r i z e into fibrils 50-200 n m long
Association of C-propeptides; tripi©-helix nucieatlon {Gly-X.Hyp)„ TripIe-hellx propagation
- Fibrils pack into fibers (stronger t h a n steel of s a m e size) VVTien d e n a t u r e d , forms gelatin (missing propeptides lead to unordered cross-linking)
ageo N- and Cpropeptides Assembly and cross-lir^ing mm of fibrilg
Figure 9. Biosynthesis and assembling of collagen fibrils.
Figure 10. Structural organization of prionfibrils,(a) TEM of prionfibrilsand (b) Selfassembling of prion peptides (from amyloidogenic region) on a solid surface.
During the multiplication of prions, the formation of fibrils in infected brain tissue causes the destruction of the brain's neuronal network. This results in the disruption of proper function as expressed by progressive psychoneurotic disease, which can be fatal [7,8]. In addition, Cu^^-binding domains were identified in the N-terminal
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portions of prion proteins. However, the physiological role of Cu^^ binding by prion proteins is not clearly understood. The affinity of fibrils to ions of various metals could contribute to the development of conductive-like elements inside the brain leading to severe disturbances in the local brain tissue conductivity. The general neuropathological sign of Alzheimer's disease is low and chaotic electrical activity of a brain in electroencephalography (EEG) observation. Thus, in situ mineralized prion fibrils may have local conductivity that disrupts normal signaling pathways in the brain. This hypothesis led to the testing of protein fibrils for the assembly of biocompatible protein-containing nanowires [8,11]. 5.3. Aj3-peptides Small Aj3-peptides have lengths from 40 to 42 amino acids and are proteolytic fragments of the huge membrane-bound amyloid precursor protein (APP) shown in Fig. 11. A nucleation domain was identified as the primary attachment of the peptides to each other and the initiation of fibril biogenesis. Short A-peptide homologous peptide fragments specifically bind to Aj3-42 and prevent fibril assembling. This is the most important evidence that the short sequence of the A/3-42 is involved in fibril assembling [8-10]. Secretase (a, P, 7) are a set of proteases, which cleaves Aj3-40-42 peptide in different positions. When Ali-40-42 peptide escapes cleavage, it is capable of forming tight fibril aggregates that are found as deposits inside destroyed brain tissue. In water, the A/?-peptide has a complicated conformation including a-heUces and j3-structures, which is modeled in Fig. 12. The assembly of the native peptide into fibrils leads to a complete transformation of the peptide from an a-helical to an entirely j3-stranded structure. Usually j3-sheet peptides interact with each other along their entire length in order to form very tight structures [9,11]. Further investigation into the structure of these peptide fibrils showed that there is a special arrangement of peptides inside the fibrils.
p-amytald precursor protein (pAPP) ^amytold (Ap)
Secretase: Cteavagesite: N
p Apt
p Apii
a Api7
y 7 Ap40 A^42
bAEFGHDSGKBWHQKLVFFAEDVGSKKGAIIGLMVGGviv'lU
-
Lipid bi layer
Schematic diagram of the p-amyloid precursor protein (pAPP)
Figure 11. Primary sequence and proteolytic sites of Aj3-40-42 peptide, partially integrated into lipid membrane. Secretase a, p and y cleavage sites are pointed along polypeptide sequence. N, NH2-terminus; C, COOH-terminus [14,15].
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c Alzhciincfs Disease
Figure 12. Computer model of Aj8-40 peptide. The peptide consist of extracellular and transmembrane parts, that is shown here. The sequence of A^40 peptide is deciphered in Fig. 11.
Figure 13. Structure and modeling of A^-peptide nanotube: (a) TEM of single Afipeptide fibril and (b) computer model of A)3-peptide nanotube.
According to X-ray analysis, peptides form water-filled nanotubes that are assembled by peptides like stick pieces resulting from interactions between short clusters of amino acid residues as shown by a model in Fig. 13b. According to a generic statistical mechanical model [12-14], the self-assembling of the peptide starts from chiral rod-like units into helical tapes, then under increasing concentration it forms into twisted ribbons or double tapes. Fibrils, as an additional step, represent
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twisted stacks of ribbons, which lead to the final stage in fibrillogenesis where fibers are produced. According to this model, the sequential transformation of peptides provides many opportunities for chemical modification, as can be seen from the morphology of A^-peptide deposits in damaged tissue [8,12,14-16]. It is hoped that understanding the further modifications that are made to peptides or proteins by metal ions may elucidate the mechanisms involved in making conductive fibers [8,12-16]. 5.4. Insulin Insulin is a protein hormone widely used in the treatment of diabetes [14]. It consists of two subunits: A and B, associated by Cys-Cys binding. It can exist in several different molecular forms such monomers, dimers, and hexamers. Zn^^ is necessary for assembling of the hexamer storage form of insulin [4,5]. The insulin dimer is primarily stabilized by ^-sheet domains located in subunit B as shown in Fig. 14. Denaturing and assembling fibrils on insulin molecules were dependent upon an acidic pH, hydration, and Zn^+ ions. As a result, a high yield of insulin nanofibers was achieved as shown in Fig. 15. In solution at neutral pH and at physiological concentrations (about 1 ng/ml), insulin exists as a monomer and is in the active form of the hormone. At higher concentrations, at acid or neutral pH, and in the absence of zinc, the insulin monomer self-associates to form dimers. In the presence of zinc, insulin forms hexamers. Studies of insulin analogues demonstrate that its activity is dependent upon the integrity of the insulin fold. Insulin domains involved in Zn-dependent hexamer and Zn-independent dimer formation have different locations. The central
A20:B19
A7:B7
Figure 14. Computer model of insulin dimer. Arrows: p strands are shown between two monomers as an interface region.
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Figure 15. TEM of insulin fibers, (a) Amorphous aggregates of insulin from acetonitrile solution, (b) Insulin fibers formed in water buffer, contained 1 mM ZnSO,. residues responsible for hexamer formation include BIO His, which binds Zn ions as well as B14 Ala, B17 Leu, B20 Gly, and A13 Leu, which are involved in close-packed hydrophobic interactions. The ability to form dimers is mediated by hydrophobic interactions involving B8 Gly B9 Ser, B12 Val, B13 Glu, B16 Tyr, B24 Phe, B25 Phe, B26 TVr, B27 Thr, and B28 Pro. The B chain of one monomer packs against the B chain of the second monomer and further stability is provided by hydrogen bonding between the anti-parallel ^-strands B24-B26 of each molecule. The packing arrangement in the dimer results in perturbations of the side-chain and main-chain structure slightly changing the resulting monomer conformation. The B25 Phe side-chain is important for stabilizing the native dimer structure. However, decreasing the pH and increasing the concentration of the protein fatally lead to insulin aggregation and fibril formation, which have been described in many studies [4]. 5.5. Sup35 yeast protein Many studies have been performed with phospholipids, tubules, DNA, bacteriophages, and cellular microtubules [1,9]. However, individual proteins are the best materials for designing conductive macromolecular units and wires due to their availabiUty, consistency, and self-assembling nature. Proteins with similar behavior, structure, and the capability to form intracellular fibrils were identified in brewer yeast by the yeast geneticist S. Lindquist. During the past decade, she discovered and studied a unique protein called Sup35. This protein is similar both in structure and function to prion-like proteins, so it has become a quite simple, safe (noninfectious), and available model for basic studies in this field. Fragments of yeast Saccharomyces cerevisiae protein Sup35 were selected for use in a conducting nanowire design [9,12]. Recombinant Sup35 protein contains N-terminal and middle region (NM) of native Sup35p. NM forms self-assembling
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j3-sheet-rich amyloid fibers that have a suitable size and shape for nanocircuitry with diameters of 9-11 nm. In water, NM is a soluble protein and with a flexible, unordered structure. Under the appropriate conditions, NM rapidly converts to insoluble amyloid fibers. This process is primed by the addition of preformed fibrils. The fibers grow into two directions and form varied fiber patterns. These fibers are also resistant to proteases and denaturing conditions and reagents, which makes them suitable for the fabrication and processing of electrical wires. The kinetics of fiber formation by NM is slow and it does not make aggregates as other amyloids and prion-like proteins. Moreover, NM fibers form under physiological conditions such as a neutral pH and at room temperature. The secondary structure of NM was determined by UV-CD and showed that in solution NM has a random coil-rich structure and a sheet-like appearance when it is in fibers. This is important because the industrial fabrication process of nanoscale devices requires mechanically stable and chemically resistant materials. NM fibers can be incubated in phosphate buffer with different ionic strengths, at temperatures from 25 °C to 98 °C, and in denaturizing agents such as 8M urea and 2M guanidinium hydrochloride. NM fibers were completely stable in the absence of salt and in 2.5 M NaCl. Moreover, NM fibers can be used after storing frozen for 3 months at -20 °C and -80 °C. Chemical stability in alkaline, acidic, and organic solvents was also demonstrated. NM fibers with variable lengths can be repeatedly produced and the average size of the fibers was approximately 500 nm. Increasing the concentration of the soluble NM increased the fiber length. NM fibers conductive properties are tailorable. They can be both good insulators or using multi-step processing, NM fibers can be converted to conducting nanowires with low Ohmic resistance. For conductivity purposes, specially engineered NM was used. By site-specific mutagenesis, the Cys derivative of NM was obtained. Moreover, this Cys residue in engineered NM remains accessible after fiber formation in the derivative NM K184C. This derivative, NM K184C, was used as the starting recombinant NM. Using monomaleimido nanogold nanoprobes, gold nanoparticles were covalently attached to the fibers. After this modification, NM K184C displayed the same morphology as the original protein. The distance between cysteine residues in the fibers was 3-5 nm where the nanogold particle diameters were only 1.4 nm. In order to bridge the particles with metal to gain conductivity, GoldEnhancer was used. This process was inefficient because some gaps remain unfilled. A special gold toning procedure was developed to create a final conductive nanowire. Nanogold particles on the labeled fibers elicited a reducing activity when reacted with silver ions. The silver-modified fibers in Fig. 16 become thicker and have diameters from 80-200 nm compared to bare fibers with diameters from 9-11 nm. The electrical behavior of NM-template metallic fibers was assessed by randomly depositing fibers. Nanogold particles on patterned electrodes were then covalently attached. Then, the gold toning procedure allowed the formation of metallically continuous gold nanowires. The 100 nm nanowires had a very low resistance of i?=86 Q. This is the first successful design of proteinaceous nanowires of natural origin with modifications inserted by genetic engineering.
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Figure 16. Metallic fiber of yeast prion-like protein. Bare and toner-labeled fibers: diameters 9-11 and 80-200 nm, respectively.
Short domains in Sup35 were also studied for forming fibers with assigned properties such as in the creation of peptide nanotubes as shown in Fig. 17. There are many successful examples of experimentally designing nanomaterials using short peptides with specific functions [3,9,17-20]. However, many studies in peptide technology for the creation of nanotools have shifted from the applications of natural polypeptides to the applications of specially designed synthetic polypeptides. A synthetic peptide is completely homogenous in its structure and could be modified or prepared for modification during its synthesis. The size of these peptide fibers provides the possibility of creating very small components for nanoelectronics. For example, the short peptide sequence GNNQQNY is capable of forming highly ordered fibers and nanotubes. However, Sup35 yeast protein was the first subject for the design of metal-filling protein nanowires [9,12,17]. The existence of sequence diversity among prion-Uke proteins capable of forming highly ordered fibrils creates an even wider range of starting materials than currently exist in the electronics industry [8,11]. All of the proteins and peptides listed in Table 1 are prion-like proteins that can be used for the creation of protein nanotubes and galvanized nanowires. It is important to note that they have very different primarily sequences, but are similar in secondary structure. Additionally, these prion-like proteins have similar aromatic amino acid residues usually located in nucleation regions where fibril formation is initiated.
From carbon nanotechnology to bionanotechnology
Xmaltered segments: ^GNNQQNY^^ 7GNNQQNYQQ^5 8NNQQNYQQ^5
'GNNOQNY «3
'QNNQQNYQQ'^ 'GNNQONYOE'5 ^NHQONYQO'^,,
715
Altered segments: ^GNNQQNYQR^s i^SQNGNQQRG^s
'^SQNGNQQRG
X-rmyi
Coital Width--280 A
Figure 17. Yeast Sup35 protein physical map and peptides active in fibrillogenesis. (a) Amyloidogenic sequences are located on the map of Sup35p in N-terminus and in the middle part of the protein. Minimal length of peptide capable to form fibrils is heptapeptide: GNNQQNY. (b) Sequences of short peptides trialed as an N-terminal nucleation domain of Sup35p [17]. Table 1 Structure and properties of fibril forming protein sequences [9,10]. Name of natural peptide
Disorder or natural condition
Short fibrillogenic sequence
1. Aj3-amyloid peptide
Alzheimer disease
KLVFFA LVFFA LPFFD KLVFFAE
2. Islet amyloid precursor protein
Type II diabetes
TNVGSNTY
3. Lactadherin
Aortic medial amyloid
NFGSVQFV
4. Gelsolin
Finnish hereditary Amyloidosis
SFNNGDCCFILD
5. Serum amyloid A
Chronic systemic inflammation amyloidosis
SFFSFLGEAFD
6. PrP^^
PHGGGWGQ
Creutzfeld-Jakob disease (CJD)
7. Sup35p yeast protein Yeast prion protein
PQGGYQQYN GNNQQNY
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6. HUMAN PATHOLOGY AND PROTEIN GALVANIZATION 6.1. Metal-filled nanowires Many studies have tried to clarify the role of metal ions in the assembly and catalytic activity of A-peptides and their fibrils. In Fig. 18, a schematic of ion modification is described where Cu^^ and Zn^^ ions are attached to Aj3-peptides. This process was termed protein galvanization [13]. Moreover, data obtained in this study support the idea that amyloid fibril galvanization is principally caused by Zn^^ released during neurotransmission as shown in Fig. 18. During active synaptic transmission, the concentration of Zn^^ ions reaches 300 |LIM. This local concentration is very high and sufficient for peptide galvanization of the localized A/?-peptides. The galvanization of Aj3-peptides leads to the precipitation of the oxidized form of the peptides and rapidly induces amyloid fibril development as shown in Fig. 18. The biogenesis of amyloid fibrils includes free radical generation and oxidation of the protein and its fragments. The main consequence of free radical generation is intra-molecular networks or fiber cross-linking. The cross-linking of generated fibrils leads to the assembly of high molecular mass aggregates and intra-tissue deposits called plaques. A similar process could be reproduced in experimental conditions [8,12,14-16]. In our opinion, Zn^^-saturated peptides become the prototype of a conductive peptide element generated during the development of disease in the human brain [4,9]. Moreover, during aggregation this protein can form bundles and branched bundles of dendrimer-like structures. 6.2. Natural protein galvanization: Zinc-binding motifs Proteins have Zn^ ^-binding sites. However, Zn^+ ions can be replaced by many other ions. In conditions where the polypeptide chain is saturated by those ions, the protein dramatically changes its properties and can then be used in nanoelectronic design. In Fig. 19, the Zn-finger motif of a DNA-binding protein is shown. This Znfinger motif usually contains His and Cys residues in a specific polypeptide loop [8]. Many proteins contain more than 10 Zn-finger motifs per molecule that bind with high affinity. Zinc-dependent galvanization of other proteins may occur in aggregates of peptides that contain out of order Cys or His residues, but that interact through interchain binding. In vitro experiments with a high molar access of Zn^^
Cu/Fe zr^Ap(Cu/fe)
H2O2
+ Zn ^ ^ Plaque
Fibrils
Toxicity
Figure 18. Metabolic galvanization of A)3-peptide [12].
H^O,
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(b) Zinc-finger motif
Figure 19. Structural organization of a DNA-binding domain and Zn-finger motif. Arrows are j8-sheet domain; cylinder is a-helix; Cys, cysteine; His, Histidine. Zn^+ binds usually to specific consensus sequence in many proteins involved in transcriptional regulation.
ions lead to saturation of the protein by tightly bound Zn^^. The same saturation binding was detected for other metals. This property of natural proteins is currently being used for the design of metal-binding short peptides as conducting wires [9]. 7. APPLICATIONS OF BIONANOTECHNOLOGY: DIAGNOSTICS, TREATMENTS, AND FUTURE DIRECTIONS Many infectious agents are rapidly being distributed throughout the world and causing epidemic outbreaks with a high death rate. Diseases such as the avian flu, SARS, Dengue, Ebola are some of the most dangerous infections. Therefore, there is a great demand for special tools that can rapidly and specifically diagnose these threats [2,9]. Many diagnostics, including enzyme-linked immunoassays, are based on an adsorption of antigens or antibodies onto the bottom of plastic plates. Using current technology, antibodies could be localized in an exact place or spot by techniques such as nanolithography. However, the main problems associated with this type of set-up are nonspecific background binding of the proteins and insufficient detection sensitivity. Nanotechnology provides a novel opportunity to construct devices for the specific detection of single viral particles. This high sensitivity is needed because only 5-10 individual HIV viral particles are enough to infect a sexual partner or drug user. Moreover, these infectious agents are rather stable and aggressive and could be transmitted in trace amounts to other hosts. Prions and other infectious amyloidoses could be transmitted on the level of fento molar concentrations. Therefore, diagnostic devices need to be extremely sensitive in order to detect single viral particles or single molecules of infectious agents. Fig. 20 shows a novel approach and sensor for the detection of single viral particles.
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Lasers are widely used in medicine for simple skin cosmetic treatments to more advanced surgical operations on the eye or brain. Specialized semiconductor lasers are used in applications such as telecommimications, informational storage, and the fabrication of integrated electrically driven devices. The problem is to understand how to combine the current silicon technology of microelectronics to other materials such as organic molecules, synthetic and natural polymers, and inorganic nanostructures. Recently, amplified stimulated emission and laser irradiation have been used in optically pumped organic systems and as inorganic nanocrystals or nanowires. For example, single-crystal cadmium sulfide nanowires were used in an electrically driven laser [22]. Small and compact distantly driven lasers are very important in medicine to replace radioactive materials in treatment of tumors and other diseases. The creation of a biocompatible type of single nanowire laser would be beneficial in the treatment of metastasis in hard to access organs of the human body [23]. The recent discovery of the heat absorptive properties of carbon nanotubes for use in laser irradiation opens up new possibilities in the treatment of tumor diseases [22-24]. Irradiation of a carbon nanotube suspension by a near-infrared laser beam leads to the heating of the solution up to 70 °C within 2 min. Electrons in the nanotubes become excited and begin releasing energy in the form of heat. By using an intrinsic property of nanotubes, a weapon that kills cancer can be developed. The main problem with this system is precise targeting. It is well known that tumor cells contain surface clusters of folate receptors and depend on a folic acid substrate for their proliferation. This difference between normal and cancer cells makes possible
Figure 20. Solid phase sensor for detection of single viral particles. Antibodies (cyan, red, green) are covalently attached to nanoelectrode. Viruses (brown) are specifically attached to antibodies and could be detected by signal registration immediately after interaction. This approach increases sensitivity of viral detection until single viral particle identification and can be used simultaneously for detection of hundred viruses [21].
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selective targeting through "labeling" of carbon nanotubes by folate. Folate-labeled nanotubes have been taken up by cancer cells with high efficiency. Then, the treated cancer cells were illuminated with a near-infrared laser, which soon heated and completely killed the cells containing the folate-labeled carbon nanotubes. This type of local heating induces the apoptosis of cancer ceUs, which is better known as hypothermal cancer treatment [22]. Many other tools for targeting can be used in further studies and for clinical applications. Thus, the combination of lasers with the intrinsic properties fullerenes and functionalized nanotubes is a rapidly growing area of research and design for novel technologies in cancer treatment. Bionanotechnology can be defined by its utility for creating devices of enhanced sensitivity or function that are useful in biomedical research, drug discovery, and clinical therapy. The successful study of human conformational pathology has opened up new opportunities in the experimental design of protein fibrils and their application as nanoparts of bionanotechnology devices for use in revolutionary technology as shown in Fig. 21. Biomolecules and their derivatives all have dimensions in the nanometer range, but there is nothing to be gained by re-classifying all of biotechnology as nanotechnology. When biomolecules are combined with manufactured nanoscale items or are significantly re-engineered, they can be considered for use in nanoelectronic tools. Likewise, some protein wires and semiconductor chips
Nano-bio interface
Current CMOS Technology Next Generation CMOS Nanofiber "—"Nanotube Nanowire — F u l l e r e n e s
Nanotube
0.1 nm
"^
Figure 21. Scheme of further development of bionanotechnology.
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now have features with dimensions smaller than 90 nm. Biotechnology and genetic engineering as well as chemical growth of nanoscale could provide the necessary complexity for the basic components of nanotools. The most important question in the further development of this imique field is to find accurate and outstanding wide spread appUcation. In the near future, nanodiagnostic devices could revolutionize the screening and detection of viruses. REFERENCES 1. S. Zhang, Nat. Biotechnol. 21 (2003) 1171-1177. 2. http://www.omedon.co.uk/influenza/influenza/. 3. L. B. Piotrovsky, Fundamental Directions in Molecular Medicine, Eds. O. I. Kiselev and U. P. Golikov, Rostok publishers, Saint-Petersburg, 2005, 197-267. 4. J. Brange, J. WhittinghamDonald, D. Edwards, Y.-S. Zhang, A. WoUmer. D. Brandenburg, G. Dodson and J. Finch, Current Science 72 (1997) 470^76. 5. M. R. H. Krebs, C. A. MacPhee, A. F Miller, I. E. Dunlop and C. M. Dobson, Proc. Natl. Acad. Sci. USA 101 (2004) 14420-14424. 6. K. Misof, G. Rapp and R Fratzl, Biophys. J. 72 (1966) 1367-1381. 7. R. Valuzzi and D. L. Kaplan, Biopolymers 53 (2000) 350-362. 8. S. B. Pruziner, Science 252 (1991) 1515-1522. 9. V. 1. Pokrovskii, O. I. Kiselev and B. L. Cherkasskii, Prions and Prion's Diseases, MedAcadem Publishers, Moscow, 2004, p. 398 (in Russian). 10. E. Gazit, A possible role for ;r-stacking in the self-assembly of amyloid fibrils, FASEB J. 16 (2002) 77-82. 11. R. W. Carrel and D. A. Lomas, Lancet 350 (1997) 94-98. 12. Sheibel, Th., R. Parthasarathy, G. Sawicki, X.-M. Lin, H. Jaeger and S. L. Lindquist, Conducting nanowires built by controlled self-assembly of amyloid fibers and selective metal deposition, Proc. Natl. Acad. Sci. USA 100 (2003) 4527-4532. 13. A. I. Bush and R. E. Tanzi, Proc. Natl. Acad. Sci. 99 (2002) 7317-7319. 14. D.-Sh. Yang, J. McLaurin, K. Qin, D. Westaway and P. E. Eraser, Eur. J. Biochem. 267 (2000) 6692-6698. 15. M. F Perutz, J. T. Finch, J. Berriman and A. Lesk, Proc. Natl. Acad. Sci. USA 99 (2002) 5591-5595. 16. A. Aggeh, I. A. Nyrkova, M. Bell, R. Harding, L. Carrick, T. C. B. McLeich, A. N. Semenov and N. Boden, Proc. Natl. Acad. Sci. USA 98 (2001) 11857-11862. 17. M. Balbimie, R. Grothe and D. Eisenberg, Proc. Natl. Acad. Sci. USA 98 (2001) 2375-2380. 18. C. Valery, F Artzner, B. Robert, T. Gulick, G. Keller, C. Grabielle-Madelmont, M.-L. Torres, R. Cherif-Cheich and M. Patemostre, Biophys. J. 86 (2004) 2484-2501. 19. M. L. De la Paz, K. Goldie, J. Zurdo, E. Lacroix, C. M. Dobson, A. Hoenger and L. Serrano, Proc. Natl. Acad. Sci. US 99 (2002) 16052-16057. 20. 1. A. Banerjee, L. Yu and H. Matsui, Proc. Natl. Acad. Sci. USA 100 (2003) 14678-14682.
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21. N. D. Lazo, M. A. Grant, M. C. Condron, A. C. Rigby and D. B. Teplow, On the nucleation of amyloid j3-protein monomer folding, Protein Science 14 (2005) 1581-1596. 22. E. Smalley, Biochip spots single viruses, Technology Research News (TRN) Oct. 20/27 (2004). http://www.tmmag.com. 23. M. Shwartz, Scientists develop nanotech-laser treatment that kills cancer cells harming healthy tissue, http://www.eurekalert.org.pub _releases. 24. X. Duan, Huang Yu, R. Agarwal and C. M. Lieber, Nature 421 (2003) 241-245.
Chapter 20 From carbon nanotechnology to bionanotechnology: Protein and peptide nanofibrils and nanowires Oleg I. Kiselev Institute of Influenza Russian Academy of Medical Sciences, Department of Molecular Virology and Genetic Engineering, Russia, Saint Petersburg 1. BIONANOTECHNOLOGY As can be seen from preceding chapters, nanotechnology is no longer a merely social talking point and is beginning to affect the lives of everyone. With the revolutionary discoveries of the C molecule and carbon nanotubes, carbon nanotechnology has become the building block of the entire field of nanotechnology. Whether it is deoxyribonucleic acid (DNA) or synthetic polymers, all are based on carbon backbones. Nature has been doing remarkably well by systematically organizing of matter at nanometer-length scale in biological systems. For example, the well-known proteins collagen and insulin form nanofibrils and the Sup35 yeast protein forms nanowires. Either by using these naturally occurring proteins or creating synthetic derivatives of these molecules, nanotools and nanoelements can be fabricated for molecular recognition, electronic transmission, or for disease diagnosis and treatment. Progress in understanding the molecular basis of human diseases has prompted a new wave of studies in bionanotechnology. Bionanotechnology is a field of science and technology that has resulted from advances in the disciplines of biochemistry, molecular biology, cytology, genetic engineering, and biotechnology. Bionanotechnology takes advantage of the unique properties of biological macromolecules such as peptides, proteins, and prions by using their self-assembling nature for the nano-engineering of molecular templates and supra-molecular structures [1]. The self-assembling of biological macromolecules including nucleic acids such as DNA is based on the recognition of complementary regions. Through complementary binding, these macromolecules naturally link and construct the body*s tissues and organs through fiber-hke structures. Many proteins are amphiphilic and maintain their structure and conformation in both hydrophilic and hydrophobic environments, which makes them attractive candidates for attachment to various materials for biocompatibility. In general, the advantages to use proteins and peptides as building blocks in bionanotechnology include, but not limit to: (1) Proteins are produced in large quantities for many purposes in mild and non-consumptive conditions; (2) Proteins and peptides are more stable and resistant to temperature, irradiation, enzymes etc.; (3) Proteins and
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peptides have many conformations and conformational transitions, which is important for the construction of molecular memory tools based on molecular plasticity; (4) Proteins and peptides undergo folding and many intermediate states; (5) Selfassembling is an intrinsic property of many proteins and peptides. Some of them are capable of forming nanofibrils and nanotubes; (6) Proteins and peptides can be galvanized by metals such as Ag, Au, Zn, Cd, Se, Cu etc.; (7) Polypeptides may have recognition patterns and specific domains for ligand binding; (8) Proteins and peptides can be used for the design of nanowires and manufacturing in very mild conditions; (9) Natural and synthetic peptides are biocompatible for use in the design of retina, neurons, wires, etc.; (10) Using peptide and protein genetic code as an operating system for other types of memory and information operation; (11) Protein-based devices may be able to replace human structures such as the eye lens and retina, skin, bones etc.; and (12) Proteins are capable of forming tight complexes with many other molecules and artificial nanomaterials. Areas of bionanotechnology where applications are beginning to be developed include the assembling of the eye's lens, retina, nerves, as well as chips for the treatment of brain disorders. Devices and approaches that are covered in this chapter include the creation of nanoscale components such as protein nanotubes, nanowires, and nanotools. Synthetic peptides and proteins are discussed as leading materials in bionanotechnology as the nearest materials to be incorporated into practical and commercial applications in medicine. 2. SIMILARITIES BETWEEN FULLERENE C AND VIRUSES Viruses are autonomous genetic machines that reproduce inside cell, and subsequently kill the cells that they infect. Because viruses are very small, difficult to detect, and highly capable of infecting a large number of people or animals, there is a threat for use of viruses as biological warfare agents. Interestingly, C^Q fuUerenes and many viruses share similar morphologies as shown in Fig. 1. Fig. 2 shows how the structure of proteins exists on many levels. For example, different types of Hemagglutinin (HA) exist, each binding to a slightly different type of host molecule. HA is like a key that allows the virus to enter those cells
(a)
(b)
(c)
(d)
Figure 1. Structure of (a) C fullerene, (b) Herpes virus, (c) model of polio virus, and (d) microscopic images of herpes virus.
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Qua ary = active form of protein Can be made of multiple polypeptides
Figure 2. Formation of secondary, tertiary, and quartemary structures by biomolecules.
with a matching lock. Bird flu HA binds to molecules found on birds' intestinal cells. Human flu HA binds to slightly different molecules on human lung cells. Birds are natural hosts to all flue strains while humans are only infected by strains that have adapted to recognize epithelium cell in upper inhalation tract and lungs. Recently humans have been infected by the bird flu H5N1. The HA associated with the virus makes it particularly lethal because people have no natural immunity to it. That is, their antibodies do not recognize this new HA antigen [2]. The protein structure of a flu virus, HA tertiary structure and its physical map are shown in Figs. 3 and 4. Many stages of viral replication are dependent on translocation across the plasma membrane. C fuUerenes are also capable of penetrating the lipid bilayer of the plasma membrane as shown in Fig. 5. In addition, fuUerenes can be used as free radical generators under light irradiation where they initially select and target proteins and membranes. Viral proteins containing the amino acids Cys, Tyr, and Met. are damaged by free radical oxidation, which leads to crosslinking, catalytic inactivation, blocking of conformational transitions, and fusion with cellular membranes. Therefore, many of the similarities between fuUerene C and viruses can be used in biology and medicine in both damaging and beneficial ways.
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®
Figure 3. The HA molecule with the sites used to bind with the host cell membrane indicated by the number one.
HAi (1-328)
HAg (1-221}
Fusion peptide a
HA
m
•s-s1 14
328
1
23 38
137
I I 18S 221
Figure 4. Influenza HA physical map.
3. FULLERENE-BINDING PEPTIDE DOMAIN Fullerenes and carbon natiotubes are the two major constituent members of the carbon family, but the two carbon materials are very different in their properties. The chemistry of fullerene molecules has provided many derivatives for a variety of purposes during the past 5 years. Now, the chemical modification of carbon nanotubes is a growing area in research and technology development. Further developments in nanotechnology will likely rely upon the manufacturing of materials that can have closer interface with biological materials. For example, fullerenes are capable of interacting with proteins [3] and computer modeling of fullerene-peptide complexes showed that the fuUerene molecule specifically interacts with the domain containing duplicated phenylalanine residues [4,5]. Unexpected data were obtained in studies of fullerene complexes with insulin fibers. In Fig. 6, the complexes have fullerene molecules clustered inside bundles of protein fibrils. These bundles
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| | = p h ,[ospholipid
airiino acid chain carbohj'-drate unit cytoplasmic face
Figure 5. Fullerenes in PVP complexes are capable of penetrating into the lipid bilayer of a cell's plasma membrane.
Figure 6. (a) Computer modeling of peptide (Lys-Leu-Val-Phe-Phe), specifically recognize single fullerene molecule and (b) interaction of insulin fibrils with fullerene clusters.
include nucleation sequences of the insulin B-subunits such as the C-terminal domain, containing duplicated phenylalanine residues (Fig. 6b). If carbon nanotubes can be functionalized to bind or recognize similar domains, a multitude of new biocompatible complexes could be formed with great potential applications.
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4. BIOLOGICAL MACROMOLECULES: PROTEINS, PEPTIDES, AND PRIONS Carbon, hydrogen, nitrogen, and oxygen, along with sulfur, phosphorus, iron, or iodine, are basic elements for generating biological macromolecules. When these elements are combined into basic amino groups and one or more acidic carboxyl groups, they can form an amino acid, which is the building block of peptides and proteins. Peptides are compounds that are composed of two or more amino acids joined by peptide bonds. Proteins are polypeptides composed of large groups of amino acids that form the human body's hormones, enzymes, antibodies, tissues, and organs. Some symptoms of defects or deficiencies in proteins are: abnormal tissue development, poor resistance to infection, and delayed healing. Proteins in the body are capable of recognizing millions of molecules, which can lead to beneficial interactions such as triggering an immune response to inactivate and destroy viruses. However, not all natural biological macromolecules are beneficial. Proteinaceous particles called prions are believed to be responsible for transmissible neurodegenerative diseases. They can cause damage to the body because they go undetected as foreign material by the immune system due to their lack of nucleic acid, which is the material responsible for genetic encoding. 5. NATURAL NANOSTRUCTURES: NANOFIBRILS AND NANOWIRES 5.1. Collagen Collagen is a rather heterogeneous protein secreted by cells into the extracellular matrix [6]. It is found throughout the human body where it forms tissues such as bones, tendons, and skin. Its conformation allows it to make long, thin, and mechanically resistant fibrils that become organized as a microfilm as shown in Fig. 7. This structure results in complicated visco-elastic mechanical behavior [6,7].
Figure 7. Transmission electron micrograph of collagen fibrils where the diameter of the collagen fibrils is 10-50 nm.
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Collagen is also a target of many post-translational modifications including ionfilling and cross-linking because it is enriched with proline and glycine amino acid residues in repeated motifs as shown in Fig. 8. These post-translational modifications result in triple-helix fibrils that are 50-200 nm in length as shown in Fig. 9. Collagen is widely used in medicine and cosmetics, but it has not been used in molecular and nano design at this point. However, understanding the structure of this protein may provide information for modeling other biomolecules with similar structures and properties. 5.2. Prions and prion-like proteins Many proteins are able to form fibrils in a wide range of physiological and pathological conditions [7-12]. Some of these proteins have native structures, but have lost their stability in a specific environment whereas others are mutants. All of these proteins are unified in groups of prion-like proteins, which are characterized by their ability to form protein fibrils [7,8]. Prions are infectious proteins having the intrinsic capability to form nanofibrils during their life cycle and biogenesis in infected cells. Fig. 10a shows an example of image of the fibrils that prions form via transmission electron microscopy (TEM) and Fig. 10b shows prion peptides from an amyloidogenic region grown on a solid surface. Prions consist of polypeptide chains that are 254 amino acids in length. The mapping of these molecules has led to the identification of rather short amyloidogenic sequences involved in conformation transformation such as: 06-KTNMKHMAGAAAAGAWGGLG-126
V^^^ NH
• - ^ ;/ /
CH2 CH2
^^"^•gfcv'
•OH
b
Pm X
0=1 " i ^
'1
N-H-
i=0
Pro Y
'^
).i.'OH
W
Gly
°i Figure 8. Primary structures of collagen fragments and inter chain hydrogen bounds. Hydrogen bonding in triple-helical collagen, (a) Ladder of intrastrand ProC^OHGly hydrogen bonds and (b) Electrostatic field of tripeptide Gly-Pro-Hyp segment of collagen molecule.
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Pathway Following exocytosis:
Synthesis in ER
- P r o p e p t i d e s cleaved off b y extracellular e n z y m e s - Triple-helix molecules p o l y m e r i z e into fibrils 50-200 n m long
Association of C-propeptides; tripi©-helix nucieatlon {Gly-X.Hyp)„ TripIe-hellx propagation
- Fibrils pack into fibers (stronger t h a n steel of s a m e size) VVTien d e n a t u r e d , forms gelatin (missing propeptides lead to unordered cross-linking)
ageo N- and Cpropeptides Assembly and cross-lir^ing mm of fibrilg
Figure 9. Biosynthesis and assembling of collagen fibrils.
Figure 10. Structural organization of prionfibrils,(a) TEM of prionfibrilsand (b) Selfassembling of prion peptides (from amyloidogenic region) on a solid surface.
During the multiplication of prions, the formation of fibrils in infected brain tissue causes the destruction of the brain's neuronal network. This results in the disruption of proper function as expressed by progressive psychoneurotic disease, which can be fatal [7,8]. In addition, Cu^^-binding domains were identified in the N-terminal
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portions of prion proteins. However, the physiological role of Cu^^ binding by prion proteins is not clearly understood. The affinity of fibrils to ions of various metals could contribute to the development of conductive-like elements inside the brain leading to severe disturbances in the local brain tissue conductivity. The general neuropathological sign of Alzheimer's disease is low and chaotic electrical activity of a brain in electroencephalography (EEG) observation. Thus, in situ mineralized prion fibrils may have local conductivity that disrupts normal signaling pathways in the brain. This hypothesis led to the testing of protein fibrils for the assembly of biocompatible protein-containing nanowires [8,11]. 5.3. Aj3-peptides Small Aj3-peptides have lengths from 40 to 42 amino acids and are proteolytic fragments of the huge membrane-bound amyloid precursor protein (APP) shown in Fig. 11. A nucleation domain was identified as the primary attachment of the peptides to each other and the initiation of fibril biogenesis. Short A-peptide homologous peptide fragments specifically bind to Aj3-42 and prevent fibril assembling. This is the most important evidence that the short sequence of the A/3-42 is involved in fibril assembling [8-10]. Secretase (a, P, 7) are a set of proteases, which cleaves Aj3-40-42 peptide in different positions. When Ali-40-42 peptide escapes cleavage, it is capable of forming tight fibril aggregates that are found as deposits inside destroyed brain tissue. In water, the A/?-peptide has a complicated conformation including a-heUces and j3-structures, which is modeled in Fig. 12. The assembly of the native peptide into fibrils leads to a complete transformation of the peptide from an a-helical to an entirely j3-stranded structure. Usually j3-sheet peptides interact with each other along their entire length in order to form very tight structures [9,11]. Further investigation into the structure of these peptide fibrils showed that there is a special arrangement of peptides inside the fibrils.
p-amytald precursor protein (pAPP) ^amytold (Ap)
Secretase: Cteavagesite: N
p Apt
p Apii
a Api7
y 7 Ap40 A^42
bAEFGHDSGKBWHQKLVFFAEDVGSKKGAIIGLMVGGviv'lU
-
Lipid bi layer
Schematic diagram of the p-amyloid precursor protein (pAPP)
Figure 11. Primary sequence and proteolytic sites of Aj3-40-42 peptide, partially integrated into lipid membrane. Secretase a, p and y cleavage sites are pointed along polypeptide sequence. N, NH2-terminus; C, COOH-terminus [14,15].
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c Alzhciincfs Disease
Figure 12. Computer model of Aj8-40 peptide. The peptide consist of extracellular and transmembrane parts, that is shown here. The sequence of A^40 peptide is deciphered in Fig. 11.
Figure 13. Structure and modeling of A^-peptide nanotube: (a) TEM of single Afipeptide fibril and (b) computer model of A)3-peptide nanotube.
According to X-ray analysis, peptides form water-filled nanotubes that are assembled by peptides like stick pieces resulting from interactions between short clusters of amino acid residues as shown by a model in Fig. 13b. According to a generic statistical mechanical model [12-14], the self-assembling of the peptide starts from chiral rod-like units into helical tapes, then under increasing concentration it forms into twisted ribbons or double tapes. Fibrils, as an additional step, represent
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twisted stacks of ribbons, which lead to the final stage in fibrillogenesis where fibers are produced. According to this model, the sequential transformation of peptides provides many opportunities for chemical modification, as can be seen from the morphology of A^-peptide deposits in damaged tissue [8,12,14-16]. It is hoped that understanding the further modifications that are made to peptides or proteins by metal ions may elucidate the mechanisms involved in making conductive fibers [8,12-16]. 5.4. Insulin Insulin is a protein hormone widely used in the treatment of diabetes [14]. It consists of two subunits: A and B, associated by Cys-Cys binding. It can exist in several different molecular forms such monomers, dimers, and hexamers. Zn^^ is necessary for assembling of the hexamer storage form of insulin [4,5]. The insulin dimer is primarily stabilized by ^-sheet domains located in subunit B as shown in Fig. 14. Denaturing and assembling fibrils on insulin molecules were dependent upon an acidic pH, hydration, and Zn^+ ions. As a result, a high yield of insulin nanofibers was achieved as shown in Fig. 15. In solution at neutral pH and at physiological concentrations (about 1 ng/ml), insulin exists as a monomer and is in the active form of the hormone. At higher concentrations, at acid or neutral pH, and in the absence of zinc, the insulin monomer self-associates to form dimers. In the presence of zinc, insulin forms hexamers. Studies of insulin analogues demonstrate that its activity is dependent upon the integrity of the insulin fold. Insulin domains involved in Zn-dependent hexamer and Zn-independent dimer formation have different locations. The central
A20:B19
A7:B7
Figure 14. Computer model of insulin dimer. Arrows: p strands are shown between two monomers as an interface region.
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Figure 15. TEM of insulin fibers, (a) Amorphous aggregates of insulin from acetonitrile solution, (b) Insulin fibers formed in water buffer, contained 1 mM ZnSO,. residues responsible for hexamer formation include BIO His, which binds Zn ions as well as B14 Ala, B17 Leu, B20 Gly, and A13 Leu, which are involved in close-packed hydrophobic interactions. The ability to form dimers is mediated by hydrophobic interactions involving B8 Gly B9 Ser, B12 Val, B13 Glu, B16 Tyr, B24 Phe, B25 Phe, B26 TVr, B27 Thr, and B28 Pro. The B chain of one monomer packs against the B chain of the second monomer and further stability is provided by hydrogen bonding between the anti-parallel ^-strands B24-B26 of each molecule. The packing arrangement in the dimer results in perturbations of the side-chain and main-chain structure slightly changing the resulting monomer conformation. The B25 Phe side-chain is important for stabilizing the native dimer structure. However, decreasing the pH and increasing the concentration of the protein fatally lead to insulin aggregation and fibril formation, which have been described in many studies [4]. 5.5. Sup35 yeast protein Many studies have been performed with phospholipids, tubules, DNA, bacteriophages, and cellular microtubules [1,9]. However, individual proteins are the best materials for designing conductive macromolecular units and wires due to their availabiUty, consistency, and self-assembling nature. Proteins with similar behavior, structure, and the capability to form intracellular fibrils were identified in brewer yeast by the yeast geneticist S. Lindquist. During the past decade, she discovered and studied a unique protein called Sup35. This protein is similar both in structure and function to prion-like proteins, so it has become a quite simple, safe (noninfectious), and available model for basic studies in this field. Fragments of yeast Saccharomyces cerevisiae protein Sup35 were selected for use in a conducting nanowire design [9,12]. Recombinant Sup35 protein contains N-terminal and middle region (NM) of native Sup35p. NM forms self-assembling
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j3-sheet-rich amyloid fibers that have a suitable size and shape for nanocircuitry with diameters of 9-11 nm. In water, NM is a soluble protein and with a flexible, unordered structure. Under the appropriate conditions, NM rapidly converts to insoluble amyloid fibers. This process is primed by the addition of preformed fibrils. The fibers grow into two directions and form varied fiber patterns. These fibers are also resistant to proteases and denaturing conditions and reagents, which makes them suitable for the fabrication and processing of electrical wires. The kinetics of fiber formation by NM is slow and it does not make aggregates as other amyloids and prion-like proteins. Moreover, NM fibers form under physiological conditions such as a neutral pH and at room temperature. The secondary structure of NM was determined by UV-CD and showed that in solution NM has a random coil-rich structure and a sheet-like appearance when it is in fibers. This is important because the industrial fabrication process of nanoscale devices requires mechanically stable and chemically resistant materials. NM fibers can be incubated in phosphate buffer with different ionic strengths, at temperatures from 25 °C to 98 °C, and in denaturizing agents such as 8M urea and 2M guanidinium hydrochloride. NM fibers were completely stable in the absence of salt and in 2.5 M NaCl. Moreover, NM fibers can be used after storing frozen for 3 months at -20 °C and -80 °C. Chemical stability in alkaline, acidic, and organic solvents was also demonstrated. NM fibers with variable lengths can be repeatedly produced and the average size of the fibers was approximately 500 nm. Increasing the concentration of the soluble NM increased the fiber length. NM fibers conductive properties are tailorable. They can be both good insulators or using multi-step processing, NM fibers can be converted to conducting nanowires with low Ohmic resistance. For conductivity purposes, specially engineered NM was used. By site-specific mutagenesis, the Cys derivative of NM was obtained. Moreover, this Cys residue in engineered NM remains accessible after fiber formation in the derivative NM K184C. This derivative, NM K184C, was used as the starting recombinant NM. Using monomaleimido nanogold nanoprobes, gold nanoparticles were covalently attached to the fibers. After this modification, NM K184C displayed the same morphology as the original protein. The distance between cysteine residues in the fibers was 3-5 nm where the nanogold particle diameters were only 1.4 nm. In order to bridge the particles with metal to gain conductivity, GoldEnhancer was used. This process was inefficient because some gaps remain unfilled. A special gold toning procedure was developed to create a final conductive nanowire. Nanogold particles on the labeled fibers elicited a reducing activity when reacted with silver ions. The silver-modified fibers in Fig. 16 become thicker and have diameters from 80-200 nm compared to bare fibers with diameters from 9-11 nm. The electrical behavior of NM-template metallic fibers was assessed by randomly depositing fibers. Nanogold particles on patterned electrodes were then covalently attached. Then, the gold toning procedure allowed the formation of metallically continuous gold nanowires. The 100 nm nanowires had a very low resistance of i?=86 Q. This is the first successful design of proteinaceous nanowires of natural origin with modifications inserted by genetic engineering.
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Figure 16. Metallic fiber of yeast prion-like protein. Bare and toner-labeled fibers: diameters 9-11 and 80-200 nm, respectively.
Short domains in Sup35 were also studied for forming fibers with assigned properties such as in the creation of peptide nanotubes as shown in Fig. 17. There are many successful examples of experimentally designing nanomaterials using short peptides with specific functions [3,9,17-20]. However, many studies in peptide technology for the creation of nanotools have shifted from the applications of natural polypeptides to the applications of specially designed synthetic polypeptides. A synthetic peptide is completely homogenous in its structure and could be modified or prepared for modification during its synthesis. The size of these peptide fibers provides the possibility of creating very small components for nanoelectronics. For example, the short peptide sequence GNNQQNY is capable of forming highly ordered fibers and nanotubes. However, Sup35 yeast protein was the first subject for the design of metal-filling protein nanowires [9,12,17]. The existence of sequence diversity among prion-Uke proteins capable of forming highly ordered fibrils creates an even wider range of starting materials than currently exist in the electronics industry [8,11]. All of the proteins and peptides listed in Table 1 are prion-like proteins that can be used for the creation of protein nanotubes and galvanized nanowires. It is important to note that they have very different primarily sequences, but are similar in secondary structure. Additionally, these prion-like proteins have similar aromatic amino acid residues usually located in nucleation regions where fibril formation is initiated.
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Xmaltered segments: ^GNNQQNY^^ 7GNNQQNYQQ^5 8NNQQNYQQ^5
'GNNOQNY «3
'QNNQQNYQQ'^ 'GNNQONYOE'5 ^NHQONYQO'^,,
715
Altered segments: ^GNNQQNYQR^s i^SQNGNQQRG^s
'^SQNGNQQRG
X-rmyi
Coital Width--280 A
Figure 17. Yeast Sup35 protein physical map and peptides active in fibrillogenesis. (a) Amyloidogenic sequences are located on the map of Sup35p in N-terminus and in the middle part of the protein. Minimal length of peptide capable to form fibrils is heptapeptide: GNNQQNY. (b) Sequences of short peptides trialed as an N-terminal nucleation domain of Sup35p [17]. Table 1 Structure and properties of fibril forming protein sequences [9,10]. Name of natural peptide
Disorder or natural condition
Short fibrillogenic sequence
1. Aj3-amyloid peptide
Alzheimer disease
KLVFFA LVFFA LPFFD KLVFFAE
2. Islet amyloid precursor protein
Type II diabetes
TNVGSNTY
3. Lactadherin
Aortic medial amyloid
NFGSVQFV
4. Gelsolin
Finnish hereditary Amyloidosis
SFNNGDCCFILD
5. Serum amyloid A
Chronic systemic inflammation amyloidosis
SFFSFLGEAFD
6. PrP^^
PHGGGWGQ
Creutzfeld-Jakob disease (CJD)
7. Sup35p yeast protein Yeast prion protein
PQGGYQQYN GNNQQNY
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6. HUMAN PATHOLOGY AND PROTEIN GALVANIZATION 6.1. Metal-filled nanowires Many studies have tried to clarify the role of metal ions in the assembly and catalytic activity of A-peptides and their fibrils. In Fig. 18, a schematic of ion modification is described where Cu^^ and Zn^^ ions are attached to Aj3-peptides. This process was termed protein galvanization [13]. Moreover, data obtained in this study support the idea that amyloid fibril galvanization is principally caused by Zn^^ released during neurotransmission as shown in Fig. 18. During active synaptic transmission, the concentration of Zn^^ ions reaches 300 |LIM. This local concentration is very high and sufficient for peptide galvanization of the localized A/?-peptides. The galvanization of Aj3-peptides leads to the precipitation of the oxidized form of the peptides and rapidly induces amyloid fibril development as shown in Fig. 18. The biogenesis of amyloid fibrils includes free radical generation and oxidation of the protein and its fragments. The main consequence of free radical generation is intra-molecular networks or fiber cross-linking. The cross-linking of generated fibrils leads to the assembly of high molecular mass aggregates and intra-tissue deposits called plaques. A similar process could be reproduced in experimental conditions [8,12,14-16]. In our opinion, Zn^^-saturated peptides become the prototype of a conductive peptide element generated during the development of disease in the human brain [4,9]. Moreover, during aggregation this protein can form bundles and branched bundles of dendrimer-like structures. 6.2. Natural protein galvanization: Zinc-binding motifs Proteins have Zn^ ^-binding sites. However, Zn^+ ions can be replaced by many other ions. In conditions where the polypeptide chain is saturated by those ions, the protein dramatically changes its properties and can then be used in nanoelectronic design. In Fig. 19, the Zn-finger motif of a DNA-binding protein is shown. This Znfinger motif usually contains His and Cys residues in a specific polypeptide loop [8]. Many proteins contain more than 10 Zn-finger motifs per molecule that bind with high affinity. Zinc-dependent galvanization of other proteins may occur in aggregates of peptides that contain out of order Cys or His residues, but that interact through interchain binding. In vitro experiments with a high molar access of Zn^^
Cu/Fe zr^Ap(Cu/fe)
H2O2
+ Zn ^ ^ Plaque
Fibrils
Toxicity
Figure 18. Metabolic galvanization of A)3-peptide [12].
H^O,
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(b) Zinc-finger motif
Figure 19. Structural organization of a DNA-binding domain and Zn-finger motif. Arrows are j8-sheet domain; cylinder is a-helix; Cys, cysteine; His, Histidine. Zn^+ binds usually to specific consensus sequence in many proteins involved in transcriptional regulation.
ions lead to saturation of the protein by tightly bound Zn^^. The same saturation binding was detected for other metals. This property of natural proteins is currently being used for the design of metal-binding short peptides as conducting wires [9]. 7. APPLICATIONS OF BIONANOTECHNOLOGY: DIAGNOSTICS, TREATMENTS, AND FUTURE DIRECTIONS Many infectious agents are rapidly being distributed throughout the world and causing epidemic outbreaks with a high death rate. Diseases such as the avian flu, SARS, Dengue, Ebola are some of the most dangerous infections. Therefore, there is a great demand for special tools that can rapidly and specifically diagnose these threats [2,9]. Many diagnostics, including enzyme-linked immunoassays, are based on an adsorption of antigens or antibodies onto the bottom of plastic plates. Using current technology, antibodies could be localized in an exact place or spot by techniques such as nanolithography. However, the main problems associated with this type of set-up are nonspecific background binding of the proteins and insufficient detection sensitivity. Nanotechnology provides a novel opportunity to construct devices for the specific detection of single viral particles. This high sensitivity is needed because only 5-10 individual HIV viral particles are enough to infect a sexual partner or drug user. Moreover, these infectious agents are rather stable and aggressive and could be transmitted in trace amounts to other hosts. Prions and other infectious amyloidoses could be transmitted on the level of fento molar concentrations. Therefore, diagnostic devices need to be extremely sensitive in order to detect single viral particles or single molecules of infectious agents. Fig. 20 shows a novel approach and sensor for the detection of single viral particles.
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Lasers are widely used in medicine for simple skin cosmetic treatments to more advanced surgical operations on the eye or brain. Specialized semiconductor lasers are used in applications such as telecommimications, informational storage, and the fabrication of integrated electrically driven devices. The problem is to understand how to combine the current silicon technology of microelectronics to other materials such as organic molecules, synthetic and natural polymers, and inorganic nanostructures. Recently, amplified stimulated emission and laser irradiation have been used in optically pumped organic systems and as inorganic nanocrystals or nanowires. For example, single-crystal cadmium sulfide nanowires were used in an electrically driven laser [22]. Small and compact distantly driven lasers are very important in medicine to replace radioactive materials in treatment of tumors and other diseases. The creation of a biocompatible type of single nanowire laser would be beneficial in the treatment of metastasis in hard to access organs of the human body [23]. The recent discovery of the heat absorptive properties of carbon nanotubes for use in laser irradiation opens up new possibilities in the treatment of tumor diseases [22-24]. Irradiation of a carbon nanotube suspension by a near-infrared laser beam leads to the heating of the solution up to 70 °C within 2 min. Electrons in the nanotubes become excited and begin releasing energy in the form of heat. By using an intrinsic property of nanotubes, a weapon that kills cancer can be developed. The main problem with this system is precise targeting. It is well known that tumor cells contain surface clusters of folate receptors and depend on a folic acid substrate for their proliferation. This difference between normal and cancer cells makes possible
Figure 20. Solid phase sensor for detection of single viral particles. Antibodies (cyan, red, green) are covalently attached to nanoelectrode. Viruses (brown) are specifically attached to antibodies and could be detected by signal registration immediately after interaction. This approach increases sensitivity of viral detection until single viral particle identification and can be used simultaneously for detection of hundred viruses [21].
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selective targeting through "labeling" of carbon nanotubes by folate. Folate-labeled nanotubes have been taken up by cancer cells with high efficiency. Then, the treated cancer cells were illuminated with a near-infrared laser, which soon heated and completely killed the cells containing the folate-labeled carbon nanotubes. This type of local heating induces the apoptosis of cancer ceUs, which is better known as hypothermal cancer treatment [22]. Many other tools for targeting can be used in further studies and for clinical applications. Thus, the combination of lasers with the intrinsic properties fullerenes and functionalized nanotubes is a rapidly growing area of research and design for novel technologies in cancer treatment. Bionanotechnology can be defined by its utility for creating devices of enhanced sensitivity or function that are useful in biomedical research, drug discovery, and clinical therapy. The successful study of human conformational pathology has opened up new opportunities in the experimental design of protein fibrils and their application as nanoparts of bionanotechnology devices for use in revolutionary technology as shown in Fig. 21. Biomolecules and their derivatives all have dimensions in the nanometer range, but there is nothing to be gained by re-classifying all of biotechnology as nanotechnology. When biomolecules are combined with manufactured nanoscale items or are significantly re-engineered, they can be considered for use in nanoelectronic tools. Likewise, some protein wires and semiconductor chips
Nano-bio interface
Current CMOS Technology Next Generation CMOS Nanofiber "—"Nanotube Nanowire — F u l l e r e n e s
Nanotube
0.1 nm
"^
Figure 21. Scheme of further development of bionanotechnology.
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now have features with dimensions smaller than 90 nm. Biotechnology and genetic engineering as well as chemical growth of nanoscale could provide the necessary complexity for the basic components of nanotools. The most important question in the further development of this imique field is to find accurate and outstanding wide spread appUcation. In the near future, nanodiagnostic devices could revolutionize the screening and detection of viruses. REFERENCES 1. S. Zhang, Nat. Biotechnol. 21 (2003) 1171-1177. 2. http://www.omedon.co.uk/influenza/influenza/. 3. L. B. Piotrovsky, Fundamental Directions in Molecular Medicine, Eds. O. I. Kiselev and U. P. Golikov, Rostok publishers, Saint-Petersburg, 2005, 197-267. 4. J. Brange, J. WhittinghamDonald, D. Edwards, Y.-S. Zhang, A. WoUmer. D. Brandenburg, G. Dodson and J. Finch, Current Science 72 (1997) 470^76. 5. M. R. H. Krebs, C. A. MacPhee, A. F Miller, I. E. Dunlop and C. M. Dobson, Proc. Natl. Acad. Sci. USA 101 (2004) 14420-14424. 6. K. Misof, G. Rapp and R Fratzl, Biophys. J. 72 (1966) 1367-1381. 7. R. Valuzzi and D. L. Kaplan, Biopolymers 53 (2000) 350-362. 8. S. B. Pruziner, Science 252 (1991) 1515-1522. 9. V. 1. Pokrovskii, O. I. Kiselev and B. L. Cherkasskii, Prions and Prion's Diseases, MedAcadem Publishers, Moscow, 2004, p. 398 (in Russian). 10. E. Gazit, A possible role for ;r-stacking in the self-assembly of amyloid fibrils, FASEB J. 16 (2002) 77-82. 11. R. W. Carrel and D. A. Lomas, Lancet 350 (1997) 94-98. 12. Sheibel, Th., R. Parthasarathy, G. Sawicki, X.-M. Lin, H. Jaeger and S. L. Lindquist, Conducting nanowires built by controlled self-assembly of amyloid fibers and selective metal deposition, Proc. Natl. Acad. Sci. USA 100 (2003) 4527-4532. 13. A. I. Bush and R. E. Tanzi, Proc. Natl. Acad. Sci. 99 (2002) 7317-7319. 14. D.-Sh. Yang, J. McLaurin, K. Qin, D. Westaway and P. E. Eraser, Eur. J. Biochem. 267 (2000) 6692-6698. 15. M. F Perutz, J. T. Finch, J. Berriman and A. Lesk, Proc. Natl. Acad. Sci. USA 99 (2002) 5591-5595. 16. A. Aggeh, I. A. Nyrkova, M. Bell, R. Harding, L. Carrick, T. C. B. McLeich, A. N. Semenov and N. Boden, Proc. Natl. Acad. Sci. USA 98 (2001) 11857-11862. 17. M. Balbimie, R. Grothe and D. Eisenberg, Proc. Natl. Acad. Sci. USA 98 (2001) 2375-2380. 18. C. Valery, F Artzner, B. Robert, T. Gulick, G. Keller, C. Grabielle-Madelmont, M.-L. Torres, R. Cherif-Cheich and M. Patemostre, Biophys. J. 86 (2004) 2484-2501. 19. M. L. De la Paz, K. Goldie, J. Zurdo, E. Lacroix, C. M. Dobson, A. Hoenger and L. Serrano, Proc. Natl. Acad. Sci. US 99 (2002) 16052-16057. 20. 1. A. Banerjee, L. Yu and H. Matsui, Proc. Natl. Acad. Sci. USA 100 (2003) 14678-14682.
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21. N. D. Lazo, M. A. Grant, M. C. Condron, A. C. Rigby and D. B. Teplow, On the nucleation of amyloid j3-protein monomer folding, Protein Science 14 (2005) 1581-1596. 22. E. Smalley, Biochip spots single viruses, Technology Research News (TRN) Oct. 20/27 (2004). http://www.tmmag.com. 23. M. Shwartz, Scientists develop nanotech-laser treatment that kills cancer cells harming healthy tissue, http://www.eurekalert.org.pub _releases. 24. X. Duan, Huang Yu, R. Agarwal and C. M. Lieber, Nature 421 (2003) 241-245.