Nanotechnology – Chad D. Paavola, Jonathan D. Trent, Suzanne L. Chan, Yi-Fen Li, R. Andrew McMillan, Hiromi Kagawa, National Aeronautics and Space Administration NASA

Abstract for “Versatile platform to nanotechnology based upon circular permutations chaperonin proteins”

The present invention allows chaperonin-polypeptides to be modified so that the N-terminal or C-terminal ends are moved from the central region of the polypeptide to different locations on the exterior. The naturally occurring N-terminal (or C-terminal) and C-terminal end of the modified chaperonin peptide are joined directly or by an interfering linker sequence. The C-terminal and N-terminal ends that have been relocated can be joined or bound to another molecule like a nucleic acids molecule, lipid, carbohydrate, second polypeptide or nanoparticle. Modified chaperonin polypeptides may be assembled into double-ringed chaperonin structure. The chaperonin structures are capable of forming higher order structures, such as nanofilaments and nanoarrays that can be used for nanodevices or nanocoatings.

Background for “Versatile platform to nanotechnology based upon circular permutations chaperonin proteins”

“The controlled organization inorganic materials into multidimensional addressable arrays forms the foundation for logic and memory devices as well as other nonlinear optical or sensing devices” (Zhirnov et. al. 2001 Computer 34: 34?43; Xia et. al. 2000 Adv. Mater. 12: 693-713). These devices are made using lithographic patterns that have improved to produce smaller and more integrated parts. However, conventional lithographic methods are reaching their theoretical and practical limits at submicron scales. Ion and electron beam printing becomes too expensive and time-consuming at scales below 100nm. Further, quantum effects fundamentally alter the properties of devices at these scales (Sato, 1997 J. Appl. Phys. 82: 696).”

“Nanoscale templates are being created using both artificial and naturally-sourced materials for constrained synthesizing, in situ deposition or direct patterning nanometer scale inorganic arrangments. Artificial materials like microphase separated block copolymers are being developed (Park et. al. 2001 Appl. Phys. Lett. 79: 257-259), and hexagonally-closed spheres (Hulteen et.al. 1995 J. Vac. Sci. Technol. For nanoscale fabrication, A (1553-1558) was used. Natural materials such as DNA (Richter et al., 2000 Adv. Mater. 12: 507-510; Keren et al., 2002 Science 297: 72-75), bacterial and archaeal surface layer proteins (S-layer proteins) (Sleytr et al., 1999 Angew. Chem. Int. Ed. 38: 1034-1054; Douglas et al., Appl. Phys. Lett. 48: 676-678; Hall et al., 2001 CHEMPHYSCHEM 3: 184-186), virus capsids (Shenton et al., 1999 Adv. Mater. 11: 253-256; Douglas et al., 1999 Adv. Mater., 679-681; Douglas et al., Nature 393: 152-155; Wang et al., 2002 Angew. Chem. Int. Ed. 41: 459-462), phage [Lee et. al. 2002 Science 296] 892-895, and some globular proteins [Yamashita I. 2001 Thin Solid Films393: 12-18] have been used in templates and other nanoscale applications.

“Nanometer-scale objects at all scales, including arrays made by non-conventional techniques, are being investigated for possible alternatives to standard lithographically printed devices. The behavior of individual nanoparticles, also called quantum dots (QDs), has been demonstrated to be isolated components for devices such as single electron transistors (Likharev K. K. 1999 Proc. IEEE 87: 606-632; Thelander et al., 2001 Appl. Phys. Lett. 79: 2106-2108). Two-dimensional arrays, with nanoscale resolution, of QDs could be the basis for future generations of electronic or photonic devices. This is according to theoreticians. These devices will function based on phenomena like coulomb charging, interdot quantum tunneling, and other coherent properties derived electronically from confinement and nanoparticle volume to surface ratio ratios (Maier S. A. and al. 2001 Adv. Mater. 13: 1501-1505; Maier et al., Phys. Rev. B 65, 193408; Zrenner, A. et al., 2002 Nature 418: 612-614; Berven et al., 2001 Adv. Mater. 13: 109-113).”

“Traditional methods for patterning ordered arrays materials onto inorganic substrates or manufacturing devices are currently used by ion beam imaging and molecular beam epitaxy. These techniques have their limitations because of the use of polymeric lights masks for pattern formation. However, there is a theoretical limitation to patterning that could eventually limit the processes in the hundreds if not thousands of nanometers.

“While there are strong incentives for developing nanoscale architectures these developments require alternative fabrication methods and new insights into materials’ behavior on nanometer scales (Nalwa H. S. 2000?Handbook of Materials and Nanotechnology? Academic Press, San Diego).

“Development and application of new methods to order nanoscale materials from the ‘bottom up. New tools will be available for creating nanostructured materials, and devices that can self-assemble or repair themselves. Because of their ability to create structures at nanometer scales with minimal or no mechanical processing, synthetic and biological polymers are gaining attention. These natural and synthetic polymers can be self-assembled or phase separated to create nanoscale materials. Biopolymers have been shown to form complex structures and assemblies that possess highly specific chemical functions. All of these elements have been used to make nanostructured materials that exhibit unique properties (J Richter, et.al. 2000 Advanced Materials 12:507-551; M G Warner, J E Hutchison and K Keren 2003 Nature Materials 2:272-277 and K Keren, et.al. 2002 Nature Materials 1:247-252) and virions (W Shenton and S-W Lee et.al. 2002 Science 296;892-895; Q Wang et.al.

“Nanoscale templates have been created from a variety of protein complexes. These templates can be genetically modified to add chemically reactive sites that bind organic materials. Chaperonin complexes, for example, can be functionalized to attach soft metals. Chaperonins in nature are protein complexes that have two stacked rings, each containing 7, 8 or 9 subunits of HSP60. Mutations to the HSP60 subunits have been made that include single cysteine residues at solvent-exposed locations, including the apical pores. These cysteine residues’ thiols act as binding sites for zinc or gold (PCT/US02/35889). These mutant HSP60 subunits are bound to gold or zinc by the chaperonin complexes. They then form two-dimensional crystals.

“Protein complexes may also be modified to contain peptide sequences with desirable binding or catalytic properties. These protein complexes include subunits that have inserted peptide sequences. The mutant subunits might not fold, assemble into complexes, or organize into higher order structures. In addition, inserting a loop into a peptide sequence may render it inactive. Fusion to one of the local termini may not be sufficient to provide enough surface accessibility. Circular permutation is used to join peptides within a protein template. Circular permutation refers to a reordering the polypeptide chain so that the N- and Cterminal ends of the polypeptide chains are joined. New termini are then created elsewhere in the protein. Without affecting subunit assembly, new peptide sequences are possible to be joined to the new termini. Studies of protein circular permutation have shown that for proteins where the native amino-carboxyl termini are close in space, there are many relocated positions available for the new termini (P T Beernink and U Heinemann, 1995 Prog Biophys Mol Biol 63:121-143; M Iwakura et. al. 2000 Nat Struct Biol 7 :580-585).

“The invention provides chaperonin-subunit polypeptides that are modified to move the native N-terminal end and C-terminal ends out of the central pore to new locations on the exterior fold modified chaperonin polypeptide. A peptide sequence acts as a reporter by joining the N- and Cterminal ends. Modified chaperonin polypeptides are formed into subunits, which self-assemble into double-ringed chaperonin structure. The chaperonin structure organizes into higher order structures like two-dimensional crystals or filaments. The reporter peptide can also be functional. These chaperonin structures can be used to produce ordered nanoscale materials or devices.

“The invention provides chaperonin-polypeptides that have been modified to include N and C-terminal end points. These ends are moved from the central region of the chaperonin protein polypeptide to different locations on the polypeptide’s exterior.”

“The C-terminal and N-terminal ends of the nucleic acids can be covalently linked to or bound with a nucleic Acid molecule, a protein, a lipid, or carbohydrate or a nanoparticle.

“In the modified chaperonin peptide, the naturally occurring N-terminal (and C-terminal) ends are joined together either directly or with an intervening linker sequence. In one embodiment, the intervening linker sequence comprises the amino acid sequence Gly-Gly-Ser-Gly-Gly-Thr.”

“Modified chaperonin is a polypeptide based on either a Group I or Group II chaperonin.

“The Group I chaperonin peptides come from Escherichia and Cyanobacteria. Mycobacteria. Coxiella. Rickettsia. Rickettsia. Chlamydia. Thermotoga. Chloroplast. Mammalian mitochondria. Or yeast mitochondria. The Group II chaperonins polypeptides come from Sulfolobales and Pyrodictium.

“In one embodiment, modified chaperonin polypeptides from Sulfolobus are an alpha, beta or gamma polypeptide.”

“In one embodiment, the modified chaperonin protein comprises a Sulfolobus ShibataeTF55 beta polypeptide that has the N-terminal end and C-terminal ends moved after any amino acids position within the range 149 to158, in particular, moved to position 153.”

“Another embodiment of the modified chaperonin protein comprises a Sulfolobus ShibataeTF55 beta polypeptide that has the N-terminal end and C-terminal ends moved after any amino acids position within the range 263-270. In particular, it is moved to position 267.”

“Another embodiment of the modified chaperonin protein comprises a Sulfolobus ShibataeTF55 beta polypeptide that has the N-terminal end and C-terminal ends moved after any amino acids position within the range 311-330. In particular, it is moved to position 316.”

“Another embodiment of the modified chaperonin protein comprises a Sulfolobus Shibatae TF55 Beta polypeptide that has the N-terminal end and C-terminal ends moved after any amino acids position in the range 472-487. In particular, it is moved to position 480.”

“Another embodiment of the modified chaperonin protein comprises a Sulfolobus Shibatae TF55 beta peptide that has the N-terminal end and C-terminal ends repositioned after any amino acids position in the range 494-508 or 499, in particular.”

“The invention also provides assembled chaperonin structure, including at least one modified chaperonin unit.”

“The chaperonin structures assembled can have one or more of the following: 2-, 3-, 4, 5, 6-, 7, 5-, 6- and 7-fold symmetry.

“The invention also provides: nucleic acids molecules encoding modified chaperonin polypeptides of the invention; vectors containing these nucleic acids molecules; and host cell vectors.”

“The present invention provides in-vivo methods of producing modified chaperonin peptides. It involves cultivating host/vector systems in conditions that allow the host to make the modified chaperonin peptide.”

“The invention also includes in vitro methods of producing assembled chaperonin structure of the invention. This involves reacting modified chaperonin peptides under conditions that allow for self-assembly.

“The invention contains chaperonin structures which organize into higher order structures like nanofilaments and nanoarrays.” These nanofilaments or nanoarrays are useful for making nanodevices and nanocoatings.

“BRIEF DESCRIPTION ABOUT FIGURES”

“FIG. 1. illustrates an end- and side view of a HSP60 chaperonin model at 2.3? resolution. The side view’s outlined area shows one subunit of HSP60.

“FIGS. “FIGS. The dot-dashed boxes contain identical residues, while blocks of similar residues are contained in a solid box. Conservative matches are enclosed within a dashed box.

“FIG. 3. The structural alignment of the archaeal chaperonin, thermosome, and the bacterial chaperonin is shown (GroEL), which indicates the structural similarities between groups I and II chaperonins. The structural alignment shows where the features of both chaperonin subunits overlap in the black areas.

“FIG. “FIG.

“FIGS. “FIGS.

“FIGS. “FIGS.

“FIGS. 7A-E shows the assembly of engineered HSP60s, heat-shock proteins, into nanotemplates to produce nanoarrays containing nanoscale materials like nanoparticles.

“FIGS. 8A-D shows gold nanoparticles bound to engineered chaperonins or chaperonin nanotemplates.

“FIGS. 9A-D are semiconductor QD nanoarrays.”

“FIGS. 10A-D shows the formation of a nanoarray with gold nanoparticles. FIG. FIG. 10(D), shows XEDS spectrum of the bare carbon film (solid lines) and the gold nanoparticle array (dashed lines) taken from the probed area indicated by a circle in FIG. 10(B), as indicated with the arrow.

“FIGS. “FIGS.

“FIG. 12 is a control experiment that shows DIC (left), and fluorescent (right), images of non-cys mutated chaperonins crystals after incubation using CdSe.ZnS QDs.

“FIG. 13 shows the Energy Filtered TEM thickness mapping of a typical 2D Protein Crystal.”

“FIG. “FIG.

“FIG. “FIG.

“FIGS. “FIGS.

“FIGS. 17A-E are models of circular permutant protein models at positions: A) 53, B) 267 and C) 361; D) 480 and E) 499. Ribbon representations of subunits of protein are shown in the left column. The middle column shows side views showing surface representations for the assembled rings. The right column displays top views of the surface representations for the assembled rings. Amino termini will be labeled with blue. Carboxyl termini will be labeled with red. The flexible linker sequences in green are marked. One subunit of the assembled rings is highlighted in yellow.

“FIG. “FIG.

“FIGS. 19A-J is transmission electron microscopy of chaperonin double ring assemblies, which include circular permutant chaperonin protein (lacking fused EYFP). Panels A-E show samples containing isolated double ring samples. These were prepared one hour after adding ATP and MgCl2 protein samples. Panels F-J show samples containing higher-order structures. These were prepared 24 hours after adding ATP and Mg2+ protein samples. A) Circular permutant at position 53 at 1 hour. F) Circular permutant in position 153 at 24 hrs. B) Circular permutant in position 267 at 1 hr. G) Circular permutant at 267 at 24 hours. C) Circular permutant in position 316 at 1 Hour. H) Circular permutant in position 316 at 24hrs. D) Circular permutant in position 480 at 1 Hour. J) Circular permutant at 480 at 24hrs. E) Circular permutant in position 499 at 1 h. J) Circular permutant at 499 at 24 Hours

“FIG. 20A-D depicts the permutant chaperonin circular proteins at positions 267, 480, joined with yellow fluorescent protein EYFP. A) Fluorescence excitation spectra (open symbols and emission spectra) for unmodified EYFP. (circles), 267 permutant EYFP fusion proteins (squares), 480 Permutant EYFP Fusion protein (triangles). B) TEM showing negatively stained rings that were formed by the 480 permutant EYFP fusion proteins. C) Fluorescence microscopy (excitation 470nm, emission 515nm) of a crystal made by 267 permutant EYFP fusion proteins using Leica filter cube 13. D) TEM showing 2-dimensional crystal made by 267 permutant EYFP fusion proteins.

“FIG. “FIG.

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“FIG. “FIG.

“FIG. “FIG.

“FIG. “FIG.

“FIG. “FIG. 26 shows the complete (A) DNA sequence (SEQID NO: 76), and (B) amino acids (SEQID NO: 77) sequences of a position267 circular permutant chaperonin?EYFP fusion proteins.

“FIG. “FIG.

“FIG. “FIG.

“FIGS. 29A and 29B are the complete (A) DNA sequence (SEQID NO:81), and (B) amino acids (SEQID NO:82). This sequence is for a position 267 ‘dwarf. deletion circular permutant chaperonin protein.”

“FIG. “FIG.

“FIG. 31 shows a distinct DNA sequence (SEQ ID No:86) for TF55 beta subunit, Sulfolobus Shibatae.

“Definitions”

“All scientific terms and technical terms used herein have the same meanings as those used in the art, unless otherwise stated. The following words and phrases are used in this application.

“Isolated” is the term used herein. “Isolated” refers to a particular nucleic acid/polypeptide or a fragment thereof in which contaminants (i.e. The specific nucleic acids or polypeptide molecules have been removed from any substances that are different from them.

“Purified” is the term used herein. A specific nucleic acid or polypeptide is an isolated nucleic acids or polypeptide or a fragment thereof in which all contaminants (i.e. Any substances that are not the same as the nucleic or polypeptide molecule have been removed from the nucleic or polypeptide.

“As used herein, the term ?naturally-occurring? Refers to a nucleic acids or polypeptide molecule that is found in nature.

“Wild type” is the term used herein. A nucleic acid or polypeptide molecular having the same sequence of nucleotide/or amino acids as a naturally occurring, non-mutant molecule.

“Modified” is the term used herein. Modified molecules are those whose amino acid and nucleotide sequences are different (mutated) than a naturally occurring sequence, i.e. wild-type amino acid sequence or nucleotide sequence. Modified molecules may retain the same structural properties of a wild-type molecule.

“Derivative” is the term used herein. Any modification or alteration to a wild-type molecular structure. A derivative can include, but is not limited to, a conservative or non-conservative substitution in an amino acid or nucleotide sequence, including substitutions by nucleotides or amino acid analogs, a deletion of one/more amino acids or nucleotides, and the insertion or removal of one/more amino acids or nucleotides. A derivative molecule may share sequence similarities and/or activity with its parent.

“A first sequence of amino acids or nucleotides is referred to as sequence?identity” To a second nucleotide sequence or amino acid sequence, respectively. A comparison of the first sequence and the second sequences reveals that they are identical.”

“A first sequence of nucleotides or amino acids is considered to be similar if it is used in this context. A second sequence is considered similar to the first sequence if they are closely related (i.e. the sequences are almost identical). Two sequences may be considered similar if they have approximately 60% to 99.99% difference in nucleotides and amino acids.

“Complementary” is the term used herein. The term “complementary” refers to nucleic acids molecules that have purine and pyrimidine nucleotide base bases. These base pairs can be formed through hydrogen bonding, thereby mediating the formation of double-stranded nucleic acids molecules. Complementarity is found in the following base pairs: guanine, cytosine, adenine, and thymine and adenine-uracil. Complementary is applicable to any base pair that contains two single-stranded molecules of nucleic acids, or to any base pair that has a single-stranded molecule of nucleic acids folded onto itself.

“Conservative” is the term used herein. A substitution of an amino acid residue with another amino acid residue with similar chemical properties is called “conservative”. An example of conservative amino acids substitution is: substituting any nonpolar hydrophobic amino amino for any hydrophobic hydrophobic amino; substituting any hydrophilic (polarized, uncharged), amino amino for any hydrophilic amino; substituting any positively charged amino for any other positively charged amino; substituting any negatively charged amino for any other negatively charged amino (TE Creighton, Proteins? WH Freeman and Company. New York. These amino acid substitutions can include substituting any one of the following: isoleucine, valine, and leucine for any other hydrophobic amino acid; aspartic (D) to replace glutamic (E) or vice versa; glutamine [Q] for asparagine [N] and vice versa, and serine (S] for threonine (?T?) and vice versa. Other substitutions may also be acceptable depending on the amino acid’s role in the protein’s three-dimensional structure. Glycine (G), alanine, or serine (S), can often be interchangeable. Alanine, valine, and glycine can also be interchangeable. Methionine, which is relatively hydrophobic can often be interchanged with leucine or isoleucine and sometimes with valine. Lysine (K), and arginine, which are both relatively hydrophobic, are often interchangeable at locations where the most important feature of an amino acid residue’s charge is not significant and the pK differences between these two amino acids are not significant. Other changes may be acceptable in certain environments.

“Nonconservative” is the term used herein. It refers to the substitution of an amino acid for another amino acid that has different chemical characteristics. Nonconservative substitutions are, among others: aspartic acid (D), being replaced by glycine [G]; asparagine [N] being replaced by lysine [K]; or alanine (?A?) being replaced (R)

“The single-letter codes of amino acid residues are: A=alanine; R=arginine; N=asparagine; D=aspartic Acid, C=cysteine; Q=Glutamine and E=Glutamic Acid, G=glycine. H=histidine. I=isoleucine. L=leucine. K=lysine. M=methionine. F=phenylalanine. P=proline.

The following description will help you to better understand the invention described herein.

“Modified Chaperonin Polypeptides

The present invention includes modified chaperonin peptides and their fragments and derivations; assembled chaperonins containing the modified chaperonin peptides of this invention; nucleic acids molecules encoding modified chaperonin peptides or fragments and derived thereof; host-vector system; methods for producing modified chaperonin peptides; methods to use the modified chaperonin peptides such as producing bio-nanoscale devices and coatings

Chaperonins are essential subcellular structures found in nature. They have 14, 16 or 18 heat-shock protein subunits (e.g. HSP60) and are arranged as two stacked rings measuring approximately 16-18 nm high by 15-17 nm wide depending on the species they come from (Hartl and al. 2002 Science 295; 1852-8). Chaperonins possess a central pores, along with apical and intermediate domains. The chaperonin’s naturally occurring N- and Cterminal ends are found in the wild-type HSP60 protein unit. FIG. FIG. 1 shows an end-side view of a chaperonin, which has 16 subunits. There are eight subunits in each ring.

“The invention provides chaperonin-subunit polypeptides and fragments or derivatives thereof. These are modified to include Nterminal and Cterminal ends that are moved from their naturally-occurring locations to other positions in the polypeptide.

“In the modified chaperonin peptide, the N and C-terminal ends of the modified chaperonin peptide are moved from the central region to a new location on the exterior fold modified chaperonin peptide.” To circumvent the constraints of space in the central pore, the positions of the N- and Cterminal ends that have been relocated are chosen to allow for joining or binding of other molecules to the modified chaperonin peptide.

“Modified chaperonin peptides according to the present invention are mutant chaperonin peptides. Modified chaperonin polypeptides have the same structural properties of naturally-occurring wild type chaperonin-subunit polypeptides. This includes folding into subunit structures with the same or similar shape to wild-type chaperonin-subunit polypeptides. To form a double-ringed chaperonin structure, the modified chaperonin foldable subunit structure can be self-assembled with other chaperonin structures (folded wild type and/or modified subunit polypeptides). To form a double-ringed chaperonin structure, the folded modified chaperonin unit structure can be self-assembled in vivo and in vitro. The double-ringed chaperonin structure of the invention, which contains at least one modified protein, can be organized into two dimensional crystals or filaments that are useful in producing nanodevices like logic and memory devices and non-linear optical device and sensing devices.

“The present invention contains modified chaperonin polypeptides or fragments or derivatives thereof. They can be derived from any source, natural, synthetic, semi-synthetic or recombinant.

“Modified chaperonin subunit peptides?”,?modified chaperonin peptides?”, and??modified polypeptides?” Modified chaperonin subunit polypeptides are those that have their N- and Cterminal ends relocated. These terms can be interchanged herein.

“Group I and II Chaperonin Polypeptides

Modified polypeptides can either be made using any group I or II chaperonin-polypeptide. Based on structural and sequence comparisons, chaperonins can be divided into Group I or II. (See, e.g., Trent et al., 1991 Nature 354: 490-493; Horwich et al., 1993 Phil. Trans R. Soc. Lond. 339: 313-326).”

“Group I chaperonins come from bacteria and bacterial-derived organelles in Eukarya (mitochondria, chloroplasts) while Group II chaperonins come from Archaea or eukaryotic cell cytosol. Trent (U.S. Patent.) discloses the description of endogenous wild-type TF55 shibatae and a comparison between a Group I chaperonin, GroEL, and the Group II chaperonin, TF55. No. 5,428,131).”

The modified chaperonin polypeptides can be made from any of the subunit proteins of Group I chaperonin. The wild-type Group I chaperonins have seven subunits within each of the two rings in the double-ring structure. Wild-type cpn60 proteins are approximately 550 to 580 amino acids long. They have been called different names in different species such as Escherichiacoli GroEL protein and Cyanobacterial GROEL analogues.

In one embodiment, for example, when using Group I chaperonins, chaperonin-polypeptides and/or mutant chaperonin-polypeptides, a cochaperonin may be used to form the higher order structures. In one example, the composition or device described in the invention also includes a co-chaperonin. The co-chaperonins can be easily identified by those skilled in the art (Harris and al. 1995 J. Structural Biol. 115: 68-77). A co-chaperonin is another example of such an embodiment. It can also be used in the production of nanofilaments. The cpn60 protein in E.coli (GroEL), is linked to a single ring structure of 10 kDa proteins, called?GroES. A GroES polypeptide is an example of a non-limiting species co-chaperonin. It can be used in conjunction with Group I chaperonins such as GroEL or GroEL derived chaperonins and chaperonins. The compositions, such as nanotemplates and nanostructures, can be made from any one or more chaperonins that have the co-chaperonin at one or both ends.

The modified chaperonin polypeptides can be made from any of the subunit proteins of Group II chaperonins. According to the organism, Group II chaperonins can be composed of identical or different subunits that are arranged in rings of eight to nine subunits. For example, eight subunits have been found in the Saccharomyces cerevisiae yeast ring (Lin and al., 1997 Proc. Natl. Acad. Sci. USA 94:10780-10785. Among the Archaea some thermophilic methanogens (e.g., Methanopyrus kandleri, Methanococcus jannaschii, Methanococcus thermolithotrophicus) have chaperonins with identical subunits (Furutani et al., 1998 J. Biol. Chem. Chem. Most of the 50 archaeal chaperonins sequences in the databases have >40% identity in amino acid sequences.

Sources of gene sequences encoding chaperonins polypeptides include, but are not limited, bacterial chaperonins genes encoding proteins such as Gro ES/Gro EL, archaeal chaperonins genes encoding proteins such as TF55, TF56 and cpn60s, mammalian chaperonins like Hsp60, TCP-1 and cpn60 as well as the homologues of these genes (1996) Heterologous genomic and cDNA libraries are also available for screening or selection of chaperonins.

“FIGS. “FIGS. S. shibatae’s TF55 beta subunit (SEQID NO. 1), E.coli (SEQID NO.2), and thermosome T. alophilum beta (SEQID NO.3). M. acetivorans (SEQID NO.5), M. tuberculosis (SEQID NO.6). The protein sequences are for S. pernix (SEQID NO.10), yeast TCP1 (SEQID NO.11), TCP1 subunit, (SEQID NO.14), TCP1 subunit, (SEQID NO.14), and TCP1 subunits (SEQID NO.1), as well as the TCP1 (SEQID NO.14), along with the TCP1 subunit, mouse TCP1 (SEQID NO.14), and consensus (SEQID NO.15). The sequence alignment is composed of white letters with black backgrounds, solid lines and dashed lines. These lines are used to surround regions containing identical residues.

“Folded Structure”

While Group I chaperonin protein subunits can possess more than 50% sequence identity (but less than 33% among subunits of Group II chaperonin proteins), it is possible to have sequence identity between them. Despite sequence differences among subunits of different species, the structural similarities between Group I and II cpn60 are significant. FIG. FIG. 3. A structural comparison of a subunit from the archaeal thermosome (Thermoplasma acidophilum), and the bacterial GroEL chaperonins (E. coli).

“The alignment was done using an algorithm that uses the iterative dynamic programming approach to protein science (Gerstein M. & Levitt M., Protein Science 7; 445-456 1998; Gerrstein M. & Levitt M, Proc. ISMB-96, pages. 59-67, 1996).”

For wild-type chaperonins or chaperonins polypeptides, this three-dimensional structural similarity illustrates that any chaperonin/chaperonin polypeptide can routinely be used as part of compositions or devices according to the invention. Modified chaperonins or chaperonins polypeptides are discussed in detail in next section. This sequence similarity provides teaching that allows routine manipulation of sequences to produce modified chaperonins polypeptides.

“The subunits have very similar structures in that they both possess an equitorial region, an intermediate, and an apical area. These two cpn60 examples are separated by sequence, which is evident by their very few similarities in sequence alignments (see FIGS. The crystal structures of these two cpn60 subunits are very similar, as evidenced by the lack of sequence alignments (see FIGS. 2A-2R). The loop of the thermosome shows that variations in structure can be tolerated in the Apical Domain, while the Equatorial Domains have similar conserved folding patterns.

“It should be noted that while chaperonins have been observed to contain seven, eight, or nine subunits per rings, the present invention provides methods, compositions, and means of exploiting chaperonins having any number of subunits per band (e.g. 7, 8, 9, or 10).

“Chaperonins can only contain one type of subunit, or they can include multiple subunits (e.g. archeal chaperonins that are alpha, beta and gamma). Because of differences in the protein sequences between subunits from different species, these subunits can be called alpha, beta, or gamma. It is common knowledge that subunits are found in different species. Chaperonin structure (Ellis and colleagues, 1998, J. Biol. Biol. This invention allows for the assembly of chaperonins using only one type of wild-type chaperonin polypeptide or modified chaperonin, or from different proportions of the various wild-type and modified chaperonin peptides.

“In one embodiment, HSP60s are heat-shock proteins found in organisms that live at high temperatures. These organisms are called?thermophiles. The source of the wild-type chaperonin polypeptides and the mutated chaperonin polypeptides are these proteins. These proteins are found in all organisms. They are also among the most abundant proteins within extreme thermophiles. For example, Pyrodictium occultum (one of the highest-temperature thermophiles), they reportedly account to 73% of total proteins (Phipps et. al., 1991 The EMBO Journal 10): 1711-1722).

“Selecting the Position for Relocated Terminal Ends”

“Using the amino acids sequence and/or predicted folded structure of any Group II wild-type chaperonin substrate polypeptide as a guide (FIGS. 2A-R, 15 and 15, one skilled in the art will be able to select the position of the amino acid to move the N- or C-terminal ends. You can choose the new terminal positions based on these criteria: The position must not be buried in the modified chaperonin protein fold; it should not be near the subunit interface; and it should not be part of the regular secondary structures.

Summary for “Versatile platform to nanotechnology based upon circular permutations chaperonin proteins”

“The controlled organization inorganic materials into multidimensional addressable arrays forms the foundation for logic and memory devices as well as other nonlinear optical or sensing devices” (Zhirnov et. al. 2001 Computer 34: 34?43; Xia et. al. 2000 Adv. Mater. 12: 693-713). These devices are made using lithographic patterns that have improved to produce smaller and more integrated parts. However, conventional lithographic methods are reaching their theoretical and practical limits at submicron scales. Ion and electron beam printing becomes too expensive and time-consuming at scales below 100nm. Further, quantum effects fundamentally alter the properties of devices at these scales (Sato, 1997 J. Appl. Phys. 82: 696).”

“Nanoscale templates are being created using both artificial and naturally-sourced materials for constrained synthesizing, in situ deposition or direct patterning nanometer scale inorganic arrangments. Artificial materials like microphase separated block copolymers are being developed (Park et. al. 2001 Appl. Phys. Lett. 79: 257-259), and hexagonally-closed spheres (Hulteen et.al. 1995 J. Vac. Sci. Technol. For nanoscale fabrication, A (1553-1558) was used. Natural materials such as DNA (Richter et al., 2000 Adv. Mater. 12: 507-510; Keren et al., 2002 Science 297: 72-75), bacterial and archaeal surface layer proteins (S-layer proteins) (Sleytr et al., 1999 Angew. Chem. Int. Ed. 38: 1034-1054; Douglas et al., Appl. Phys. Lett. 48: 676-678; Hall et al., 2001 CHEMPHYSCHEM 3: 184-186), virus capsids (Shenton et al., 1999 Adv. Mater. 11: 253-256; Douglas et al., 1999 Adv. Mater., 679-681; Douglas et al., Nature 393: 152-155; Wang et al., 2002 Angew. Chem. Int. Ed. 41: 459-462), phage [Lee et. al. 2002 Science 296] 892-895, and some globular proteins [Yamashita I. 2001 Thin Solid Films393: 12-18] have been used in templates and other nanoscale applications.

“Nanometer-scale objects at all scales, including arrays made by non-conventional techniques, are being investigated for possible alternatives to standard lithographically printed devices. The behavior of individual nanoparticles, also called quantum dots (QDs), has been demonstrated to be isolated components for devices such as single electron transistors (Likharev K. K. 1999 Proc. IEEE 87: 606-632; Thelander et al., 2001 Appl. Phys. Lett. 79: 2106-2108). Two-dimensional arrays, with nanoscale resolution, of QDs could be the basis for future generations of electronic or photonic devices. This is according to theoreticians. These devices will function based on phenomena like coulomb charging, interdot quantum tunneling, and other coherent properties derived electronically from confinement and nanoparticle volume to surface ratio ratios (Maier S. A. and al. 2001 Adv. Mater. 13: 1501-1505; Maier et al., Phys. Rev. B 65, 193408; Zrenner, A. et al., 2002 Nature 418: 612-614; Berven et al., 2001 Adv. Mater. 13: 109-113).”

“Traditional methods for patterning ordered arrays materials onto inorganic substrates or manufacturing devices are currently used by ion beam imaging and molecular beam epitaxy. These techniques have their limitations because of the use of polymeric lights masks for pattern formation. However, there is a theoretical limitation to patterning that could eventually limit the processes in the hundreds if not thousands of nanometers.

“While there are strong incentives for developing nanoscale architectures these developments require alternative fabrication methods and new insights into materials’ behavior on nanometer scales (Nalwa H. S. 2000?Handbook of Materials and Nanotechnology? Academic Press, San Diego).

“Development and application of new methods to order nanoscale materials from the ‘bottom up. New tools will be available for creating nanostructured materials, and devices that can self-assemble or repair themselves. Because of their ability to create structures at nanometer scales with minimal or no mechanical processing, synthetic and biological polymers are gaining attention. These natural and synthetic polymers can be self-assembled or phase separated to create nanoscale materials. Biopolymers have been shown to form complex structures and assemblies that possess highly specific chemical functions. All of these elements have been used to make nanostructured materials that exhibit unique properties (J Richter, et.al. 2000 Advanced Materials 12:507-551; M G Warner, J E Hutchison and K Keren 2003 Nature Materials 2:272-277 and K Keren, et.al. 2002 Nature Materials 1:247-252) and virions (W Shenton and S-W Lee et.al. 2002 Science 296;892-895; Q Wang et.al.

“Nanoscale templates have been created from a variety of protein complexes. These templates can be genetically modified to add chemically reactive sites that bind organic materials. Chaperonin complexes, for example, can be functionalized to attach soft metals. Chaperonins in nature are protein complexes that have two stacked rings, each containing 7, 8 or 9 subunits of HSP60. Mutations to the HSP60 subunits have been made that include single cysteine residues at solvent-exposed locations, including the apical pores. These cysteine residues’ thiols act as binding sites for zinc or gold (PCT/US02/35889). These mutant HSP60 subunits are bound to gold or zinc by the chaperonin complexes. They then form two-dimensional crystals.

“Protein complexes may also be modified to contain peptide sequences with desirable binding or catalytic properties. These protein complexes include subunits that have inserted peptide sequences. The mutant subunits might not fold, assemble into complexes, or organize into higher order structures. In addition, inserting a loop into a peptide sequence may render it inactive. Fusion to one of the local termini may not be sufficient to provide enough surface accessibility. Circular permutation is used to join peptides within a protein template. Circular permutation refers to a reordering the polypeptide chain so that the N- and Cterminal ends of the polypeptide chains are joined. New termini are then created elsewhere in the protein. Without affecting subunit assembly, new peptide sequences are possible to be joined to the new termini. Studies of protein circular permutation have shown that for proteins where the native amino-carboxyl termini are close in space, there are many relocated positions available for the new termini (P T Beernink and U Heinemann, 1995 Prog Biophys Mol Biol 63:121-143; M Iwakura et. al. 2000 Nat Struct Biol 7 :580-585).

“The invention provides chaperonin-subunit polypeptides that are modified to move the native N-terminal end and C-terminal ends out of the central pore to new locations on the exterior fold modified chaperonin polypeptide. A peptide sequence acts as a reporter by joining the N- and Cterminal ends. Modified chaperonin polypeptides are formed into subunits, which self-assemble into double-ringed chaperonin structure. The chaperonin structure organizes into higher order structures like two-dimensional crystals or filaments. The reporter peptide can also be functional. These chaperonin structures can be used to produce ordered nanoscale materials or devices.

“The invention provides chaperonin-polypeptides that have been modified to include N and C-terminal end points. These ends are moved from the central region of the chaperonin protein polypeptide to different locations on the polypeptide’s exterior.”

“The C-terminal and N-terminal ends of the nucleic acids can be covalently linked to or bound with a nucleic Acid molecule, a protein, a lipid, or carbohydrate or a nanoparticle.

“In the modified chaperonin peptide, the naturally occurring N-terminal (and C-terminal) ends are joined together either directly or with an intervening linker sequence. In one embodiment, the intervening linker sequence comprises the amino acid sequence Gly-Gly-Ser-Gly-Gly-Thr.”

“Modified chaperonin is a polypeptide based on either a Group I or Group II chaperonin.

“The Group I chaperonin peptides come from Escherichia and Cyanobacteria. Mycobacteria. Coxiella. Rickettsia. Rickettsia. Chlamydia. Thermotoga. Chloroplast. Mammalian mitochondria. Or yeast mitochondria. The Group II chaperonins polypeptides come from Sulfolobales and Pyrodictium.

“In one embodiment, modified chaperonin polypeptides from Sulfolobus are an alpha, beta or gamma polypeptide.”

“In one embodiment, the modified chaperonin protein comprises a Sulfolobus ShibataeTF55 beta polypeptide that has the N-terminal end and C-terminal ends moved after any amino acids position within the range 149 to158, in particular, moved to position 153.”

“Another embodiment of the modified chaperonin protein comprises a Sulfolobus ShibataeTF55 beta polypeptide that has the N-terminal end and C-terminal ends moved after any amino acids position within the range 263-270. In particular, it is moved to position 267.”

“Another embodiment of the modified chaperonin protein comprises a Sulfolobus ShibataeTF55 beta polypeptide that has the N-terminal end and C-terminal ends moved after any amino acids position within the range 311-330. In particular, it is moved to position 316.”

“Another embodiment of the modified chaperonin protein comprises a Sulfolobus Shibatae TF55 Beta polypeptide that has the N-terminal end and C-terminal ends moved after any amino acids position in the range 472-487. In particular, it is moved to position 480.”

“Another embodiment of the modified chaperonin protein comprises a Sulfolobus Shibatae TF55 beta peptide that has the N-terminal end and C-terminal ends repositioned after any amino acids position in the range 494-508 or 499, in particular.”

“The invention also provides assembled chaperonin structure, including at least one modified chaperonin unit.”

“The chaperonin structures assembled can have one or more of the following: 2-, 3-, 4, 5, 6-, 7, 5-, 6- and 7-fold symmetry.

“The invention also provides: nucleic acids molecules encoding modified chaperonin polypeptides of the invention; vectors containing these nucleic acids molecules; and host cell vectors.”

“The present invention provides in-vivo methods of producing modified chaperonin peptides. It involves cultivating host/vector systems in conditions that allow the host to make the modified chaperonin peptide.”

“The invention also includes in vitro methods of producing assembled chaperonin structure of the invention. This involves reacting modified chaperonin peptides under conditions that allow for self-assembly.

“The invention contains chaperonin structures which organize into higher order structures like nanofilaments and nanoarrays.” These nanofilaments or nanoarrays are useful for making nanodevices and nanocoatings.

“BRIEF DESCRIPTION ABOUT FIGURES”

“FIG. 1. illustrates an end- and side view of a HSP60 chaperonin model at 2.3? resolution. The side view’s outlined area shows one subunit of HSP60.

“FIGS. “FIGS. The dot-dashed boxes contain identical residues, while blocks of similar residues are contained in a solid box. Conservative matches are enclosed within a dashed box.

“FIG. 3. The structural alignment of the archaeal chaperonin, thermosome, and the bacterial chaperonin is shown (GroEL), which indicates the structural similarities between groups I and II chaperonins. The structural alignment shows where the features of both chaperonin subunits overlap in the black areas.

“FIG. “FIG.

“FIGS. “FIGS.

“FIGS. “FIGS.

“FIGS. 7A-E shows the assembly of engineered HSP60s, heat-shock proteins, into nanotemplates to produce nanoarrays containing nanoscale materials like nanoparticles.

“FIGS. 8A-D shows gold nanoparticles bound to engineered chaperonins or chaperonin nanotemplates.

“FIGS. 9A-D are semiconductor QD nanoarrays.”

“FIGS. 10A-D shows the formation of a nanoarray with gold nanoparticles. FIG. FIG. 10(D), shows XEDS spectrum of the bare carbon film (solid lines) and the gold nanoparticle array (dashed lines) taken from the probed area indicated by a circle in FIG. 10(B), as indicated with the arrow.

“FIGS. “FIGS.

“FIG. 12 is a control experiment that shows DIC (left), and fluorescent (right), images of non-cys mutated chaperonins crystals after incubation using CdSe.ZnS QDs.

“FIG. 13 shows the Energy Filtered TEM thickness mapping of a typical 2D Protein Crystal.”

“FIG. “FIG.

“FIG. “FIG.

“FIGS. “FIGS.

“FIGS. 17A-E are models of circular permutant protein models at positions: A) 53, B) 267 and C) 361; D) 480 and E) 499. Ribbon representations of subunits of protein are shown in the left column. The middle column shows side views showing surface representations for the assembled rings. The right column displays top views of the surface representations for the assembled rings. Amino termini will be labeled with blue. Carboxyl termini will be labeled with red. The flexible linker sequences in green are marked. One subunit of the assembled rings is highlighted in yellow.

“FIG. “FIG.

“FIGS. 19A-J is transmission electron microscopy of chaperonin double ring assemblies, which include circular permutant chaperonin protein (lacking fused EYFP). Panels A-E show samples containing isolated double ring samples. These were prepared one hour after adding ATP and MgCl2 protein samples. Panels F-J show samples containing higher-order structures. These were prepared 24 hours after adding ATP and Mg2+ protein samples. A) Circular permutant at position 53 at 1 hour. F) Circular permutant in position 153 at 24 hrs. B) Circular permutant in position 267 at 1 hr. G) Circular permutant at 267 at 24 hours. C) Circular permutant in position 316 at 1 Hour. H) Circular permutant in position 316 at 24hrs. D) Circular permutant in position 480 at 1 Hour. J) Circular permutant at 480 at 24hrs. E) Circular permutant in position 499 at 1 h. J) Circular permutant at 499 at 24 Hours

“FIG. 20A-D depicts the permutant chaperonin circular proteins at positions 267, 480, joined with yellow fluorescent protein EYFP. A) Fluorescence excitation spectra (open symbols and emission spectra) for unmodified EYFP. (circles), 267 permutant EYFP fusion proteins (squares), 480 Permutant EYFP Fusion protein (triangles). B) TEM showing negatively stained rings that were formed by the 480 permutant EYFP fusion proteins. C) Fluorescence microscopy (excitation 470nm, emission 515nm) of a crystal made by 267 permutant EYFP fusion proteins using Leica filter cube 13. D) TEM showing 2-dimensional crystal made by 267 permutant EYFP fusion proteins.

“FIG. “FIG.

“FIG. “FIG.

“FIG. “FIG.

“FIG. “FIG.

“FIG. “FIG.

“FIG. “FIG. 26 shows the complete (A) DNA sequence (SEQID NO: 76), and (B) amino acids (SEQID NO: 77) sequences of a position267 circular permutant chaperonin?EYFP fusion proteins.

“FIG. “FIG.

“FIG. “FIG.

“FIGS. 29A and 29B are the complete (A) DNA sequence (SEQID NO:81), and (B) amino acids (SEQID NO:82). This sequence is for a position 267 ‘dwarf. deletion circular permutant chaperonin protein.”

“FIG. “FIG.

“FIG. 31 shows a distinct DNA sequence (SEQ ID No:86) for TF55 beta subunit, Sulfolobus Shibatae.

“Definitions”

“All scientific terms and technical terms used herein have the same meanings as those used in the art, unless otherwise stated. The following words and phrases are used in this application.

“Isolated” is the term used herein. “Isolated” refers to a particular nucleic acid/polypeptide or a fragment thereof in which contaminants (i.e. The specific nucleic acids or polypeptide molecules have been removed from any substances that are different from them.

“Purified” is the term used herein. A specific nucleic acid or polypeptide is an isolated nucleic acids or polypeptide or a fragment thereof in which all contaminants (i.e. Any substances that are not the same as the nucleic or polypeptide molecule have been removed from the nucleic or polypeptide.

“As used herein, the term ?naturally-occurring? Refers to a nucleic acids or polypeptide molecule that is found in nature.

“Wild type” is the term used herein. A nucleic acid or polypeptide molecular having the same sequence of nucleotide/or amino acids as a naturally occurring, non-mutant molecule.

“Modified” is the term used herein. Modified molecules are those whose amino acid and nucleotide sequences are different (mutated) than a naturally occurring sequence, i.e. wild-type amino acid sequence or nucleotide sequence. Modified molecules may retain the same structural properties of a wild-type molecule.

“Derivative” is the term used herein. Any modification or alteration to a wild-type molecular structure. A derivative can include, but is not limited to, a conservative or non-conservative substitution in an amino acid or nucleotide sequence, including substitutions by nucleotides or amino acid analogs, a deletion of one/more amino acids or nucleotides, and the insertion or removal of one/more amino acids or nucleotides. A derivative molecule may share sequence similarities and/or activity with its parent.

“A first sequence of amino acids or nucleotides is referred to as sequence?identity” To a second nucleotide sequence or amino acid sequence, respectively. A comparison of the first sequence and the second sequences reveals that they are identical.”

“A first sequence of nucleotides or amino acids is considered to be similar if it is used in this context. A second sequence is considered similar to the first sequence if they are closely related (i.e. the sequences are almost identical). Two sequences may be considered similar if they have approximately 60% to 99.99% difference in nucleotides and amino acids.

“Complementary” is the term used herein. The term “complementary” refers to nucleic acids molecules that have purine and pyrimidine nucleotide base bases. These base pairs can be formed through hydrogen bonding, thereby mediating the formation of double-stranded nucleic acids molecules. Complementarity is found in the following base pairs: guanine, cytosine, adenine, and thymine and adenine-uracil. Complementary is applicable to any base pair that contains two single-stranded molecules of nucleic acids, or to any base pair that has a single-stranded molecule of nucleic acids folded onto itself.

“Conservative” is the term used herein. A substitution of an amino acid residue with another amino acid residue with similar chemical properties is called “conservative”. An example of conservative amino acids substitution is: substituting any nonpolar hydrophobic amino amino for any hydrophobic hydrophobic amino; substituting any hydrophilic (polarized, uncharged), amino amino for any hydrophilic amino; substituting any positively charged amino for any other positively charged amino; substituting any negatively charged amino for any other negatively charged amino (TE Creighton, Proteins? WH Freeman and Company. New York. These amino acid substitutions can include substituting any one of the following: isoleucine, valine, and leucine for any other hydrophobic amino acid; aspartic (D) to replace glutamic (E) or vice versa; glutamine [Q] for asparagine [N] and vice versa, and serine (S] for threonine (?T?) and vice versa. Other substitutions may also be acceptable depending on the amino acid’s role in the protein’s three-dimensional structure. Glycine (G), alanine, or serine (S), can often be interchangeable. Alanine, valine, and glycine can also be interchangeable. Methionine, which is relatively hydrophobic can often be interchanged with leucine or isoleucine and sometimes with valine. Lysine (K), and arginine, which are both relatively hydrophobic, are often interchangeable at locations where the most important feature of an amino acid residue’s charge is not significant and the pK differences between these two amino acids are not significant. Other changes may be acceptable in certain environments.

“Nonconservative” is the term used herein. It refers to the substitution of an amino acid for another amino acid that has different chemical characteristics. Nonconservative substitutions are, among others: aspartic acid (D), being replaced by glycine [G]; asparagine [N] being replaced by lysine [K]; or alanine (?A?) being replaced (R)

“The single-letter codes of amino acid residues are: A=alanine; R=arginine; N=asparagine; D=aspartic Acid, C=cysteine; Q=Glutamine and E=Glutamic Acid, G=glycine. H=histidine. I=isoleucine. L=leucine. K=lysine. M=methionine. F=phenylalanine. P=proline.

The following description will help you to better understand the invention described herein.

“Modified Chaperonin Polypeptides

The present invention includes modified chaperonin peptides and their fragments and derivations; assembled chaperonins containing the modified chaperonin peptides of this invention; nucleic acids molecules encoding modified chaperonin peptides or fragments and derived thereof; host-vector system; methods for producing modified chaperonin peptides; methods to use the modified chaperonin peptides such as producing bio-nanoscale devices and coatings

Chaperonins are essential subcellular structures found in nature. They have 14, 16 or 18 heat-shock protein subunits (e.g. HSP60) and are arranged as two stacked rings measuring approximately 16-18 nm high by 15-17 nm wide depending on the species they come from (Hartl and al. 2002 Science 295; 1852-8). Chaperonins possess a central pores, along with apical and intermediate domains. The chaperonin’s naturally occurring N- and Cterminal ends are found in the wild-type HSP60 protein unit. FIG. FIG. 1 shows an end-side view of a chaperonin, which has 16 subunits. There are eight subunits in each ring.

“The invention provides chaperonin-subunit polypeptides and fragments or derivatives thereof. These are modified to include Nterminal and Cterminal ends that are moved from their naturally-occurring locations to other positions in the polypeptide.

“In the modified chaperonin peptide, the N and C-terminal ends of the modified chaperonin peptide are moved from the central region to a new location on the exterior fold modified chaperonin peptide.” To circumvent the constraints of space in the central pore, the positions of the N- and Cterminal ends that have been relocated are chosen to allow for joining or binding of other molecules to the modified chaperonin peptide.

“Modified chaperonin peptides according to the present invention are mutant chaperonin peptides. Modified chaperonin polypeptides have the same structural properties of naturally-occurring wild type chaperonin-subunit polypeptides. This includes folding into subunit structures with the same or similar shape to wild-type chaperonin-subunit polypeptides. To form a double-ringed chaperonin structure, the modified chaperonin foldable subunit structure can be self-assembled with other chaperonin structures (folded wild type and/or modified subunit polypeptides). To form a double-ringed chaperonin structure, the folded modified chaperonin unit structure can be self-assembled in vivo and in vitro. The double-ringed chaperonin structure of the invention, which contains at least one modified protein, can be organized into two dimensional crystals or filaments that are useful in producing nanodevices like logic and memory devices and non-linear optical device and sensing devices.

“The present invention contains modified chaperonin polypeptides or fragments or derivatives thereof. They can be derived from any source, natural, synthetic, semi-synthetic or recombinant.

“Modified chaperonin subunit peptides?”,?modified chaperonin peptides?”, and??modified polypeptides?” Modified chaperonin subunit polypeptides are those that have their N- and Cterminal ends relocated. These terms can be interchanged herein.

“Group I and II Chaperonin Polypeptides

Modified polypeptides can either be made using any group I or II chaperonin-polypeptide. Based on structural and sequence comparisons, chaperonins can be divided into Group I or II. (See, e.g., Trent et al., 1991 Nature 354: 490-493; Horwich et al., 1993 Phil. Trans R. Soc. Lond. 339: 313-326).”

“Group I chaperonins come from bacteria and bacterial-derived organelles in Eukarya (mitochondria, chloroplasts) while Group II chaperonins come from Archaea or eukaryotic cell cytosol. Trent (U.S. Patent.) discloses the description of endogenous wild-type TF55 shibatae and a comparison between a Group I chaperonin, GroEL, and the Group II chaperonin, TF55. No. 5,428,131).”

The modified chaperonin polypeptides can be made from any of the subunit proteins of Group I chaperonin. The wild-type Group I chaperonins have seven subunits within each of the two rings in the double-ring structure. Wild-type cpn60 proteins are approximately 550 to 580 amino acids long. They have been called different names in different species such as Escherichiacoli GroEL protein and Cyanobacterial GROEL analogues.

In one embodiment, for example, when using Group I chaperonins, chaperonin-polypeptides and/or mutant chaperonin-polypeptides, a cochaperonin may be used to form the higher order structures. In one example, the composition or device described in the invention also includes a co-chaperonin. The co-chaperonins can be easily identified by those skilled in the art (Harris and al. 1995 J. Structural Biol. 115: 68-77). A co-chaperonin is another example of such an embodiment. It can also be used in the production of nanofilaments. The cpn60 protein in E.coli (GroEL), is linked to a single ring structure of 10 kDa proteins, called?GroES. A GroES polypeptide is an example of a non-limiting species co-chaperonin. It can be used in conjunction with Group I chaperonins such as GroEL or GroEL derived chaperonins and chaperonins. The compositions, such as nanotemplates and nanostructures, can be made from any one or more chaperonins that have the co-chaperonin at one or both ends.

The modified chaperonin polypeptides can be made from any of the subunit proteins of Group II chaperonins. According to the organism, Group II chaperonins can be composed of identical or different subunits that are arranged in rings of eight to nine subunits. For example, eight subunits have been found in the Saccharomyces cerevisiae yeast ring (Lin and al., 1997 Proc. Natl. Acad. Sci. USA 94:10780-10785. Among the Archaea some thermophilic methanogens (e.g., Methanopyrus kandleri, Methanococcus jannaschii, Methanococcus thermolithotrophicus) have chaperonins with identical subunits (Furutani et al., 1998 J. Biol. Chem. Chem. Most of the 50 archaeal chaperonins sequences in the databases have >40% identity in amino acid sequences.

Sources of gene sequences encoding chaperonins polypeptides include, but are not limited, bacterial chaperonins genes encoding proteins such as Gro ES/Gro EL, archaeal chaperonins genes encoding proteins such as TF55, TF56 and cpn60s, mammalian chaperonins like Hsp60, TCP-1 and cpn60 as well as the homologues of these genes (1996) Heterologous genomic and cDNA libraries are also available for screening or selection of chaperonins.

“FIGS. “FIGS. S. shibatae’s TF55 beta subunit (SEQID NO. 1), E.coli (SEQID NO.2), and thermosome T. alophilum beta (SEQID NO.3). M. acetivorans (SEQID NO.5), M. tuberculosis (SEQID NO.6). The protein sequences are for S. pernix (SEQID NO.10), yeast TCP1 (SEQID NO.11), TCP1 subunit, (SEQID NO.14), TCP1 subunit, (SEQID NO.14), and TCP1 subunits (SEQID NO.1), as well as the TCP1 (SEQID NO.14), along with the TCP1 subunit, mouse TCP1 (SEQID NO.14), and consensus (SEQID NO.15). The sequence alignment is composed of white letters with black backgrounds, solid lines and dashed lines. These lines are used to surround regions containing identical residues.

“Folded Structure”

While Group I chaperonin protein subunits can possess more than 50% sequence identity (but less than 33% among subunits of Group II chaperonin proteins), it is possible to have sequence identity between them. Despite sequence differences among subunits of different species, the structural similarities between Group I and II cpn60 are significant. FIG. FIG. 3. A structural comparison of a subunit from the archaeal thermosome (Thermoplasma acidophilum), and the bacterial GroEL chaperonins (E. coli).

“The alignment was done using an algorithm that uses the iterative dynamic programming approach to protein science (Gerstein M. & Levitt M., Protein Science 7; 445-456 1998; Gerrstein M. & Levitt M, Proc. ISMB-96, pages. 59-67, 1996).”

For wild-type chaperonins or chaperonins polypeptides, this three-dimensional structural similarity illustrates that any chaperonin/chaperonin polypeptide can routinely be used as part of compositions or devices according to the invention. Modified chaperonins or chaperonins polypeptides are discussed in detail in next section. This sequence similarity provides teaching that allows routine manipulation of sequences to produce modified chaperonins polypeptides.

“The subunits have very similar structures in that they both possess an equitorial region, an intermediate, and an apical area. These two cpn60 examples are separated by sequence, which is evident by their very few similarities in sequence alignments (see FIGS. The crystal structures of these two cpn60 subunits are very similar, as evidenced by the lack of sequence alignments (see FIGS. 2A-2R). The loop of the thermosome shows that variations in structure can be tolerated in the Apical Domain, while the Equatorial Domains have similar conserved folding patterns.

“It should be noted that while chaperonins have been observed to contain seven, eight, or nine subunits per rings, the present invention provides methods, compositions, and means of exploiting chaperonins having any number of subunits per band (e.g. 7, 8, 9, or 10).

“Chaperonins can only contain one type of subunit, or they can include multiple subunits (e.g. archeal chaperonins that are alpha, beta and gamma). Because of differences in the protein sequences between subunits from different species, these subunits can be called alpha, beta, or gamma. It is common knowledge that subunits are found in different species. Chaperonin structure (Ellis and colleagues, 1998, J. Biol. Biol. This invention allows for the assembly of chaperonins using only one type of wild-type chaperonin polypeptide or modified chaperonin, or from different proportions of the various wild-type and modified chaperonin peptides.

“In one embodiment, HSP60s are heat-shock proteins found in organisms that live at high temperatures. These organisms are called?thermophiles. The source of the wild-type chaperonin polypeptides and the mutated chaperonin polypeptides are these proteins. These proteins are found in all organisms. They are also among the most abundant proteins within extreme thermophiles. For example, Pyrodictium occultum (one of the highest-temperature thermophiles), they reportedly account to 73% of total proteins (Phipps et. al., 1991 The EMBO Journal 10): 1711-1722).

“Selecting the Position for Relocated Terminal Ends”

“Using the amino acids sequence and/or predicted folded structure of any Group II wild-type chaperonin substrate polypeptide as a guide (FIGS. 2A-R, 15 and 15, one skilled in the art will be able to select the position of the amino acid to move the N- or C-terminal ends. You can choose the new terminal positions based on these criteria: The position must not be buried in the modified chaperonin protein fold; it should not be near the subunit interface; and it should not be part of the regular secondary structures.

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