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WO2005005464A2 - Segment de liaison peptidique - Google Patents

Segment de liaison peptidique Download PDF

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Publication number
WO2005005464A2
WO2005005464A2 PCT/GB2004/002939 GB2004002939W WO2005005464A2 WO 2005005464 A2 WO2005005464 A2 WO 2005005464A2 GB 2004002939 W GB2004002939 W GB 2004002939W WO 2005005464 A2 WO2005005464 A2 WO 2005005464A2
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Prior art keywords
peptide
belt
braces
linker according
peptide linker
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PCT/GB2004/002939
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English (en)
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WO2005005464A3 (fr
Inventor
Derek Woolfson
Maxim Gennadievich Ryadnov
Bülent CEYHAN
Christof Niemeyer
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University Of Sussex
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Publication of WO2005005464A3 publication Critical patent/WO2005005464A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)

Definitions

  • the present invention relates to peptide linkers capable of linking components together.
  • the present invention also relates to methods of forming peptide linkers and linking components together.
  • Biomolecular recognition systems such as nucleic-acid hybridization and protein-ligand interactions, are being utilized in biosensor (1) and microarray technology (27).
  • biomolecules as components in supramolecular self-assembly to generate novel components for the development of advanced functional materials and molecular electronics (8, 9).
  • a key driver in these fields is the demand for smaller, cheaper and more-complex components. It is widely accepted that such improvements will not be fully realized using conventional "top-down” methods (e.g. photolithography), but will require fundamentally new approaches for the fabrication of electronic and other components (8). Consequently, complementary "bottom-up” technologies are being explored as potential ways for fabricating nanometer-sized devices.
  • Biomolecules have been established as effective tools for mediating supramolecular assembly of molecular and collodial components.
  • DNA oligonucleotides having unique and predictable self-recognition capabilities that make them promising starting materials for constructing well-defined nanostructures and assemblies of metal and semiconductor nanoparticles (8, 13, 14).
  • Fig 1A is an illustration of the assembly of nanoparticles using biomolecular linkers.
  • Nanoparticles are functionalized with individual recognition groups that are complementary for a separate molecular linker. Addition of the linker drives assembly of the particles to form extended networks, which, in some cases, grow to macroscopic materials.
  • Mirkin's group has pioneered this approach using oligonucleotide-based recognition motifs and has successfully applied it to solution-phase assembly in nanoparticles, (15-21) and the directed immobilization of nanoparticles on solid substrates, which allows for the fast and sensitive detection of nucleic acid analytes (22-25).
  • peptide-based linking systems introduce the possibility of recombinant protein production and, with it, the possibility of producing functional protein fusions as building blocks for new bioinspired materials.
  • the present invention provides a self-assembling peptide linker comprising a peptide belt and a plurality of peptide braces, the peptide belt comprising a plurality of peptide monomer units arranged as a strand, and each of the peptide braces comprising a plurality of peptide monomer units arranged as a strand, wherein the plurality of monomer units of the peptide braces are aligned with the plurality of monomer units in the peptide belt to form a substantially blunt ended coiled coil structure.
  • Such a linker allows easy assembly of molecular or colloidal components for use in various aspects of nanotechnology.
  • the linker allows components to be linked together.
  • the peptide linker is self-assembling, meaning that its elements will associate together to form a stable structure.
  • the linker comprises a peptide belt, which acts as a template for the assembly of the linker, bringing together the plurality of peptide braces. When mixed under appropriate conditions the peptide belt and peptide braces will automatically assemble to form the linker. Appropriate conditions for allowing the self-assembly of such peptides are well known in the art.
  • the peptide belt and peptide braces are peptides that together form a coiled coil.
  • Coiled coils are ⁇ -helical bundles with strands arranged in parallel, antiparallel or mixed topologies.
  • the linker forms a leucine zipper.
  • a leucine zipper is a type of coiled coil with two parallel strands. Coiled coil structures, and especially leucine zippers, are well known to those skilled in the art.
  • the peptide linker provided by the present invention introduces a new concept over what has gone before, namely that one full size peptide (belt) can template the assembly of smaller peptides (braces). In the absence of the belt, the braces do not associate. This is different from standard coiled-coil assembly, and has several advantages, including the fact that assembly of peptide can be initiated simply by adding the belts to the braces.
  • peptide refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the term "peptide". Equally the terms “protein” and “oligopeptide” are deemed to have the same meaning. The terms also do not exclude post-expression modifications of the peptide, for example, glycosylations, acetylations and phosphorylations. Included in the definition are peptides containing one or more analogs of an amino acid (including for example, unnatural amino acids), peptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and synthesised.
  • the peptide belt and peptide braces are made up of peptide monomer units, which comprise a series of amino acids selected to bring about the self assembly of the peptide belt and peptide braces into the linker.
  • the monomer units of the peptide belt and peptide braces comprise a heptad repeat motif (abcdefg), which is a 7 - residue repeat of hydrophobic (H) and polar (P) residues, arranged in the pattern HPPHPPP.
  • the residues in the repeat motif in the peptide belt are chosen so as to attract residues in the repeat motif in the peptide braces.
  • the monomer units may comprise a hendecad repeat motif (abcdefghijk).
  • the monomer units consist of the heptad or hendecad repeat motif. Such heptad and hendecad repeat motifs are well known to those skilled in the art and are described in WOO 1/21646.
  • the monomer units of the peptide braces are aligned with those in the peptide belt. This means that the first monomer unit in the peptide belt is aligned with the first monomer unit in one of the peptide braces, and the last monomer unit in the peptide belt is aligned with the last monomer unit in another peptide brace. Also, the first amino acid in each monomer unit in the peptide belt is aligned with the first amino acid in each of the corresponding monomer units in the peptide braces.
  • an amino acid in one position of the heptad (or hendecad) in the peptide belt is in line with an amino acid in the corresponding position in the heptad (or hendecad) in the peptide braces, a with a, b with b, c with c etc.
  • the linker is substantially blunt ended, which means that the ends of the peptide belt and peptide braces at each end of the linker are substantially flush with each other. That is to say, the peptide belt and peptide braces do not substantially over hang one another. If there is an overhang it is so small that the free end is not capable of interacting with the free end of another linker, thereby preventing the linkers of the present invention joining together to form an extended structure. Preferably there is less than 4 amino acids overhanging, more preferably less than 3 amino acids. Most preferably there is no overhang. This prevents the linkers from associating together.
  • the sum of the length of the plurality of braces is preferably substantially equal to the length of the belt.
  • the belt and the sum of the braces differ in length by no more than 4 amino acids, more preferably no more than 2 amino acids.
  • the residues at positions a, d, e and g preferably constitute the interface between the peptide belt and the peptide braces.
  • the amino acids at the a positions are preferably either isoleucine or asparagine. More preferably all but one of the residues at the a position in the peptide belt are isoleucine, and the remainder is asparagine. Where there is an asparagine in an a position in the peptide belt, there is preferably also an asparagine at the "corresponding position" in at least one of the peptide braces. "Corresponding position" means at the position in the peptide brace aligned with the asparagine in the peptide belt when the linker is formed. The remainder of a positions are preferably isoleucine.
  • the e and g positions in the peptide belt are preferably negatively charged residues, more preferably glutamic acids.
  • the e and g positions in the peptide braces are preferably basic residues, more preferably lysine. This helps to prevent autonomous folding of the peptide belt and peptide braces and encourages tertiary assembly.
  • the amino acids in the b and c positions are preferably alanine, glutamic acid, glutamine or lysine.
  • both b and c may be alanine, or b may be alanine, and c glutamic acid.
  • the amino acids in the /positions are also preferably alanine, glutamic acid, glutamine or lysine, but with one position in each strand being a modified amino acid, such as a tyrosine chromophore.
  • the peptide belt is composed of from 4 to 10 units, preferably 6 to 8, most preferably 6 units.
  • the peptide braces may or may not be equal in length. There are preferably 2 to 5 peptide braces, most preferably 2 peptide braces. In the case of 2 peptide braces, the peptide braces are preferably half the length of the peptide belt, and are equal in length.
  • the peptide braces are preferably composed of between 2 and 5 units, more preferably 3 and 4, most preferably 3 units. The sum of the number of units in the peptide braces is preferably equal to the number of units in the peptide belt.
  • the belt is preferably negatively charged, and the braces are preferably positively charged, or vice versa.
  • the linker of the present invention comprises a peptide belt and a first and a second peptide brace.
  • the N terminus of the first peptide brace is preferably aligned with the N terminus of the peptide belt.
  • a CysGlyGly tag is preferably added to the N terminus of the first peptide brace.
  • the C terminus of the second peptide brace is preferably aligned with the C terminus of the peptide belt.
  • a GlyGlyCys tag is preferably added to the C terminus of the second peptide brace. This allows derivatisation. Except for the C terminus of the first peptide brace, and the N terminus of the second peptide brace, the termini are preferably capped.
  • the peptide belt preferably has the sequence: IAALEKEIAALEQEIAALEKEIAALEYENAALEKEIAALEQE
  • the first peptide brace preferably has the sequence: IAALKQKIAALKQKIAALKYK
  • the second peptide brace preferably has the sequence: IAALKQKN-AALKQKIAALKYK
  • the linker allows components to be linked together, each component being attached to a separate peptide brace of the linker, or to the peptide belt, and the peptide belt and peptide braces together forming a reversible, stable structure.
  • the components are attached to separate peptide braces.
  • the components may be, for example, molecules, such as molecules in solution, peptides or proteins and peptide bound cargo, such as pharmaceuticals or analytical reagents.
  • the term "components" also encompasses surfaces, for example gold surfaces.
  • a method of producing peptide linkers comprising providing a mixture of peptide belt and peptide braces which associate to form a self-assembling peptide linker comprising a peptide belt and a plurality of peptide braces, the peptide belt comprising a plurality of peptide monomer units arranged as a strand, and each of the peptide braces comprising a plurality of peptide monomer units arranged as a strand, wherein the plurality of monomer units of the peptide braces are aligned with the plurality of monomer units in the peptide belt to form a substantially blunt ended coiled coil structure.
  • the peptide belt and peptide braces may be made by any standard procedure known to those skilled in the art, for example synthetically or recombinantly.
  • the invention also provides a kit for making a peptide linker according to the present invention comprising a peptide belt and peptide braces which associate to form the peptide linker according to the invention.
  • each of the components to be linked are separately coupled to, or include, the different elements of the linker (i.e. the peptide belt and the plurality of peptide braces).
  • the peptide belt and peptide braces are then allowed to associate and form the linker, thereby linking the components together.
  • each component is linked to a separate peptide brace.
  • the components and linker are formed separately, and the method includes the step of coupling the components to the peptide belt or peptide braces, prior to formation of the linker.
  • the peptide belt or peptide braces may be made as part of the components. This is particularly true of protein components.
  • the invention also provides a method of linking a protein to another component, which may or may not be another protein, wherein the protein is recombinantly formed and includes the peptide belts or the braces of a peptide linker of the invention.
  • the method of linking the components is preferably performed in solution.
  • the invention provides a method of linking a component to a surface (i.e. a solid support) using the peptide linker according to the invention.
  • a surface i.e. a solid support
  • the peptide belt is coupled to the surface
  • the peptide braces are coupled to other components.
  • the method can particularly be used for coupling components to gold surfaces, for use, for example, in biosensors.
  • the invention further provides a method of spacing components comprising coupling components to a peptide linker according to the invention.
  • the size of the linker is accurately known, hence by attaching the components to the linker their position in relation to each other can be accurately predicted.
  • the peptide linker according to the invention may also be used as a ruler or spacer, to measure distances in nanotechnology.
  • One linker can be used to space two or three components, or a plurality of linkers can be used to space more components, for example linearly, or forming a grid. This can be achieved by coupling each component to more than one belt or brace, so that each component will be coupled to more than one linker. When the linkers assemble they form a network.
  • a scaffold comprising a peptide linker according the invention.
  • the peptide linker can be used as a bridge between components in order to form a molecular scaffold.
  • the peptide linker can also be used to form hydrogels.
  • Figure 1 shows linker-mediated assembly of nanoparticles.
  • Figure IB illustrates peptide linkers in accordance with the invention, showing preferred linear sequences of the belt (A) and braces (B N and Be). Designed inter-peptide electrostatic interactions are depicted by double-headed arrows. The break between the BN and B c peptides is indicated by scissors. In (C) the sequences are drawn out on a 3.5-residue-per-turn helical wheel to show how the interfacial a, d, e and g positions of the heptad repeats come together. The break between the B N and Be peptides occurs between K 2 and 1 2 5, which are highlighted bold.
  • FIG. 2 is an illustration of a peptide assembly including peptide linkers in accordance with the invention probed by CD spectroscopy.
  • A Spectra for the individual first strand (broken line), a 1:1 mixture of the second and third strands (dotted line) and the 1:1:1 mixture of all three strands (solid discs). The open discs show the spectrum of the latter after thermal unfolding and cooling.
  • B Spectra for one of the two-component mixtures, namely BN plus the first strand in 1 :1 (dotted line) and 2:1 (dashed line) ratios, solid discs are as in (A). Samples were 100 ⁇ M in each peptide, 10 mM MOPS, 1 mM DTT, pH7 and at 5°C.
  • Figure 3 is a thermal unfolding image of a 20 ⁇ M peptide linker in accordance with the invention. Open discs show the raw CD data and the broken line gives the first derivative.
  • Figure 4 is an illustration of pH stability of a 20 ⁇ M peptide linker in accordance with the invention.
  • A CD spectra for 1:1:1 mixtures of the peptides recorded at pH 5.4 (solid line), 7 (crosses) and 8.6 (dashed line).
  • B [ ⁇ ] 222 versus pH.
  • FIG. 5 is an illustration of on-surface assembly of peptide linkers in accordance with the invention followed by SPR
  • A shows step-wise assembly of a peptide linker on a sensor chip starting with the coupling of Be followed by the addition of A and then B N *.
  • B shows coupling of BN followed by washing and addition of a 1:1 mix of A + Be*.
  • C shows peptide-concentration dependencies of the coupling of the cysteine-containing braces (open discs) and subsequent complex formation using the sequential (solid triangles for A, and open triangles for BN) and the alternative methods (solid discs).
  • N represents asparagines. Conditions: except for the experiments shown in panel D where peptide concentrations were varied, all peptide injections were 50 ⁇ L of 20 ⁇ M of each peptide in HEPES buffered saline at pH 7.4 and room temperature.
  • Figure 6 shows a UV-visible spectra for the 1 :1 BN AU :B C AU mixture before (solid line) and after (open circles) adding a stoichiometric amount of peptide A, the first strand.
  • Figure 7 shows direct observation of peptide-mediated nanoparticle assembly.
  • D a typical colloidal material from a mixture of the linker with one brace (BN AU or Bc Au ) in 1:1 or 1:2 ratios.
  • Figure 8 shows folding of the nanoparticle-bound peptides.
  • coiled-coil assemblies are relatively simple and sequence-to-structure relationships are available that permit their prediction and design.
  • coiled coils are bundles of between two and five amphiphatic helices, which assemble through their hydrophobic faces, Fig IC.
  • the nature of the residues at the interface determines coiled-coil oligomer state, helix orientation and partner selection.
  • the simplest and best understood coiled coils are the two-stranded, parallel leucine zippers.
  • the belt comprised six heptads. whilst the braces had * three each.
  • the braces had * three each.
  • the e and g positions of the belt were all made charged (glutamic acid), whilst the corresponding positions of the braces were all made basic (lysine).
  • the central a position of the C-terminal brace (Be) was made asparagine, as was its complement, the fifth a position in the belt; though destabilizing, the resulting asparagine-asparagine interaction is highly specific (40, 43, 48, 49).
  • the remaining b, c and /positions were made combinations of alanine, glutamic acid, and lysine; one /position in each peptide was reserved for a tyrosine chromophore.
  • CysGlyGly and GlyGlyCys tags were added to the N and C-termini of B N and Be, respectively.
  • the termini of all the peptides were capped.
  • CD experiments were conducted on a JASCO J-715 spectropolarimeter fitted with a Peltier temperature controller (Tokyo, Japan). SPR experiments were carried out on BIACORE 2000 instrument (Biacore AB, Uppsala, Sweden). TEM data were acquired using Hitachi 7 100 transmission electron microscope (Tokyo, Japan), fitted with a charge-coupled device camera from Digital Pixel Co. Ltd. (Brighton, UK) and software from Kinetic Imaging Ltd. (Liverpool, UK).
  • Peptide synthesis was carried out by the combination of standard Fmoc/tBu solid phase protocols with TBTU/DIPEA as coupling reagents on a PEG-PS-resin for carboxyl-free peptides and using a PAL linker for peptide amides.
  • a 95% TFA mixture (95:2.5:2.5 TFA/water/TIS) was used as the post-synthesis cleavage cocktail for non-cysteine peptides.
  • TFA mixture 93.5:2.5:2.5:1.5 TFA water/EDT/TIS
  • Sedimentation equilibrium experiments were conducted at 5°C. 100 ⁇ L samples of 100 ⁇ M peptide A or mixtures of the braces, and the belt plus both braces, with initial A 28 o values being of 0.15, 0.147 and 0.144, respectively, in the 1.2 cni path length cells used, were buffered to pH 7 with 10 mM MOPS containing 1 mM DTT and 100 m.M sodium chloride. Samples were equilibrated for 48 h at 30000, 37500 and 55000 rpm. Sedimentation equilibrium curves were measured by the absorbance at 28C resulting data were fitted simultaneously using routines in the Beckman Optima )M-A/XL-I da-a software (version 4.0).
  • the density of the buffer at 5°C was taken as 1.005 mg/mL. Based on the amino acid composition the averaged partial specific volume for the peptides was calculated to be 0.75 mL/mg for peptide A, 0.774 mL/mg for B N /B C , and 0.77 mL/mg for the equimolar mixture of A, BN and Be.
  • Circular dichroism spectroscopy All data for peptide samples prepared in 10 mM MOPS (pH 7) were collected in 1-mm quartz cuvettes. Data points for CD spectra were recorded at 1-nm intervals using a 1-nm bandwidth and 4-16-s response times. After baseline correction, ellipticities in mdeg were converted to molar ellipticities (deg cm 2 dmol res "1 ) by normalizing for the concentration of peptide bonds. Data points for the thermal unfolding curves were recorded through l°C/min ramps using a 2-nm bandwidth, averaging the signal for 8s at 1 °C intervals. Data for pH experiments were obtained for peptide samples in 10 mM EPPS (basic pH) and MES (acidic pH) buffers.
  • Gold nanoparticle conjugates were synthesized by treating 15 nm gold nanoparticles (1 ml of a 1 nM aqueous solution) separately with peptides Be and BN (25 ycl of 3 mM solutions, to give final peptide concentrations of 73 ⁇ M). After standing overnight the solution was centrifuged at 14000 rpm. The supernatant was removed and the red sediment washed twice and resuspended in water. «65 - 80% of the original Au nanoparticle concentration was recovered.
  • UV/Nis spectra were recorded on a Hitachi U3000 spectrometer. Quantification of the Au-peptide conjugates and spectral measurements of the aggregation experiments were carried out at room temperature in water.
  • Circular dichroism (CD) spectroscopy provides a convenient probe of ⁇ -helical structure in leucine-zipper systems (50). Consistent with the design concept, none the individual belt-and-braces peptides nor the combination of the two braces showed appreciable ⁇ -helix in solution by CD spectroscopy, ( Figure 2A). However, equimolar mixtures of all three peptides gave CD spectra indicative of considerable ⁇ -helix formation, Figure 2A.
  • the amount of helix was concentration dependent: based on the CD signal at 222 nm ([ ⁇ ]222), the percent helix for samples of 20 ⁇ M, 100 ⁇ M and 200 ⁇ M of each peptide were «70%, ⁇ 80% and «85%, respectively; these values were consistent with TFE-induced helix formation, which gave a benchmark for the upper limit of the [ ⁇ ] 22 in our system of «-32,000 deg cm 2 dmol "1 (45, 52).
  • the oligomerisation states of the belt-and-braces peptides were probed by sedimentation equilibrium analysis in an analytical ultracentrifuge. In these experiments all samples were 100 ⁇ M in each peptide, 10 mM MOPS, 1 mM DTT, pH 7 and at 5°C sedimentation equilibrium data were fitted assuming a single ideal species.
  • the returned molecular weights for the belt alone and for the 1 :1 mixture of the braces were consistent with the design and the foregoing CD data; that is, all peptides behaved as monomers and fitted the models well; the experimental molecular weights were 4875 Da (95% confidence limits 4592 and 5157) for the belt, and 2690 Da (2518 and 2857) for the braces, respectively, compared with the calculated relative molecular masses of 4624 for the belt, and 2570 and 2528 for the braces.
  • the belt-and-braces system comprises oppositely charged peptide strands - at neutral pH
  • the belt is negatively charged and the complementary braces are positive - we probed the ⁇ -helical content of the 20 ⁇ M 1:1 :1 complex in the pH range 5.4 and 8.6, Figure 4.
  • CD spectra in this range all showed a ⁇ -helix, Figure 4 A, whereas spectra of the individual peptides as well as the 1 :1 mixture of indicated random coil.
  • the helical content of the 1 :1:1 mixtures fell off rapidly outside the pH range 6.8 - 7.4,
  • Figure 5 shows typical SPR experiments in which the individual braces, and their binary combinations with the belt were passed over a Biacore sensor chip and the binding monitored. These were continuous-flow experiments, in which the instrument was first equilibrated with standard running buffer. Peptide-containing solutions were then injected, and after some defined time flow of the running buffer was resumed to wash away any unbound material. As changes in refractive index of the different buffers and solutions affect the SPR signal, only equilibrium signals - for example, the starting (t s ) and final (t F ) values - can be compared reliably. These signals, quoted in resonance units (RU), were used to gauge the mass of material bound to the surfaces and the percentage of complex formation on the chips.
  • RU resonance units
  • the belt-and-braces coiled coil would span 6 to 7 nm.
  • the system has potential as a self-assembling nanoscale linker of defined length. We tested this through the assembly of gold nanoparticles to extend the concept of DNA-based nanoparticles assembly, Figure 1A.
  • the brace peptides, BN and Bc were separately coupled to 15-nm colloidal gold particles to give BN AU and Bc Au . After removal of excess, unbound peptide, the derivatized particles were combined. The characteristic red color of the gold suspensions did not change either after peptide coupling, or mixing.
  • UV-Vis spectroscopy the conjugates showed no significant changes in gold-absorbance spectra compared with unmodified gold particles (data not shown). This is consistent with no assembly taking place and the designs of the belt-and-braces system.
  • flocculation assays revealed that both of the Au-peptide conjugates were more stable to increased salt (up to 1 M NaCl) than the bare An colloids.
  • brace peptides are positively charged and, so, inhibit particle aggregation and growth.
  • addition of the belt peptide to the mixture of brace-peptide-derivatized gold nanoparticles caused a color change and a precipitation of aggregated particles, which settled in the reaction vessel.
  • the surface plasmon resonance absorption band associated with the gold nanoparticles for this sample was broadened and red shifted (from 529 to 545 nm) compared with mixtures lacking the belt peptide, Figure 6.
  • Nanoparticle assemblies were visualized directly by transmission electron microscopy (TEM), Figure 7. As seen by others working with colloidal precipitates, we observed both 3-D and 2-D networks, Figures 7 A, and B & C, respectively. The former could not be analyzed with any certainty, but the 2-D networks showed uniformly separated particles at distances of 7.22 ⁇ 0.34 nm. These measurements are consistent with a folded six-heptad coiled-coil linker. The networks were found exclusively in preparations containing all three belt-and-braces components, B N AU , B c Au and peptide A: under similar conditions, bare gold colloids displayed only closely aggregated particles without distinct spacings indicative of non-specific particle-growth processes. Similarly, in the mixtures of peptide A with BN AU or Bc Au single particles and non-uniform aggreagtes dominated, Figure 7D.
  • TEM transmission electron microscopy
  • the system comprises three leucine-zipper peptides of de novo design: the longer peptide, A or "the belt”, acts as a template for the co-assembly of two half-sized peptides, B N and Be, also termed "the braces".
  • the basic features of the design were confirmed in solution by CD spectroscopy and analytical ultracentrifugation, which indicated a predominantly a-helical, ternary assembly. These assemblies were thermally stable and unfolded reversibly. Although specific positive and negative features were included in the design, some promiscuous belt-brace interactions were observed. However, these were not cooperatively folded structures, and were thermally labile.
  • belt-and-braces is a novel concept in coiled-coil assembly, and the first example of employing rationally designed peptides to guide nanoparticle assembly, which, hitherto, has been achieved using DNA-based linkers of some description.(18, 12-25)
  • peptides potentially offer some advantages over DNA in such applications.
  • synthetic genes for peptide sequences can be synthesized and cloned for use in recombinant DNA technologies.
  • the braces need not be added chemically, but brace-protein fusions could be engineered recombinantly. This possibility may unlock exciting new routes to enable the generation of novel and functional protein-based supramolecular and nanostructured assemblies and biosensors (58).

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Abstract

L'invention concerne un segment de liaison peptidique à autoassemblage qui comprend un peptide de pleine dimension structurant un ensemble de peptides plus petits.
PCT/GB2004/002939 2003-07-08 2004-07-08 Segment de liaison peptidique WO2005005464A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2592103A1 (fr) 2011-11-08 2013-05-15 Adriacell S.p.A. Dérivés d'aldéhyde de polymère
WO2022225987A1 (fr) * 2021-04-19 2022-10-27 University Of Florida Research Foundation, Incorporated Agents pour la production de peptides de co-assemblage

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* Cited by examiner, † Cited by third party
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AU695679B2 (en) * 1995-10-06 1998-08-20 Pence, Inc. Coiled-coil heterodimer methods and compositions for the detection and purification of expressed proteins
GB9922013D0 (en) * 1999-09-17 1999-11-17 Univ Sussex Peptides

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2592103A1 (fr) 2011-11-08 2013-05-15 Adriacell S.p.A. Dérivés d'aldéhyde de polymère
WO2013068117A1 (fr) 2011-11-08 2013-05-16 Adriacell S.P.A. Dérivés aldéhydes polymères
WO2022225987A1 (fr) * 2021-04-19 2022-10-27 University Of Florida Research Foundation, Incorporated Agents pour la production de peptides de co-assemblage

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