Hydroxyproline-free Single Composition ABC Collagen Heterotrimer

Hydroxyproline plays a major role in stabilizing collagenous domains in eukaryotic organisms. Lack of this modification is associated with significant lowering in thermal stability of the collagen triple helix and may also affect fibrillogenesis and folding of the peptide chains. In contrast, even though bacterial collagens lack hydroxyproline, their thermal stability is comparable to fibrillar collagen. This has been attributed to the high frequency of charged amino acids found in bacterial collagen. Here we report a thermally stable hydroxyproline-free ABC heterotrimeric collagen mimetic system composed of decapositive and decanegative peptides and a zwitterionic peptide. None of the peptides contain hydroxyproline and furthermore the zwitterionic peptide does not even contain proline. The heterotrimer is electrostatically stabilized via multiple interpeptide lysine-aspartate and lysine-glutamate salt-bridges and maintains good thermal stability with a melting temperature of 37 °C. The ternary peptide mixture also populates a single composition ABC heterotrimer as confirmed by circular dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy. This system illustrates the power of axial salt-bridges to direct and stabilize the self-assembly of a triple helix and may be useful in analogous designs in expression systems where the incorporation of hydroxyproline is challenging

Comparative NMR analysis of collagen triple helix organization from N- to C-termini

The collagen triple helix consists of three supercoiled solvent-exposed polypeptide chains, and by dry weight it is the most abundant fold in mammalian tissues. Many factors affecting the structure and stability of collagen have been determined through the use of short synthetically prepared peptides, generally called collagen-mimetic peptides (CMPs). NMR (nuclear magnetic resonance spectroscopy) investigations into the molecular structure of CMPs have suffered from large amounts of signal overlap and degeneracy because of collagen’s repetitive primary sequence, the close and symmetric packing of the three chains and the identical peptide sequences found in homotrimers. In this paper a peptide library is prepared in which a single isotopic 15N-Gly label is moved sequentially along the peptide backbone. Our approach allows for a more explicit examination of local topology than available in past reports. This reveals larger regions of disorder at the C-terminus than previously detected by crystallographic or NMR studies, and here C-terminal fraying is seen to extend for six amino acids in a (POG)10 sequence. Furthermore, small sequence changes at the N-terminus greatly influence the degree of this localized disorder and may be useful in the future design of CMPs to maximize collagen’s interstrand hydrogen bonding pattern. Our approach and data serves as a reference for future CMP characterizations to determine the quality and extent of folding

Rational Design of a Non-canonical “Sticky-Ended” Collagen Triple Helix

In a canonical collagen triple helix, three peptides self-assemble into a supercoiled motif with a one-amino-acid offset between the peptide chains. Design of triple helices that contain more than one residue offset is lucrative, as it leaves the non-covalent interactions unsatisfied at the termini and renders the termini “sticky” to further self-assemble into collagen-like nanofibers. Here we use lysine–glutamate axial salt-bridges to design a heterotrimeric collagen triple helix, ABC-1, containing a non-canonical offset of four residues between the peptide chains. The four-residue offset is necessary to prevent aggregation, which would prevent characterization of the non-canonical chain arrangement at the molecular level by NMR spectroscopy. A second heterotrimer, ABC-2, also stabilized by axial salt-bridges, is designed containing a canonical one-amino-acid offset to facilitate comparison of structure and stability by CD and NMR. ABC-1 and ABC-2 demonstrate our ability to modulate chain offset in a collagen triple helix. This lays the groundwork to design longer, and therefore stickier, offsets allowing access to a new class of collagen-related nanostructures

Rational Design of Single-Composition ABC Collagen Heterotrimers

Design of heterotrimeric ABC collagen triple helices is challenging due to the large number of competing species that may be formed. Given the required one amino acid stagger between adjacent peptide strands in this fold, a ternary mixture of peptides can form as many as 27 triple helices with unique composition or register. Previously we have demonstrated that electrostatic interactions can be used to bias the helix population toward a desired target. However, homotrimeric assemblies have always remained the most thermally stable species in solution and therefore comprised a significant component of the peptide mixture. In this work we incorporate complementary modifications to this triple-helical design strategy to destabilize an undesirable competing state while compensating for this destabilization in the desired ABC composition. The result of these modifications is a new ABC triple-helical system with high thermal stability and control over composition, as observed by NMR. An additional set of modifications, which exchanges aspartate for glutamate, results in an overall lowering of stability of the ABC triple helix yet shows further improvement in the system’s specificity. This rationally designed system helps to elucidate the rules governing the self-assembly of synthetic collagen triple helices and sheds light on the biological mechanisms of collagen assembly

Keplerate cluster (Mo-132) mediated electrostatic assembly of nanoparticles

Gooch J, Jalan AA, Jones S, et al. Keplerate cluster (Mo-132) mediated electrostatic assembly of nanoparticles. Journal of Colloid and Interface Science. 2014;432:144-150.The electrostatic assembly between a series of differently charged Mo-132-type Keplerates present in the compounds (NH4)(42)[{(Mo-VI)(Mo5O21)-O-VI(H2O)(6)}(12) {(MO2O4)-O-V(CH3COO)}(30)].ca. {300 H2O + 10 CH3COONH4} (Mo-132a), (NH4)(72-n)[{(H2O)(81-n) + (NH4)(n)} {(Mo-VI)(Mo5O21)-O-VI(H2O)(6)}(12) {(Mo2O4)-O-V(SO4)}(30)].ca. 200 H2O (Mo-132b), and Na-10(NH4)(62)[{(Mo-VI)(Mo5O21)-O-VI(H2O2)(6)}(12) {(Mo2O4)-O-V(HPO4)}(30). ca. {300H(2)O + 2Na(+) + 2NH(4)(+) + 4H(2)PO(4)(-)) (Mo-132c) with cationic gold nanoparticles (AuNPs) was investigated for the first time. The rapid electrostatic assembly from nanoscopic entities to micron scale aggregates was observed upon precipitation, which closely matched the point of aggregate electroneutrality. Successful assembly was demonstrated using UV-vis, DLS, TEM, and zeta-potential analysis. Results indicate that the point at which precipitation occurs is related to charge balance or electroneutrality, and that counterions at both the Mo-132 and AuNP play a significant role in assembly. (C) 2014 Elsevier Inc. All rights reserved

Chain alignment of collagen I deciphered using computationally designed heterotrimers.

The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register

Highly Angiogenic Peptide Nanofibers

Major limitations of current tissue regeneration approaches using artificial scaffolds are fibrous encapsulation, lack of host cellular infiltration, unwanted immune responses, surface degradation preceding biointegration, and artificial degradation byproducts. Specifically, for scaffolds larger than 200–500 μm, implants must be accompanied by host angiogenesis in order to provide adequate nutrient/waste exchange in the newly forming tissue. In the current work, we design a peptide-based self-assembling nanofibrous hydrogel containing cell-mediated degradation and proangiogenic moieties that specifically address these challenges. This hydrogel can be easily delivered by syringe, is rapidly infiltrated by cells of hematopoietic and mesenchymal origin, and rapidly forms an extremely robust mature vascular network. Scaffolds show no signs of fibrous encapsulation and after 3 weeks are resorbed into the native tissue. These supramolecular assemblies may prove a vital paradigm for tissue regeneration and specifically for ischemic tissue disease