Collagen is the most abundant protein in animals and the major component of connective tissues. Although collagen isolated from natural sources has long served as the basis for some biomaterials, natural collagen is difficult to modify and can engender pathogenic and immunological side effects. Collagen comprises a helix of three strands. Triple helices derived from synthetic peptides are much shorter (<10 nm) than natural collagen (Ϸ300 nm), limiting their utility. Here, we describe the synthesis of short collagen fragments in which the three strands are held in a staggered array by disulfide bonds. Data from CD spectroscopy, dynamic light scattering, analytical ultracentrifugation, atomic force microscopy, and transmission electron microscopy indicate that these ''sticky-ended'' fragments self-assemble via intermolecular triple-helix formation. The resulting fibrils resemble natural collagen, and some are longer (>400 nm) than any known collagen. We anticipate that our self-assembly strategy can provide synthetic collagen-mimetic materials for a variety of applications.biomaterial ͉ coiled-coil ͉ nanotechnology ͉ cystine knot ͉ peptide C ollagen constitutes one-third of the human proteome, including three-quarters of the dry weight of human skin. Its high natural abundance and intrinsic plasticity have spurred the development of collagen as a biomaterial (1, 2). The most common source of clinical collagen is now Bos taurus, the domestic cow. Unfortunately, bovine collagen can illicit deleterious pathological and immunological effects when transplanted into humans (3-5). Moreover, the preparation of enriched solutions of natural collagen is problematic (6), and its sitespecific covalent modification is not feasible. We suspected that synthetic chemistry could offer a solution to these problems.The quaternary structure of collagen comprises three strands that wrap around one another to form a triple helix (7). Each strand has the repeating sequence XaaYaaGly, with the most abundant triplet being ProHypGly [Hyp ϭ (2 S,4R)-4-hydroxyproline] (8). The folding of (XaaYaaGly) nՅ10 peptides into blunt-ended triple helices has been investigated thoroughly (7). Although such triple helices are biomaterial candidates (9-12), they are limited to the length of a synthetic peptide (Ͻ10 nm), which is much shorter than natural collagen (Ϸ300 nm). The polymerization of (XaaYaaGly) 10 peptides has afforded long strands that adopt triple-helical structure but have high polydispersity (13).Molecular self-assembly underlies the ''bottom-up'' approach to macromolecular design, wherein a desirable structure forms spontaneously through noncovalent interactions (14, 15). Selfassembling peptides and proteins have been designed to serve as materials for biological and nanotechnological applications (16,17). Using the self-assembly approach, Woolfson and coworkers (18,19) have produced fibers with a design based on a dimeric coiled-coil structure. Likewise, fibrous peptides based on the natural protein elastin (20) and de novo building b...

The lipophilic nucleoside, G 1, extracts Pb(2+) picrate from water into organic solvents to give structures based on the hydrogen-bonded G-quartet. Crystal structures indicate important differences between (G 1)(8)-Pb(2+) and (G 1)(8)-K(+). The divalent Pb(2+) templates a smaller G(8) cage than does K(+), as judged by the M-O6 bond length, O6-O6 diagonal distance, and inter-tetramer separation. The more compact Pb(2+) octamer correlates with NMR data indicating that N2-N7 hydrogen bonds in (G 1)(8)-Pb(2+) are kinetically more stable than in (G 1)(8)-K(+).

The ion transport activity of calix[4]arene tetrabutylamide 1,3-alt 2 was studied in liposomes, planar lipid bilayers, and HEK-293 cells. These experiments, when considered together with (1)H NMR and X-ray crystallography data, indicate that calix[4]arene tetrabutylamide 2 (1) forms ion channels in bilayer membranes, (2) mediates ion transport across cell membranes at positive holding potential, (3) alters the pH inside liposomes experiencing a Cl(-) gradient, and (4) shows a significant Cl(-)/SO(4)(2)(-) transport selectivity. An analogue, calix[4]arene tetramethylamide 1, self-assembles in the presence of HCl to generate solid-state structures with chloride-filled and water-filled channels. Structureminus signactivity studies indicate that the hydrophobicity, amide substitution, and macrocyclic framework of the calixarene are essential for HCl binding and transport. Calix[4]arene tetrabutylamide 2 is a rare example of an anion-dependent, synthetic ion channel.

No abstract

Nucleic acid quadruplexes are composed of guanine quartets stabilized by specific metal ions. X-ray diffraction can provide high-resolution information on the structure and metal binding properties of quadruplexes, but only if they can be crystallized. NMR can provide detailed information on the solution structure of such quadruplexes but little quantitative data concerning the metal binding site. Here we apply extended X-ray absorption fine structure (EXAFS) measurements to characterize the metal ion binding site, in frozen solution, of the unimolecular quadruplex formed by the thrombin binding aptamer, d(G 2 T 2 G 2 TGTG 2 T 2 G 2 ) (TBA), in the presence of Pb 2+ ions. The Pb L III -edge X-ray absorption spectrum of this metal-DNA complex is very similar to that we obtain for a Pb 2+ -stabilized quartet system of known structure constructed from a modified guanine nucleoside (G1). The Fourier transforms of the Pb 2+ complexes with both TBA and G1 show a first-shell interaction at about 2.6 Å, and a weaker, broader shell at 3.5-4.0 Å. Quantitative analysis of the EXAFS data reveals the following: (i) very close agreement between interatomic distances at the metal coordination site for the Pb 2+ -G1 complex determined by EXAFS and by X-ray crystallography; (ii) similarly close agreement between interatomic distances measured by EXAFS for the Pb 2+ -G1 and Pb 2+ -TBA complexes. These results provide strong evidence for binding of the Pb 2+ ion in the region between the two quartets in the Pb 2+ -TBA complex, coordinated to the eight surrounding guanine O6 atoms. The specific binding of Pb 2+ to DNA examined here may be relevant to the genotoxic effects of this environmentally important heavy metal. Furthermore, these results demonstrate the utility of EXAFS as a method for quantitative characterization of specific metal binding sites in nucleic acids in solution.Metal ion-DNA interactions are important in nature, often impacting the genetic material's structure and function. To better understand the molecular details of these interactions, it is essential that we have a reliable picture of the metal ion coordination site. This is not always so straightforward. Thus, X-ray diffraction can provide high-resolution information on the structure and metal binding properties of nucleic acids, but only if diffraction-quality crystals can be obtained. NMR can provide detailed information on solution structure, but usually little quantitative data concerning the metal binding site. This paper describes how extended X-ray absorption fine structure (EXAFS) 1 measurements can be used to gain detailed information about a Pb 2+ binding site within a folded DNA oligonucleotide.DNA and RNA can form quadruplex structures in the presence of certain metal ions, based on the guanine quartet, which is shown in Figure 1A. Only a small number of X-ray crystal structure determinations on quadruplexes have been reported, and these have located the metal binding site in the region between two quartet stacks, or coplanar with one...

Prolyl 4-hydroxylases install a hydroxyl group in the 4R configuration on the γ-carbon atom of certain (2S)-proline (Pro) residues in tropocollagen, elastin, and other proteins and other proteins to form (2S,4R)-4-hydroxyproline (Hyp). The gauche effect arising from this prevalent post-translational modification enforces a Cγ-exo ring pucker and stabilizes the collagen triple helix. The Hyp diastereomer (2S,4S)-4-hydroxyproline (hyp) has not been observed in a protein, despite the ability of electronegative 4S substituents to enforce the more common Cγ-endo ring pucker of Pro. Here, we use density functional theory, spectroscopy, crystallography, and calorimetry to explore the consequences of hyp incorporation on protein stability using a collagen model system. We find that the 4S-hydroxylation of Pro to form hyp does indeed enforce a Cγ-endo ring pucker, but a transannular hydrogen bond between the hydroxyl moiety and the carbonyl of hyp distorts the main-chain torsion angles that typically accompany a Cγ-endo ring pucker. This same transannular hydrogen bond enhances an n→π* interaction that stabilizes the trans conformation of the peptide bond preceding hyp, endowing hyp with the unusual combination of a Cγ-endo ring pucker and high trans:cis ratio. O-Methylation of hyp to form (2S,4S)-4-methoxyproline (mop) eliminates the transannular hydrogen bond and restores a prototypical Cγ-endo pucker. mop residues endow the collagen triple helix with much more conformational stability than do hyp residues. These findings highlight the critical importance of the configuration of the hydroxyl group installed on Cγ of proline residues.

In this paper, we report on the formation and properties of a water-stabilized dimer comprising calix[4]arene-guanosine conjugate cG 2. The 1,3-alternate calixarene cG 2 was poorly soluble in dry CDCl(3) and gave an ill-resolved NMR spectrum, consistent with its nonspecific aggregation. The compound was much more soluble in water-saturated CDCl(3). Two sets of well-resolved (1)H NMR signals for the guanosine residues in cG 2, present in a 1:1 ratio, indicated that the compound's D(2) symmetry had been broken and provided the first hint that cG 2 dimerizes in water-saturated CDCl(3). The resulting dimer, (cG 2)(2).(H(2)O)(n)(), has a unique property: it extracts alkali halide salts from water into organic solution. This dimer is a rare example of a self-assembled ion pair receptor. The identity of the (cG 2)(2).NaCl.(H(2)O)(n)() dimer was confirmed by comparing its self-diffusion coefficient in CDCl(3), determined by pulsed-field gradient NMR, with that of control compound cA 3, an adenosine analogue. The dimer's stoichiometry was also confirmed by quantitative measurement of the cation and anion using ion chromatrography. Two-dimensional NMR and ion-induced NMR shifts indicated that the cation binding site is formed by an intermolecular G-quartet and the anion binding site is provided by the 5'-amide NH groups. Once bound, the salt increases the dimer's thermal stability. Both (1)H NMR and ion chromatography measurements indicated that the cG 2 dimer has a modest selectivity for extracting K(+) over Na(+) and Br(-) over Cl(-). The anion's identity also influences the association process: NaCl gives a soluble, discrete dimer whereas addition of NaBPh(4) to (cG 2)(2).(H(2)O)(n)() leads to extensive aggregation and precipitation. This study suggests a new direction for ion pair receptors, namely, the use of molecular self-assembly. The study also highlights water's ability to stabilize a functional noncovalent assembly.

This contribution describes the discovery and properties of a synthetic, low-molecular weight compound that transports Cl- across bilayer membranes. Such compounds have potential as therapeutics for cystic fibrosis and cancer. The H+/Cl- co-transport activities of acyclic tetrabutylamides 1-6 were compared by using a pH-stat assay with synthetic EYPC liposomes. The ion transport activity of the most active compound, trimer 3, was an order of magnitude greater than that of calix[4]arene tetrabutylamide C1 a macrocycle known to function as a synthetic ion channel. Trimer 3 has an unprecedented function for a synthetic compound, as it induces a stable potential in liposomes experiencing a transmembrane Cl-/SO42- gradient. Data from both pH-stat and 35Cl NMR experiments indicate that 3 co-transports H+/Cl-. Although 3 transports both Cl- and H+ the overall process is not electrically silent. Thus, trimer 3 induces a stable potential in LUVs due to a transmembrane anionic gradient. The ability of trimer 3 to transport Cl-, to maintain a transmembrane potential, along with its high activity at uM concentrations, its low molecular weight, and its simple preparation, make this compound a valuable lead in drug development for diseases caused by Cl- transport malfunction.