Responsive hydrogels from the intramolecular folding and self-assembly of a designed peptide

A general peptide design is presented that links the pH-dependent intramolecular folding of beta-hairpin peptides to their propensity to self-assemble, affording hydrogels rich in beta-sheet. Chemical responsiveness has been specifically engineered into the material by linking intramolecular folding to changes in solution pH, and mechanical responsiveness, by linking hydrogelation to self-assembly. Circular dichroic and infrared spectroscopies show that at low pH individual peptides are unstructured, affording a low-viscosity aqueous solution. Under basic conditions, intramolecular folding takes place, affording amphiphilic beta-hairpins that intermolecularly self-assemble. Rheology shows that the resulting hydrogel is rigid but is shear-thinning. However, quick mechanical strength recovery after cessation of shear is observed due to the inherent self-assembled nature of the scaffold. Characterization of the gelation process, from the molecular level up through the macroscopic properties of the material, suggests that by linking the intramolecular folding of small designed peptides to their ability to self-assemble, responsive materials can be prepared. Cryo-transmission electron and laser scanning confocal microscopies reveal a water-filled porous scaffold on both the nano- and microscale. The environmental responsiveness, morphology, and peptidic nature make this hydrogel a possible material candidate for biomedical and engineering technology.     

Inherent antibacterial activity of a peptide-based beta-hairpin hydrogel

Among several important considerations for implantation of a biomaterial, a main concern is the introduction of infection. We have designed a hydrogel scaffold from the self-assembling peptide, MAX1, for tissue regeneration applications whose surface exhibits inherent antibacterial activity. In experiments where MAX1 gels are challenged with bacterial solutions ranging in concentrations from 2 x 10(3) colony forming units (CFUs)/dm2 to 2 x 10(9) CFUs/dm2, gel surfaces exhibit broad-spectrum antibacterial activity. Results show that the hydrogel surface is active against Gram-positive (Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, all prevalent in hospital settings. Live-dead assays employing laser scanning confocal microscopy show that bacteria are killed when they engage the surface. In addition, the surface of MAX1 hydrogels was shown to cause inner and outer membrane disruption in experiments that monitor the release of beta-galactosidase from the cytoplasm of lactose permease-deficient E. coli ML-35. These data suggest a mechanism of antibacterial action that involves membrane disruption that leads to cell death upon cellular contact with the gel surface. Although the hydrogel surface exhibits bactericidal activity, co-culture experiments indicate hydrogel surfaces show selective toxicity to bacterial versus mammalian cells. Additionally, gel surfaces are nonhemolytic toward human erythrocytes, which maintain healthy morphologies when in contact with the surface. These material attributes make MAX1 gels attractive candidates for use in tissue regeneration, even in nonsterile environments.     

Thermally reversible hydrogels via intramolecular folding and consequent self-assembly of a de novo designed peptide

A small de novo designed peptide (MAX3) is described that exhibits complete thermoreversible self-assembly into a hydrogel network. Importantly, a prerequisite to hydrogelation is that the peptide must first fold into a conformation conducive to self-assembly. At ambient temperature, MAX3 is unfolded, resulting in a low viscosity aqueous solution. On increasing the temperature, the peptide undergoes a unimolecular folding event, affording an amphiphilic beta-hairpin that consequently self-assembles into a hydrogel network. Increasing the temperature serves to dehydrate the nonpolar residues of the unfolded peptide and trigger folding via hydrophobic collapse. Cooling the resultant hydrogel results in beta-hairpin unfolding and consequent complete dissolution of the hydrogel. The temperature at which folding and consequent self-assembly into a rigid hydrogel occur can be tuned by altering the hydrophobicity of the peptides.     

Design and application of basic amino acids displaying enhanced hydrophobicity

Three noncoding basic amino acids, mono-, di-, and trimethyldiaminopropionic acid (mmdap, dmdap, and tmdap), have been synthesized for use in protein design. Covalent modification of a diaminopropionic acid (dap) side chain with an increasing number of methyl moieties results in a family of residues displaying short basic side chains with varying degrees of enhanced hydrophobic character. These residues may be used to introduce charged/polar interactions into the confining hydrophobic interior or interfacial spaces of proteins. As a demonstration of their utility, the ability of these residues to promote interior salt bridge formation at the helix/helix interface of GCN4-p1, a dimeric two-stranded coiled coil, was assessed. Heterodimerization mediated by buried salt bridge formation between a GCN4-based peptide containing either mmdap, dmdap, or tmdap at position 16 and an analogous peptide containing aspartic acid at the same position was studied. Mmdap-derived heterodimers are 0.5 kcal/mol more stable than the corresponding dap-derived heterodimers. This result indicates that the addition of one methyl group to the dap side chain can stabilize the heterodimeric fold. The stabilization can most likely be attributed to a decrease in the desolvation penalty incurred upon folding as well as enhanced van der Waals contacts in the folded state. The addition of three methyl groups to the dap side chain results in heterodimers that are significantly less stable than the corresponding dap-derived heterodimers, suggesting that increased steric bulk is not well accommodated in the interior of this protein. Unexpectedly, the addition of two methyl groups leads to homotrimerization of the dmdap-peptide. The resulting trimer is relatively stable (DeltaG(37)( degrees )(C) degrees = 11.8 kcal/mol) and undergoes cooperative thermal unfolding. The GCN4-p1 system exemplifies how small incremental changes in size and hydrophobicity can alter the folding preferences of a protein. Generally, this versatile suite of residues can be utilized in any protein and offer new options to the protein chemist.     

Effects of lipopolysaccharide-induced inflammation on expression of growth-associated genes by corticospinal neurons

Background  

Inflammation around cell bodies of primary sensory neurons and retinal ganglion cells enhances expression of neuronal growth-associated genes and stimulates axonal regeneration. We have asked if inflammation would have similar effects on corticospinal neurons, which normally show little response to spinal cord injury. Lipopolysaccharide (LPS) was applied onto the pial surface of the motor cortex of adult rats with or without concomitant injury of the corticospinal tract at C4. Inflammation around corticospinal tract cell bodies in the motor cortex was assessed by immunohistochemistry for OX42 (a microglia and macrophage marker). Expression of growth-associated genes c-jun, ATF3, SCG10 and GAP-43 was investigated by immunohistochemistry or in situ hybridisation.     

Beta-delayed proton emitter kr

The nucleide ⁷³Kr has been identified by on-line mass separation as a precursor of β-delayed proton emission. The proton branch is (6.8 ±1.2) × 10⁻³ proton/decay. The protons populate the ground state and also the first excited 2⁺ state at 866 keV in ⁷²Se with a relative intensity of (35±9) %. The value of Q_EC−B_p, where B_p is the proton separation energy for the nucleus ⁷³Br, is found to be 4.85 ±0.30 MeV based on the fraction of proton events preceded by positron decay.