Session H18: De Novo Designed Peptides as Building Nanostructural Blocks

Responsive Polypeptide-based Block Copolymer Assemblies

Amphiphilic block copolymers of poly(butadiene) and poly(L- lysine) (PB-P(Lys)) as well as poly(propylene oxide) and P(Lys) (PPO-P(Lys)) were synthesized and their solution properties studied using dynamic light scattering and transmission electron microscopy. We exploit secondary structure changes that occur in the P(Lys) chain to observe changes in solution morphology as a function of solution conditions. At high pH, the P(Lys) chain assumes either an $\alpha$-helical or a $\beta$-sheet conformation depending on temperature, while at lower pH the side chains become protonated, resulting in an expanded coil configuration. In these studies, we explore the pH and temperature responsiveness for a series of block copolymers with varying morphology.   

Early Stages of De Novo Designed Beta-Hairpin Peptide Self-Assembly

In aqueous solution, MAX 1 peptide is unfolded and does not self-assemble. The peptide intramolecularly folds into a beta-hairpin when the electrostatic interactions between charged residues are screened through increasing the ionic strength at neutral pH. Beta-hairpin molecules supramolecularly assemble via hydrophobic collapse and hydrogen bonding into a 3-D hydrogel network. By combining the results of CD, cryo-TEM, DLS, and oscillatory rheology, we understand that the self-assembly proceeds by nucleation of monodisperse (3 nm wide) beta-sheet fibrils, which elongate, branch and cross-link to form clusters of fibrils. Assembly kinetics at this early stage indicates power law growth with assembly time. Eventually, clusters of fibrils interpenetrate to form a percolated network, as evidenced by the increasing network rigidity. The early stage assembly process will be discussed and compared to published gelation models.   

Effect of Strand Symmetry on the Nanostructure and Material Properties in Beta-Hairpin Peptide Hydrogels

Hydrogels have been established as promising biomaterials for applications such as scaffolds for tissue engineering, controlled drug delivery and cell encapsulation. De novo designed beta hairpin peptides, capable of undergoing self assembly and hydrogel formation, were investigated that contain asymmetric beta strand arms surrounding a turn sequence. The stimuli responsive self assembly of the hydrogels occurs via an intramolecular folding and strand interdigitation mechanism. CD and FTIR indicate a beta sheet secondary structure. WAXS shows a fibril structure reminiscent of the cross beta spine. SANS has been employed to globally quantify the local structure as being rod-like. Modification of the strand registry results in fibrils with non-twisting, laminated vs. twisted nanostructure. Fibril dimensions as measured by TEM and AFM corroborate the interdigitated assembly. Bulk material properties of these hydrogels studied using oscillatory rheology vary significantly for the different morphologies. Differences in the peptide registry that drive hydrogel nanostructure and the consequent material properties can be potentially utilized for usage in specific biomaterial applications.   

Self-assembling, bioactive protein hydrogels via engineered coiled-coil aggregation.

We describe associating triblock proteins with that self-assemble into reversible, nanostructured hydrogels with a regular network structure and specific biofunctional attributes. These fibrilar, telechelic designs consist of a hydrophilic random coil (denoted R) flanked by associating coiled-coil end domains (denoted A, B, C). The central R domain also encodes specific cell binding and signaling functions of extracellular matrix (ECM) constituents. We will discuss a series of proteins with complimentary associating end blocks that preferentially form heterotrimer aggregates of A, B, and C domains. Mixtures of symmetric triblocks ARA, BRB, and CRC in aqueous solution self assemble into reversible viscoelastic network structures, which we characterize using microscopy, light scattering techniques and computer simulations. Supporting circular dichroism and analytical ultracentrifugation studies of the secondary structure and association behavior of the A, B, C domains will also be presented. Through the use of microscopic and cell proliferation assays, we also show that these hydrogels are capable of inducing biomimetic responses of ECM constituents in cell culture experiments.   

Planar peptide processing

Spatial manipulation on small length scales of biological materials, in particular peptide based substances, is important both for implementing assays and for exploiting the properties of the materials set. In this talk we describe methods for patterning peptides in planar manipulations much as is exercised with materials in semiconductor processing: Controlled deposition into small length-scale patterns is accomplished through selective adhesion to patterned substrates or deposition through patterned masks, and removal of peptide films can be achieved through wet or dry etching techniques. These methods are shown to be applicable to at least the micron scale, and this technique summary presents an elemental tool-box for planar processing of this set of biological films. Collectively these techniques provide a ``toolbox'' of methods to accomplish rudimentary planar processing with peptides.   

Self-Assembling Octa-peptides

In this work we have focused on examining systematically the effect of hydrophobicity, charge distribution and size of amino acid on the self assembly behavior of a series of octa-peptides that have been synthesized in our laboratory: AEAEAKAK, AEAKAEAK, FEFKFEFK. FEFKFEFK, FDFDFRFR, FDFRFDFR, FDFDFKFK, FDFKFDFK, FKFDFDFK and FDFKFKFD. The structure of our systems have been elucidated using a combination of Fourier transform infra-red spectroscopy, atomic force microscopy and small angle neutron scattering. This work has shown that the peptides form beta-sheet rich fibrils that have circa 4-6 nm in diameter, and these can associate further along their length scales depending on the amino acid sequence. In some cases these fibrils, or thicker fibers, then become physically entangled to give rise to a 3-dimensional fibrillar hydrogel that does not flow upon inversion of the sample vial. The mechanical properties of all resulting hydrogels have been explored using oscillatory rheometry and results related back to hydrogel structure across the length scales. Here we will present phases diagrams, propose a generalized gelation mechanism and link molecular structure to macroscopic properties.   

Sequence Dependent Peptide Self-Assembly and Beta-Sheet Fibrils as Templates for Inorganic Material

Synthetic peptides have been designed to self-assemble into beta-sheet fibrils of varying morphology depending on the peptide sequence. Incorporation of a diproline sequence between two beta-sheet forming strands is used to affect peptide conformation and thus the self-assembly mechanism and resulting fibrillar morphology (e.g. twisted vs. untwisted). Peptide length, proline stereochemistry, diproline sequence position, and assembly kinetics are shown to significantly affect fibril morphology. Furthermore, fibrils of varying morphology are employed as templates for inorganic material such as amorphous silica. In addition, metal nanoparticles were synthesized and functionalized to interact with the fibrils resulting in laterally spaced, linear particle arrays.   

Incorporation of Designed Extended Chromophores into Amphiphilic 4-helix Bundle Peptides for Biomolecular Materials

De novo designed peptides, together with synthetic non-biological cofactors, could lead to peptide-based~systems with novel properties not exhibited by biological systems. Extended chromophores can be designed and tailored, with appropriate donors, acceptors and constituents,~to exhibit selected nonlinear optical responses and light-induced electron transport and/or proton~translocation over large distances. Designed extended chromophores can be incorporated into the amphiphilic 4-helix bundle peptides via bis-histidyl ligation. Amphiphilic 4-helix bundle peptide monolayer, both the apo- and holo-form, can be oriented vectorially at the air/water interface. Nanoporous thin films made from diblock copolymers are ideal templates to assemble the artificial proteins with laterally hexagonal order. We will also discuss the efforts on re-designing the artificial proteins and incorporate them into block copolymer based nanoporous templates.   

Turning protein into room temperature molecular magnet

Metallothionein-2 (MT-2) is a cysteine-rich protein that binds seven divalent transition metal ions avidly via its metal-thiol linkages. A magnetic MT-2 containing two Mn and five Cd (Mn,Cd-MT-2) has been synthesized by protein refolding process. No trace of Fe was detected by ICP mass spectroscopy. The uniform size distribution, tested by dynamic light scattering, indicated that each Mn,Cd-MT-2 molecule is a single molecular magnet. Its coercive field of ferromagnetic signals changed slightly from 50 to 300K, but dropped rapidly when the temperature rose from 330 to 395 K. The blocking temperature T$_{B}$ is around 410K, in powder form. These results indicated that the un-paired electron of both Mn$^{2+}$ might be aligned by electron hoping of the bridging sulfurs in the $\beta $-metal binding cluster of MT-2 and when the protein deformed at 410K the ferromagnetic signals disappear correspondingly. This engineered molecule exhibits both molecular magnetization and bio-compatibility. These features make Mn,Cd-MT-2, a good candidate for biological applications and sensing sources of new nano-devices.   

Interaction of the synthetic polypeptide poly(FFDD) with single-walled carbon nanotubes

Dispersion of bulk-synthesized single-walled carbon nanotubes (SWCNT) and their subsequent assembly into beneficial structures, especially in aqueous medium, requires the interaction of amphiphilic moieties. Among these, proteins as well as \textit{de-novo} polypeptides have been found to provide useful functional SWCNT dispersions. The synthetic polypeptides reported so far have rather elaborate sequences, which are deemed necessary for the specific conformations that successfully interact with the SWCNT surface. We have sought to study simple oligo-peptides and their basic interactions with SWCNTs in water. An oligo-peptide: poly(FFDD) [F=phenyl alanine, D = aspartic acid] with 30 amino-acid units, exhibiting and alternating hydrophobic/hydrophilic motif, was synthesized and used successfully to disperse SWCNTS. Small-angle neutron scattering (SANS) measurements with contrast variations were performed in different D$_{2}$O/H$_{2}$O mixtures. The SANS patterns show that poly(FFDD) alone in water assembles into a complex structure. However, an open conformation which is loosely attached to the SWCNT surface is indicated by SANS.   

Investigating the specificity of adsorption of onto gold by gold-binding peptides using molecular dynamics simulations

It is possible to engineer artificial peptide sequences showing high specificity of adsorption for surfaces like gold, platinum or other solid materials. However, the reasons behind that high specificity are not clear. We investigate the adsorption of a genetically engineered peptide with high gold specificity using all-atom molecular dynamics simulations. Accurate Lennard-Jones parameters describing the interactions of gold with both water and amino acids are not currently available, so thus we discuss assignment of appropriate values. Two sets of simulations are presented: one using peptides made of a gold-binding motif (MHGKTQATSGTIQS) and another using peptides made of a non gold-binding motif (AIRRDVNCIGASMH). Adsorption onto the (111) and the (100) crystalline faces of gold is investigated. We discuss our results in light of the features of the peptide (sequence, charge, structure, nature of the amino acids) that may be responsible for the specificity of the gold-binding motif for gold.   

Direct Assembly of Periplasmic Binding Proteins on Gold Surfaces

We present a new and very promising approach to protein based biosensor design, which uses a technique called nanografting to immobilize proteins at addressable locations on Au surfaces. In nanografting, an Atomic Force Microscope (AFM) tip is used to disrupt a preexisting monolayer of alkanethiol molecules on a gold surface, thereby facilitating exchange with alternative thiol-linked proteins from the surrounding solution. This technique opens the possibility of preparing highly ordered, nanometer size protein arrays that can be patterned at different addressable locations on the surface. We also use the AFM to monitor the ligand-induced conformational changes of periplasmic binding proteins nanografted on Au substrates.