Anatomy of a selectively coassembled ß-sheet peptide nanofiber.

Peptide self-assembly, wherein molecule A associates with other A molecules to form fibrillar ß-sheet structures, is common in nature and widely used to fabricate synthetic biomaterials. Selective coassembly of peptide pairs A and B with complementary partial charges is gaining interest due to its potential for expanding the form and function of biomaterials that can be realized. It has been hypothesized that charge-complementary peptides organize into alternating ABAB-type arrangements within assembled ß-sheets, but no direct molecular-level evidence exists to support this interpretation. We report a computational and experimental approach to characterize molecular-level organization of the established peptide pair, CATCH. Discontinuous molecular dynamics simulations predict that CATCH(+) and CATCH(-) peptides coassemble but do not self-assemble. Two-layer ß-sheet amyloid structures predominate, but off-pathway ß-barrel oligomers are also predicted. At low concentration, transmission electron microscopy and dynamic light scattering identified nonfibrillar ∼20-nm oligomers, while at high concentrations elongated fibers predominated. Thioflavin T fluorimetry estimates rapid and near-stoichiometric coassembly of CATCH(+) and CATCH(-) at concentrations ≥100 µM. Natural abundance 13C NMR and isotope-edited Fourier transform infrared spectroscopy indicate that CATCH(+) and CATCH(-) coassemble into two-component nanofibers instead of self-sorting. However, 13C-13C dipolar recoupling solid-state NMR measurements also identify nonnegligible AA and BB interactions among a majority of AB pairs. Collectively, these results demonstrate that strictly alternating arrangements of ß-strands predominate in coassembled CATCH structures, but deviations from perfect alternation occur. Off-pathway ß-barrel oligomers are also suggested to occur in coassembled ß-strand peptide systems.

Side-Chain Chemistry Governs Hierarchical Order of Charge-Complementary ß-sheet Peptide Coassemblies.

Self-assembly of proteinaceous biomolecules into functional materials with ordered structures that span length scales is common in nature yet remains a challenge with designer peptides under ambient conditions. This report demonstrates how charged side-chain chemistry affects the hierarchical co-assembly of a family of charge-complementary ß-sheet-forming peptide pairs known as CATCH(X+/Y-) at physiologic pH and ionic strength in water. In a concentration-dependent manner, the CATCH(6K+) (Ac-KQKFKFKFKQK-Am) and CATCH(6D-) (Ac-DQDFDFDFDQD-Am) pair formed either ß-sheet-rich microspheres or ß-sheet-rich gels with a micron-scale plate-like morphology, which were not observed with other CATCH(X+/Y-) pairs. This hierarchical order was disrupted by replacing D with E, which increased fibril twisting. Replacing K with R, or mutating the N- and C-terminal amino acids in CATCH(6K+) and CATCH(6D-) to Qs, increased observed co-assembly kinetics, which also disrupted hierarchical order. Due to the ambient assembly conditions, active CATCH(6K+)-green fluorescent protein fusions could be incorporated into the ß-sheet plates and microspheres formed by the CATCH(6K+/6D-) pair, demonstrating the potential to endow functionality.

Optimisation of root traits to provide enhanced ecosystem services in agricultural systems: A focus on cover crops.

Roots are the interface between the plant and the soil and play a central role in multiple ecosystem processes. With intensification of agricultural practices, rhizosphere processes are being disrupted and are causing degradation of the physical, chemical and biotic properties of soil. However, cover crops, a group of plants that provide ecosystem services, can be utilised during fallow periods or used as an intercrop to restore soil health. The effectiveness of ecosystem services provided by cover crops varies widely as very little breeding has occurred in these species. Improvement of ecosystem service performance is rarely considered as a breeding trait due to the complexities and challenges of belowground evaluation. Advancements in root phenotyping and genetic tools are critical in accelerating ecosystem service improvement in cover crops. In this study, we provide an overview of the range of belowground ecosystem services provided by cover crop roots: (1) soil structural remediation, (2) capture of soil resources and (3) maintenance of the rhizosphere and building of organic matter content. Based on the ecosystem services described, we outline current and promising phenotyping technologies and breeding strategies in cover crops that can enhance agricultural sustainability through improvement of root traits.

Post-assembly α-helix to ß-sheet structural transformation within SAF-p1/p2a peptide nanofibers.

We report an unanticipated helix-to-sheet structural transformation within an assembly of SAF-p1 and SAF-p2a designer peptides. Solid-state NMR spectroscopic data support the assembled structure that was targeted by rational peptide design: an α-helical coiled-coil co-assembly of both peptides. Subsequent to assembly, however, the system converts to a ß-sheet structure that continues to exhibit nearest-neighbor interactions between the two peptide components. The structural transition occurs at pH 7.4 and exhibits strongly temperature-dependent kinetics between room temperature (weeks) and 40 °C (minutes). We further observed evidence of reversibility on the timescale of months at 4 °C. The structural conversion from the anticipated structure to an unexpected structure highlights an important aspect to the challenge of designing peptide assemblies. Furthermore, the conformational switching mechanism mediated by a prerequisite α-helical nanostructure represents a previously unknown route for ß-sheet designer peptide assembly.

Parallel ß-Sheet Structure and Structural Heterogeneity Detected within Q11 Self-Assembling Peptide Nanofibers.

Q11 peptide nanofibers are used as a biomaterial for applications such as antigen presentation and tissue engineering, yet detailed knowledge of molecular-level structure has not been reported. The Q11 peptide sequence was designed using heuristics-based patterning of hydrophobic and polar amino acids with oppositely charged amino acids placed at opposite ends of the sequence to promote antiparallel ß-sheet formation. In this work, we employed solid-state nuclear magnetic resonance spectroscopy (NMR) to evaluate whether the molecular organization within Q11 self-assembled peptide nanofibers is consistent with the expectations of the peptide designers. We discovered that Q11 forms a distribution of molecular structures. NMR data from two-dimensional (2D) 13C-13C dipolar-assisted rotational resonance indicate that the K3 and E9 residues between Q11 ß-strands are spatially proximate (within ∼0.6 nm). Frequency-selective rotational echo double resonance (fsREDOR) on K3 Nζ and E9 Cδ-labeled sites showed that approximately 9% of the sites are close enough for salt bridge formation to occur. Surprisingly, dipolar recoupling measurements revealed that Q11 peptides do not assemble into antiparallel ß-sheets as expected, and structural analysis using Fourier-transform infrared spectroscopy and 2D NMR alone can be misleading. 13C PITHIRDS-CT dipolar recoupling measurements showed that the most abundant structure consists of parallel ß-sheets, in contrast to the expected antiparallel ß-sheet structure. Structural heterogeneity was detected from 15N{13C} REDOR measurements, with approximately 22% of ß-strands having antiparallel nearest neighbors. We cannot propose a complete structural model of Q11 nanofibers because of the complexity involved when examining structurally heterogeneous samples using NMR. Altogether, our results show that while heuristics-based patterning is effective in promoting ß-sheet formation, designing a peptide sequence to form a targeted ß-strand arrangement remains challenging.

De novo design of peptides that coassemble into ß sheet-based nanofibrils.

Peptides' hierarchical coassembly into nanostructures enables controllable fabrication of multicomponent biomaterials. In this work, we describe a computational and experimental approach to design pairs of charge-complementary peptides that selectively coassemble into ß-sheet nanofibers when mixed together but remain unassembled when isolated separately. The key advance is a peptide coassembly design (PepCAD) algorithm that searches for pairs of coassembling peptides. Six peptide pairs are identified from a pool of ~106 candidates via the PepCAD algorithm and then subjected to DMD/PRIME20 simulations to examine their co-/self-association kinetics. The five pairs that spontaneously aggregate in kinetic simulations selectively coassemble in biophysical experiments, with four forming ß-sheet nanofibers and one forming a stable nonfibrillar aggregate. Solid-state NMR, which is applied to characterize the coassembling pairs, suggests that the in silico peptides exhibit a higher degree of structural order than the previously reported CATCH(+/−) peptides.

Engineering ß-Sheet Peptide Coassemblies for Biomaterial Applications.

Peptide coassembly, wherein at least two different peptides interact to form multicomponent nanostructures, is an attractive approach for generating functional biomaterials. Current efforts seek to design pairs of peptides, A and B, that form nanostructures (e.g., ß-sheets with ABABA-type ß-strand patterning) while resisting self-assembly (e.g., AAAAA-type or BBBBB-type ß-sheets). To confer coassembly behavior, most existing designs have been based on highly charged variants of known self-assembling peptides; like-charge repulsion limits self-assembly while opposite-charge attraction promotes coassembly. Recent analyses using solid-state NMR and coarse-grained simulations reveal that preconceived notions of structure and molecular organization are not always correct. This perspective highlights recent advances and key challenges to understanding and controlling peptide coassembly.

CATCH Peptides Coassemble into Structurally Heterogeneous ß-Sheet Nanofibers with Little Preference to ß-Strand Alignment.

Coassembling peptides offer an additional degree of freedom in the design of nanostructured biomaterials when compared to analogous self-assembling peptides. Yet, our understanding of how amino acid sequences encodes coassembled nanofiber structure is limited. Prior work on a charge-complementary pair, CATCH+ and CATCH- peptides, detected like-peptide nearest neighbors (CATCH+:CATCH+ and CATCH-:CATCH-) within coassembled ß-sheet nanofibers; these self-associated peptide pairs marked a departure from an "ideal" coassembled structure. In this work, we employ solid-state NMR, isotope-edited FTIR, and coarse-grained molecular dynamics simulations to evaluate the alignment of ß-strands within CATCH peptide nanofibers. Both experimental and computational results suggest that CATCH molecules coassemble into structurally heterogeneous nanofibers, which is consistent with our observations in another coassembling system, the King-Webb peptides. Within ß-sheet nanofibers, ß-strands were found to have nearest neighbors aligned in-register parallel, in-register antiparallel, and out-of-register. In comparison to the King-Webb peptides, CATCH nanofibers exhibit a greater degree of structural heterogeneity. By comparing the amino acid sequences of CATCH and King-Webb peptides, we can begin to unravel sequence-to-structure relationships, which may encode more precise coassembled ß-sheet nanostructures.

Rational Design of Antigen Incorporation Into Subunit Vaccine Biomaterials Can Enhance Antigen-Specific Immune Responses.

Peptide subunit vaccines increase safety by reducing the risk of off-target responses and improving the specificity of the induced adaptive immune response. The immunogenicity of most soluble peptides, however, is often insufficient to produce robust and lasting immunity. Many biomaterials and delivery vehicles have been developed for peptide antigens to improve immune response while maintaining specificity. Peptide nanoclusters (PNC) are a subunit peptide vaccine material that has shown potential to increase immunogenicity of peptide antigens. PNC are comprised only of crosslinked peptide antigen and have been synthesized from several peptide antigens as small as 8 amino acids in length. However, as with many peptide vaccine biomaterials, synthesis requires adding residues to the peptide and/or engaging amino acids within the antigen epitope covalently to form a stable material. The impact of antigen modifications made to enable biomaterial incorporation or formation is rarely investigated, since the goal of most studies is to compare the soluble antigen with biomaterial form of antigen. This study investigates PNC as a platform vaccine biomaterial to evaluate how peptide modification and biomaterial formation with different crosslinking chemistries affect epitope-specific immune cell presentation and activation. Several types of PNC were synthesized by desolvation from the model peptide epitope SIINFEKL, which is derived from the immunogenic protein ovalbumin. SIINFEKL was altered to include extra residues on each end, strategically chosen to enable multiple conjugation chemistry options for incorporation into PNC. Several crosslinking methods were used to control which functional groups were used to stabilize the PNC, as well as the reducibility of the crosslinking. These variations were evaluated for immune responses and biodistribution following in vivo immunization. All modified antigen formulations still induced comparable immune responses when incorporated into PNC compared to unmodified soluble antigen alone. However, some crosslinking methods led to a significant increase in desirable immune responses while others did not, suggesting that not all PNC were processed the same. These results help guide future peptide vaccine biomaterial design, including PNC and a wide variety of conjugated and self-assembled peptide antigen materials, to maximize and tune the desired immune response.

Charge guides pathway selection in ß-sheet fibrillizing peptide co-assembly.

Peptide co-assembly is attractive for creating biomaterials with new forms and functions. Emergence of these properties depends on the peptide content of the final assembled structure, which is difficult to predict in multicomponent systems. Here using experiments and simulations we show that charge governs content by affecting propensity for self- and co-association in binary CATCH(+/-) peptide systems. Equimolar mixtures of CATCH(2+/2-), CATCH(4+/4-), and CATCH(6+/6-) formed two-component ß-sheets. Solid-state NMR suggested the cationic peptide predominated in the final assemblies. The cationic-to-anionic peptide ratio decreased with increasing charge. CATCH(2+) formed ß-sheets when alone, whereas the other peptides remained unassembled. Fibrillization rate increased with peptide charge. The zwitterionic CATCH parent peptide, "Q11", assembled slowly and only at decreased simulation temperature. These results demonstrate that increasing charge draws complementary peptides together faster, favoring co-assembly, while like-charged molecules repel. We foresee these insights enabling development of co-assembled peptide biomaterials with defined content and predictable properties.

Molecular complementarity and structural heterogeneity within co-assembled peptide ß-sheet nanofibers.

Self-assembling peptides have garnered an increasing amount of interest as a functional biomaterial for medical and biotechnological applications. Recently, ß-sheet peptide designs utilizing complementary pairs of peptides composed of charged amino acids positioned to impart co-assembly behavior have expanded the portfolio of peptide aggregate structures. Structural characterization of these charge-complementary peptide co-assemblies has been limited. Thus, it is not known how the complementary peptides organize on the molecular level. Through a combination of solid-state NMR measurements and discontinuous molecular dynamics simulations, we investigate the molecular organization of King-Webb peptide nanofibers. KW+ and KW- peptides co-assemble into near stoichiometric two-component ß-sheet structures as observed by computational simulations and 13C-13C dipolar couplings. A majority of ß-strands are aligned with antiparallel nearest neighbors within the ß-sheet as previously suggested by Fourier transform infrared spectroscopy measurements. Surprisingly, however, a significant proportion of ß-strand neighbors are parallel. While charge-complementary peptides were previously assumed to organize in an ideal (AB)n pattern, dipolar recoupling measurements on isotopically diluted nanofiber samples reveal a non-negligible amount of self-associated (AA and BB) pairs. Furthermore, computational simulations predict these different structures can coexist within the same nanofiber. Our results highlight structural disorder at the molecular level in a charge-complementary peptide system with implications on co-assembling peptide designs.