Peptoid nanosheets exhibit a new secondary-structure motif.

A promising route to the synthesis of protein-mimetic materials that are capable of complex functions, such as molecular recognition and catalysis, is provided by sequence-defined peptoid polymers--structural relatives of biologically occurring polypeptides. Peptoids, which are relatively non-toxic and resistant to degradation, can fold into defined structures through a combination of sequence-dependent interactions. However, the range of possible structures that are accessible to peptoids and other biological mimetics is unknown, and our ability to design protein-like architectures from these polymer classes is limited. Here we use molecular-dynamics simulations, together with scattering and microscopy data, to determine the atomic-resolution structure of the recently discovered peptoid nanosheet, an ordered supramolecular assembly that extends macroscopically in only two dimensions. Our simulations show that nanosheets are structurally and dynamically heterogeneous, can be formed only from peptoids of certain lengths, and are potentially porous to water and ions. Moreover, their formation is enabled by the peptoids' adoption of a secondary structure that is not seen in the natural world. This structure, a zigzag pattern that we call a Σ('sigma')-strand, results from the ability of adjacent backbone monomers to adopt opposed rotational states, thereby allowing the backbone to remain linear and untwisted. Linear backbones tiled in a brick-like way form an extended two-dimensional nanostructure, the Σ-sheet. The binary rotational-state motif of the Σ-strand is not seen in regular protein structures, which are usually built from one type of rotational state. We also show that the concept of building regular structures from multiple rotational states can be generalized beyond the peptoid nanosheet system.

Conformations of peptoids in nanosheets result from the interplay of backbone energetics and intermolecular interactions.

The conformations adopted by the molecular constituents of a supramolecular assembly influence its large-scale order. At the same time, the interactions made in assemblies by molecules can influence their conformations. Here we study this interplay in extended flat nanosheets made from nonnatural sequence-specific peptoid polymers. Nanosheets exist because individual polymers can be linear and untwisted, by virtue of polymer backbone elements adopting alternating rotational states whose twists oppose and cancel. Using molecular dynamics and quantum mechanical simulations, together with experimental data, we explore the design space of flat nanostructures built from peptoids. We show that several sets of peptoid backbone conformations are consistent with their being linear, but the specific combination observed in experiment is determined by a combination of backbone energetics and the interactions made within the nanosheet. Our results provide a molecular model of the peptoid nanosheet consistent with all available experimental data and show that its structure results from a combination of intra- and intermolecular interactions.

Cooperative Intramolecular Hydrogen Bonding Strongly Enforces cis-Peptoid Folding.

Sequence-defined peptoids, or N-substituted glycines, are an attractive class of bioispired polymer due to their biostability and efficient synthesis. However, the de novo design of folded peptoids with precise three-dimensional structures has been hindered by limited means to deterministically control backbone conformation. Peptoid folds are generally destabilized by the cis/trans backbone-amide isomerization, and few side-chains are capable of enforcing a specific amide conformation. Here, we show that a novel class of cationic alkyl ammonium ethyl side-chains demonstrates significant enforcement of the cis-amide backbone (Kcis/trans up to 70) using an unexpected ensemble of weak intramolecular CH-O and/or NH-O hydrogen bonds between the side-chain and the backbone carbonyl moieties. These interactions are evidenced by X-ray crystallography, variable-temperature NMR spectroscopy, and DFT calculations. Moreover, these side-chains are inexpensive, structurally diverse, hydrophilic, and can be integrated into longer peptoid sequences via solid-phase synthesis. Notably, we extended these concepts to synthesize a water-soluble peptoid 10-mer that adopts one predominant fold in solution, as determined by multidimensional NMR spectroscopy. This decamer, to the best of our knowledge, is the longest linear peptoid sequence atomically characterized to retain a well-folded structure. These findings fill a critical gap in peptoid folding and should propel the development of peptoid applications in a broad range of contexts, from pharmaceutical to material sciences.

Stereochemistry of polypeptoid chain configurations.

Like polypeptides, peptoids, or N-substituted glycine oligomers, have intrinsic conformational preferences due to their amide backbones and close spacing of side chain substituents. However, the conformations that peptoids adopt are distinct from polypeptides due to several structural differences: the peptoid backbone is composed of tertiary amide bonds that have trans and cis conformers similar in energy, they lack a backbone hydrogen bond donor, and have an N-substituent. To better understand how these differences manifest in actual peptoid structures, we analyzed 46 high quality, experimentally determined peptoid structures reported in the literature to extract their backbone conformational preferences. One hundred thirty-two monomer dihedral angle pairs were compared to the calculated energy landscape for the peptoid Ramachandran plot, and were found to fall within the expected minima. Interestingly, only two regions of the backbone dihedral angles ϕ and ψ were found to be populated that are mirror images of each other. Furthermore, these two conformers are present in both cis and trans forms. Thus, there are four primary conformers that are sufficient to describe almost all known backbone conformations for peptoid oligomers, despite conformational constraints imposed by a variety of side chains, macrocyclization, or crystal packing forces. Because these conformers are predominant in peptoid structure, and are distinct from those found in protein secondary structures, we propose a simple naming system to aid in the description and classification of peptoid structure.

Unconstrained peptoid tetramer exhibits a predominant conformation in aqueous solution.

Conformational control in peptoids, N-substituted glycines, is crucial for the design and synthesis of biologically-active compounds and atomically-defined nanomaterials. While there are a growing number of structural studies in solution, most have been performed with conformationally-constrained short sequences (e.g., sterically-hindered sidechains or macrocyclization). Thus, the inherent degree of heterogeneity of unconstrained peptoids in solution remains largely unstudied. Here, we explored the folding landscape of a series of simple peptoid tetramers in aqueous solution by NMR spectroscopy. By incorporating specific 13 C-probes into the backbone using bromoacetic acid-2-13 C as a submonomer, we developed a new technique for sequential backbone assignment of peptoids based on the 1,n-Adequate pulse sequence. Unexpectedly, two of the tetramers, containing an N-(2-aminoethyl)glycine residue (Nae), had preferred conformations. NMR and molecular dynamics studies on one of the tetramers showed that the preferred conformer (52%) had a trans-cis-trans configuration about the three amide bonds. Moreover, >80% of the ensemble contained a cis amide bond at the central amide. The backbone dihedral angles observed fall directly within the expected minima in the peptoid Ramachandran plot. Analysis of this compound against similar peptoid analogs suggests that the commonly used Nae monomer plays a key role in the stabilization of peptoid structure via a side-chain-to-main-chain interaction. This discovery may offer a simple, synthetically high-yielding approach to control peptoid structure, and suggests that peptoids have strong intrinsic conformational preferences in solution. These findings should facilitate the predictive design of folded peptoid structures, and accelerate application in areas ranging from drug discovery to biomimetic nanoscience.

Stabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET Hydrolysis.

Cutinases are polyester hydrolases that show a remarkable capability to hydrolyze polyethylene terephthalate (PET) to its monomeric units. This revelation has stimulated research aimed at developing sustainable and green cutinase-catalyzed PET recycling methods. Leaf and branch compost cutinase (LCC) is particularly suited toward these ends given its relatively high PET hydrolysis activity and thermostability. Any practical enzymatic PET recycling application will require that the protein have kinetic stability at or above the PET glass transition temperature (Tg, i.e., 70 °C). This paper elucidates the thermodynamics and kinetics of LCC conformational and colloidal stability. Aggregation emerged as a major contributor that reduces LCC kinetic stability. In its native state, LCC is prone to aggregation owing to electrostatic interactions. Further, with increasing temperature, perturbation of LCC's tertiary structure and corresponding exposure of hydrophobic domains leads to rapid aggregation. Glycosylation was employed in an attempt to impede LCC aggregation. Owing to the presence of three putative N-glycosylation sites, expression of native LCC in Pichia pastoris resulted in the production of glycosylated LCC (LCC-G). LCC-G showed improved stability to native state aggregation while increasing the temperature for thermal induced aggregation by 10 °C. Furthermore, stabilization against thermal aggregation resulted in improved catalytic PET hydrolysis both at its optimum temperature and concentration.

Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation.

Cutinase thermostability is important so that the enzymes can function above the glass transition of what are often rigid polymer substrates. A detailed thermal inactivation analysis was performed for two well-characterized cutinases, Aspergillus oryzae Cutinase (AoC) and Thiellavia terrestris Cutinase (TtC). Both AoC and TtC are prone to thermal aggregation upon unfolding at high temperature, which was found to be a major reason for irreversible loss of enzyme activity. Our study demonstrates that glycosylation stabilizes TtC expressed in Pichia pastoris by inhibiting its thermal aggregation. Based on the comparative thermal inactivation analyses of non-glycosylated AoC, glycosylated (TtC-G), and non-glycosylated TtC (TtC-NG), a unified model for thermal inactivation is proposed that accounts for thermal aggregation and may be applicable to other cutinase homologues. Inspired by glycosylated TtC, we successfully employed glycosylation site engineering to inhibit AoC thermal aggregation. Indeed, the inhibition of thermal aggregation by AoC glycosylation was greater than that achieved by conventional use of trehalose under a typical condition. Collectively, this study demonstrates the excellent potential of implementing glycosylation site engineering for thermal aggregation inhibition, which is one of the most common reasons for the irreversible thermal inactivation of cutinases and many proteins. Biotechnol. Bioeng. 2017;114: 63-73. © 2016 Wiley Periodicals, Inc.

Toward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stability.

Cutinases are powerful hydrolases that can cleave ester bonds of polyesters such as poly(ethylene terephthalate) (PET), opening up new options for enzymatic routes for polymer recycling and surface modification reactions. Cutinase from Aspergillus oryzae (AoC) is promising owing to the presence of an extended groove near the catalytic triad which is important for the orientation of polymeric chains. However, the catalytic efficiency of AoC on rigid polymers like PET is limited by its low thermostability; as it is essential to work at or over the glass transition temperature (Tg) of PET, that is, 70 °C. Consequently, in this study we worked toward the thermostabilization of AoC. Use of Rosetta computational protein design software in conjunction with rational design led to a 6 °C improvement in the thermal unfolding temperature (Tm) and a 10-fold increase in the half-life of the enzyme activity at 60 °C. Surprisingly, thermostabilization did not improve the rate or temperature optimum of enzyme activity. Three notable findings are presented as steps toward designing more thermophilic cutinase: (a) surface salt bridge optimization produced enthalpic stabilization, (b) mutations to proline reduced the entropy loss upon folding, and (c) the lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the Tm of the global structure.

Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris cutinase biochemical characteristics.

Cutinases are esterases of industrial importance for applications in recycling and surface modification of polyesters. The cutinase from Thielavia terrestris (TtC) is distinct in terms of its ability to retain its stability and activity in acidic pH. Stability and activity in acidic pHs are desirable for esterases as the pH of the reaction tends to go down with the generation of acid. The pH stability and activity are governed by the charged state of the residues involved in catalysis or in substrate binding. In this study, we performed the detailed structural and biochemical characterization of TtC coupled with surface charge analysis to understand its acidic tolerance. The stability of TtC in acidic pH was rationalized by evaluating the contribution of charge interactions to the Gibbs free energy of unfolding at varying pHs. The activity of TtC was found to be limited by substrate binding affinity, which is a function of the surface charge. Additionally, the presence of glycosylation affects the biochemical characteristics of TtC owing to steric interactions with residues involved in substrate binding.

De novo structure prediction and experimental characterization of folded peptoid oligomers.

Peptoid molecules are biomimetic oligomers that can fold into unique three-dimensional structures. As part of an effort to advance computational design of folded oligomers, we present blind-structure predictions for three peptoid sequences using a combination of Replica Exchange Molecular Dynamics (REMD) simulation and Quantum Mechanical refinement. We correctly predicted the structure of a N-aryl peptoid trimer to within 0.2 Å rmsd-backbone and a cyclic peptoid nonamer to an accuracy of 1.0 Å rmsd-backbone. X-ray crystallographic structures are presented for a linear N-alkyl peptoid trimer and for the cyclic peptoid nonamer. The peptoid macrocycle structure features a combination of cis and trans backbone amides, significant nonplanarity of the amide bonds, and a unique "basket" arrangement of (S)-N(1-phenylethyl) side chains encompassing a bound ethanol molecule. REMD simulations of the peptoid trimers reveal that well folded peptoids can exhibit funnel-like conformational free energy landscapes similar to those for ordered polypeptides. These results indicate that physical modeling can successfully perform de novo structure prediction for small peptoid molecules.

A rotamer library to enable modeling and design of peptoid foldamers.

Peptoids are a family of synthetic oligomers composed of N-substituted glycine units. Along with other "foldamer" systems, peptoid oligomer sequences can be predictably designed to form a variety of stable secondary structures. It is not yet evident if foldamer design can be extended to reliably create tertiary structure features that mimic more complex biomolecular folds and functions. Computational modeling and prediction of peptoid conformations will likely play a critical role in enabling complex biomimetic designs. We introduce a computational approach to provide accurate conformational and energetic parameters for peptoid side chains needed for successful modeling and design. We find that peptoids can be described by a "rotamer" treatment, similar to that established for proteins, in which the peptoid side chains display rotational isomerism to populate discrete regions of the conformational landscape. Because of the insufficient number of solved peptoid structures, we have calculated the relative energies of side-chain conformational states to provide a backbone-dependent (BBD) rotamer library for a set of 54 different peptoid side chains. We evaluated two rotamer library development methods that employ quantum mechanics (QM) and/or molecular mechanics (MM) energy calculations to identify side-chain rotamers. We show by comparison to experimental peptoid structures that both methods provide an accurate prediction of peptoid side chain placements in folded peptoid oligomers and at protein interfaces. We have incorporated our peptoid rotamer libraries into ROSETTA, a molecular design package previously validated in the context of protein design and structure prediction.

The Proteome Folding Project: proteome-scale prediction of structure and function.

The incompleteness of proteome structure and function annotation is a critical problem for biologists and, in particular, severely limits interpretation of high-throughput and next-generation experiments. We have developed a proteome annotation pipeline based on structure prediction, where function and structure annotations are generated using an integration of sequence comparison, fold recognition, and grid-computing-enabled de novo structure prediction. We predict protein domain boundaries and three-dimensional (3D) structures for protein domains from 94 genomes (including human, Arabidopsis, rice, mouse, fly, yeast, Escherichia coli, and worm). De novo structure predictions were distributed on a grid of more than 1.5 million CPUs worldwide (World Community Grid). We generated significant numbers of new confident fold annotations (9% of domains that are otherwise unannotated in these genomes). We demonstrate that predicted structures can be combined with annotations from the Gene Ontology database to predict new and more specific molecular functions.

Conformational preferences of peptide-peptoid hybrid oligomers.

Peptomers are oligomeric molecules composed of both α-amino acids and N-substituted glycine monomers, thus creating a hybrid of peptide and peptoid units. Peptomers have been used in several applications such as antimicrobials, protease inhibitors, and antibody mimics. Despite the considerable promise of peptomers as chemically diverse molecular scaffolds, we know little about their conformational tendencies. This lack of knowledge limits the ability to implement computational approaches for peptomer design. Here we computationally evaluate the local structural propensities of the peptide-peptoid linkage. We find some general similarities between the peptide residue conformational preferences and the Ramachandran distribution of residues that precede proline in folded protein structures. However, there are notable differences. For example, several ß-turn motifs are disallowed when the i+2 residue is also a peptoid monomer. Significantly, the lowest energy geometry, when dispersion forces are accounted for, corresponds to a "cis-Pro touch-turn" conformation, an unusual turn motif that has been observed at protein catalytic centers and binding sites. The peptomer touch-turn thus represents a useful design element for the construction of folded oligomers capable of molecular recognition and as modules in the assembly of structurally complex peptoid-protein hybrid macromolecules.

Structural Elucidation of a Polypeptoid Chain in a Crystalline Lattice Reveals Key Morphology-Directing Role of the N-Terminus.

The ability to engineer synthetic polymers with the same structural precision as biomacromolecules is crucial to enable the de novo design of robust nanomaterials with biomimetic function. Peptoids, poly(N-substituted) glycines, are a highly controllable bio-inspired polymer family that can assemble into a variety of functional, crystalline nanostructures over a wide range of sequences. Extensive investigation on the molecular packing in these lattices has been reported; however, many key atomic-level details of the molecular structure remain underexplored. Here, we use cryo-TEM 3D reconstruction to directly visualize the conformation of an individual polymer chain within a peptoid nanofiber lattice in real space at 3.6 Å resolution. The backbone in the N-decylglycine hydrophobic core is shown to clearly adopt an extended, all-cis-sigma strand conformation, as previously suggested in many peptoid lattice models. We also show that packing interactions (covalent and noncovalent) at the solvent-exposed N-termini have a dominant impact on the local chain ordering and hence the ability of the chains to pack into well-ordered lattices. Peptoids in pure water form fibers with limited growth in the a direction (<14 molecules in width), whereas in the presence of formamide, they grow to over microns in length in the a direction. This dependence points to the significant role of the chain terminus in determining the long-range order in the packing of peptoid lattices and provides an opportunity to modulate lattice stability and nanoscale morphology by the addition of exogenous small molecules. These findings help resolve a major challenge in the de novo structure-based design of sequence-defined biomimetic nanostructures based on crystalline domains and should accelerate the design of functional nanostructures.

Atomic-Scale Corrugations in Crystalline Polypeptoid Nanosheets Revealed by Three-Dimensional Cryogenic Electron Microscopy.

Amphiphilic molecules that can crystallize often form molecularly thin nanosheets in aqueous solutions. The possibility of atomic-scale corrugations in these structures has not yet been recognized. We have studied the self-assembly of amphiphilic polypeptoids, a family of bio-inspired polymers that can self-assemble into various crystalline nanostructures. Atomic-scale structure of the crystals in these systems has been inferred using both X-ray diffraction and electron microscopy. Here we use cryogenic electron microscopy to determine the in-plane and out-of-plane structures of a crystalline nanosheet. Data were collected as a function of tilt angle and analyzed using a hybrid single-particle crystallographic approach. The analysis reveals that adjacent rows of peptoid chains, which are separated by 4.5 Å in the plane of the nanosheet, are offset by 6 Å in the direction perpendicular to the plane of the nanosheet. These atomic-scale corrugations lead to a doubling of the unit cell dimension from 4.5 to 9 Å. Our work provides an alternative interpretation for the observed Å X-ray diffraction peak often reported in polypeptoid crystals.

A statistical framework for high-content phenotypic profiling using cellular feature distributions.

High-content screening (HCS) uses microscopy images to generate phenotypic profiles of cell morphological data in high-dimensional feature space. While HCS provides detailed cytological information at single-cell resolution, these complex datasets are usually aggregated into summary statistics that do not leverage patterns of biological variability within cell populations. Here we present a broad-spectrum HCS analysis system that measures image-based cell features from 10 cellular compartments across multiple assay panels. We introduce quality control measures and statistical strategies to streamline and harmonize the data analysis workflow, including positional and plate effect detection, biological replicates analysis and feature reduction. We also demonstrate that the Wasserstein distance metric is superior over other measures to detect differences between cell feature distributions. With this workflow, we define per-dose phenotypic fingerprints for 65 mechanistically diverse compounds, provide phenotypic path visualizations for each compound and classify compounds into different activity groups.

Tryptophanyl-tRNA synthetase Urzyme: a model to recapitulate molecular evolution and investigate intramolecular complementation.

We substantiate our preliminary description of the class I tryptophanyl-tRNA synthetase minimal catalytic domain with details of its construction, structure, and steady-state kinetic parameters. Generating that active fragment involved deleting 65% of the contemporary enzyme, including the anticodon-binding domain and connecting peptide 1, CP1, a 74-residue internal segment from within the Rossmann fold. We used protein design (Rosetta), rather than phylogenetic sequence alignments, to identify mutations to compensate for the severe loss of modularity, thus restoring stability, as evidenced by renaturation described previously and by 70-ns molecular dynamics simulations. Sufficient solubility to enable biochemical studies was achieved by expressing the redesigned Urzyme as a maltose-binding protein fusion. Michaelis-Menten kinetic parameters from amino acid activation assays showed that, compared with the native full-length enzyme, TrpRS Urzyme binds ATP with similar affinity. This suggests that neither of the two deleted structural modules has a strong influence on ground-state ATP binding. However, tryptophan has 10(3) lower affinity, and the Urzyme has comparably reduced specificity relative to the related amino acid, tyrosine. Molecular dynamics simulations revealed how CP1 may contribute significantly to cognate amino acid specificity. As class Ia editing domains are nested within the CP1, this finding suggests that this module enhanced amino acid specificity continuously, throughout their evolution. We call this type of reconstructed protein catalyst an Urzyme (Ur prefix indicates original, primitive, or earliest). It establishes a model for recapitulating very early steps in molecular evolution in which fitness may have been enhanced by accumulating entire modules, rather than by discrete amino acid sequence changes.

Computational design of the sequence and structure of a protein-binding peptide.

The de novo design of protein-binding peptides is challenging because it requires the identification of both a sequence and a backbone conformation favorable for binding. We used a computational strategy that iterates between structure and sequence optimization to redesign the C-terminal portion of the RGS14 GoLoco motif peptide so that it adopts a new conformation when bound to Gα(i1). An X-ray crystal structure of the redesigned complex closely matches the computational model, with a backbone root-mean-square deviation of 1.1 Å.

Peptoid atropisomers.

We report the isolation of N-aryl peptoid oligomers that adopt chiral folds, despite the absence of chiral centers. Peptoid monomers incorporating ortho-substituted N-aryl side chains are identified that exhibit axial chirality. We observe significant energy barriers to rotation about the stereogenic carbon-nitrogen bond, allowing chromatographic purification of stable atropisomeric forms. We study the atropisomerism of N-aryl peptoid oligomers by computational modeling, NMR, X-ray crystallography, dynamic HPLC, and circular dichroism. The results demonstrate a new approach to promote the conformational ordering of this important class of foldamer compounds.