Anchor extension: a structure-guided approach to design cyclic peptides targeting enzyme active sites
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99 GENERAL AND MISCELLANEOUS↗
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Designing Sequence-Defined Peptoids for Biomimetic Control over Inorganic Crystallization
Crystallization defines what the world is all about, ranging from biomineralization in living organisms to various materials in our lives. During these processes, biomineralization set a superexcellent example of the additive-controlled crystallization. However, the high complexity and low stability of peptides and proteins preclude their wide applications in controlling inorganic crystallization beyond the biological systems. Peptoids are one type of sequence-defined biomimetic polymers with a simple structure, high stability, protein-like molecular recognition, and distinctive self-assembly properties. They provide a promising alternative to mimic peptides and proteins for controlling inorganic crystallization and extend its application to predictive materials synthesis. In this review article, we extract the main concepts of peptoid engineering and highlight the recent advances in peptoid-controlled inorganic crystallization. Our focus is to understand the principles of sequence engineering that lead to the predictable physiochemical properties of these substances, which will give insights into other inorganic systems.
36 MATERIALS SCIENCE↗
Cyclic Peptides for Lanthanide Binding
Lanthanide ions are difficult to separate from one another due to their similar chemical properties. The discovery of lanthanide-binding peptides and proteins in nature has led to an increased interest in the possibility of utilizing the strong binding of peptides to lanthanide ions for their separations; as such, there has been an effort to identify or design peptides with improved lanthanide binding and selectivity toward particular lanthanide ions. Here, in this study, we designed and characterized lanthanide-binding cyclic peptides (LBCPs) with molecular dynamics simulations, electronic structure calculations, and emission spectroscopy. Luminescent decay measurements were done to determine the number of water molecules coordinated to the Eu 3+ ion in Eu-LBCP complexes and compare to the predicted number of water molecules by computation to assess the lanthanide-binding affinity of LBCPs. Measured stability constants show binding of the LBCPs to the Eu 3+ ion with stronger than micromolar affinity. We were able to identify multiple peptides that selectively bind to middle lanthanides. We describe the structural basis of the lanthanide-binding selectivity trend with strongest binding to the middle lanthanides, followed by the heavier lanthanides, and finally to the lighter ions.
ions↗
Computational design of mixed chirality peptide macrocycles with internal symmetry
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Mulligan, Vikram Khipple↗
Design of amyloidogenic peptide traps
Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein–peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid β 1–42 (Aβ42). The Aβ binders block the assembly of Aβ fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aβ42 species.
59 BASIC BIOLOGICAL SCIENCES↗
Closing the Gap Between Modeling and Experiments in the Self-assembly of Biomolecules at Interfaces and in Solution
Molecular self-assembly is a powerful tool in materials design, wherein non-covalent interactions like electrostatic, hydrophobic, hydrogen bonding, and van der Waals can be exploited to produce supramolecular nanostructures that are functional and highly tunable. Biomolecules are attractive building blocks, as they are biocompatible, biodegradable and adopt a wide array of higher order structures. Moreover, naturally occurring protein systems display a manifold of structures and interactions that can be replicated in synthetic biomolecules. In this perspective, we highlight advances in multiscale simulation techniques across broad spatiotemporal scales that can aid in characterizing self-assembly of hybrid and hierarchical bionanomaterial systems, with an emphasis on physics-based simulation approaches currently employed to study biomolecules at mineral interfaces. The power of these approaches is highlighted across a few recent areas where molecular simulations have advanced our understanding of self-assembly spanning peptides to protein self-assembly. Looking forward, we discuss how in the near future emerging methods in statistical and machine learning will advance this research field in all areas from expanding the capabilities of physics-based simulation methods to enabling new analyses of high throughput experiments. These advances will pave the way for understanding the molecular recognition patterns in systems that are dictated by self-assembly - biomineralizing peptides, hierarchical peptoids, and large protein assemblies, and will aid in the development of a new synthesis science for achieving precise molecular control in materials design
Sampath, Janani↗
Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate
SETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.
36 MATERIALS SCIENCE↗
Peptide Inhibitors of Bacterial Protein Synthesis with Broad Spectrum and SbmA-Independent Bactericidal Activity against Clinical Pathogens
Abstract not provided
Mardirossian, Mario↗
Phase Control of Nanocrystalline Inclusions in Bioprecipitated Titania with a Panel of Mutant Silica-Binding Proteins
The biomimetic route to inorganic synthesis presents an opportunity to produce complex materials with superior properties under ambient conditions and from nontoxic precursors. While there has been significant progress in using solid-binding peptides (SBPs), proteins, and organisms to produce a variety of inorganic and hybrid structures, it has been more challenging to understand the interplay of solution conditions and solid-binding peptide (SBP) sequence, structure, and self-association on synthetic outcomes. Here, we show that fusing the Car9 silica-binding peptide—but not the silaffin-derived R5 peptide—to superfolder green fluorescent protein (sfGFP) enhances the ability of micromolar concentrations of protein to induce rapid titania (TiO 2 ) precipitation from acidified solutions of tetrakis(di-lactato)-oxo-titanate (TiBALDH). TiO 2 is produced stoichiometrically and although predominantly amorphous, contains nanosized anatase and monoclinic TiO 2 (B) inclusions. Remarkably, the phase of these nanocrystallites can be tuned from about 80% TiO 2 (B) to about 65% anatase by using Car9 mutants impaired in their ability to drive the formation of higher-order sfGFP-Car9 oligomers. Our results suggest that the presentation of multiple basic side chains in an extended plane formed by SBP self-association is critical to template the formation of monoclinic crystallites and underscore the subtle influence that single or dual substitutions in dodecameric SBPs can exert on the yield and crystallinity of biomineralized inorganics.
mutagenesis↗
Native Chemical Ligation of Peptoid Oligomers
Bioorganic chemists are inspired by natural biopolymers to design peptidomimetic oligomers that can exhibit sequence-structure-function relationships. Biomimetic polymers can be synthesized to incorporate a specific sequence of nonbiological monomer units using a variety of iterative solution-phase or solid-phase reaction schemes. These protocols generally provide access to a vast diversity of oligomeric compounds but are limited with respect to their ability to attain protein-like chain lengths. This constraint can preclude access to sequence-defined synthetic macromolecules with sufficient sizes required to exhibit tertiary structure and other protein-mimetic attributes. In contrast, peptide chemists have overcome this limitation by developing convergent synthetic methods, such as native chemical ligation, to join individual, smaller peptide chains together to make larger peptides or full proteins. A similar convergent approach is needed to establish efficient synthetic routes to non-natural sequence-defined macromolecules. Herein, we adapt the peptide native chemical ligation method to peptoid oligomers, demonstrating how short chains can be conjoined to create sequence-defined peptoid macromolecules. Nanosheet-forming peptoid polymers with distinct surface loop display domains were generated by sequential ligation of several discrete fragments. This method provides a reliable convergent ligation route for sequence-defined polypeptoids that results in a native amide bond joining the fragments. We envision that this strategy will be useful in synthesizing peptoid-based proteomimetics that incorporate diverse chemical features.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Target–decoy false discovery rate estimation using Crema
Assigning statistical confidence estimates to discoveries produced by a tandem mass spectrometry proteomics experiment is critical to enabling principled interpretation of the results and assessing the cost/benefit ratio of experimental follow-up. The most common technique for computing such estimates is to use target-decoy competition (TDC), in which observed spectra are searched against a database of real (target) peptides and a database of shuffled or reversed (decoy) peptides. TDC procedures for estimating the false discovery rate (FDR) at a given score threshold have been developed for application at the level of spectra, peptides, or proteins. Although these techniques are relatively straightforward to implement, it is common in the literature to skip over the implementation details or even to make mistakes in how the TDC procedures are applied in practice. Here we present Crema, an open-source Python tool that implements several TDC methods of spectrum-, peptide- and protein-level FDR estimation. Crema is compatible with a variety of existing database search tools and provides a straightforward way to obtain robust FDR estimates.
59 BASIC BIOLOGICAL SCIENCES↗
Structural Characterization of Disulfide-Linked p53-Derived Peptide Dimers
Disulfide bonds provide a convenient method for chemoselective alteration of peptide and protein structure and function. We previously reported that mild oxidation of a p53-derived bisthiol peptide (CTFANLWRLLAQNC) under dilute non-denaturing conditions led to unexpected disulfide-linked dimers as the exclusive product. The dimers were antiparallel, significantly α-helical, resistant to protease degradation, and easily reduced back to the original bisthiol peptide. Here, in this work, we examine the intrinsic factors influencing peptide dimerization using a combination of amino acid substitution, circular dichroism (CD) spectroscopy, and X-ray crystallography. CD analysis of peptide variants suggests critical roles for Leu6 and Leu10 in the formation of stable disulfide-linked dimers. The 1.0 Å resolution crystal structure of the peptide dimer supports these data, revealing a leucine-rich LxxLL dimer interface with canonical knobs-into-holes packing. Two levels of higher-order oligomerization are also observed in the crystal: an antiparallel “dimer of dimers” mediated by Phe3 and Trp7 residues in the asymmetric unit and a tetramer of dimers mediated by Trp7 and Leu10. In CD spectra of Trp-containing peptide variants, minima at 227 nm provide evidence for the dimer of dimers in dilute aqueous solution. Importantly, and in contrast to the original dimer model, the canonical leucine-rich core and robust dimerization of most peptide variants suggests a tunable molecular architecture to target various proteins and evaluate how folding and oligomerization impact various properties, such as cell permeability.
36 MATERIALS SCIENCE↗
Recombinant expression of computationally designed peptide-bundlemers in Escherichia coli
Computational design of fully artificial peptides is extensively researched by material scientists and engineers for the construction of novel nanostructures and biomaterials. Such design has yielded a peptide-based building block or bundlemer, a coiled coil peptide assembly that undergoes further physical-covalent interactions to form 1D, 2D and, potentially, 3D hierarchical assemblies and displays targeted and biomimetic material properties. Recombinant expression is a convenient, flexible tool to synthesize such artificial and modified peptides in large quantities while also enabling economical synthesis of isotopically labeled peptides and longer protein-like artificial peptides. This report describes the protocol for recombinant expression of a 31-amino acid, computationally designed bundlemer-forming peptide in Escherichia coli. Peptide yields of 10 mgs per liter of media were achieved which highlights complementary advantages of recombinant expression technique relative to conventional laboratory-scale synthesis, such as solid-phase peptide synthesis.
36 MATERIALS SCIENCE↗
Aggregation Methods for Quantifying PTM and Structural Changes in Bottom-Up Proteomics
Bottom-up proteomic workflows rely on sequential preprocessing steps, commonly including peptide-to-protein aggregation (“roll-up”), to enhance data reliability and interpretability. While roll-up is effective for protein-centered analyses, it may be suboptimal for applications focused on post-translational modifications (PTMs) or protein structural changes, such as limited proteolysis–mass spectrometry (LiP-MS). Here, we investigate how different roll-up strategies influence site-level quantification in PTM differential analysis. Moreover, we introduce a novel site-centric roll-up approach tailored for LiP-MS, which quantifies proteolytic fragments rather than solely tryptic peptides. We benchmark these methods through simulation studies, comparing their sensitivity and specificity in detecting structural and PTM-driven changes. We found that the median and mean roll-up methods outperform the sum method in both PTM and LiP proteomics, and site-level quantification in LiP outperforms peptide-level quantification. Our findings offer the first systematic, data-driven guidance for selecting roll-up techniques in site-level proteomic analyses, with implications for both PTM-focused and structural proteomics studies.
aggregation↗
Drug discovery efforts at George Mason University
With over 39,000 students, and research expenditures in excess of $200 million, George Mason University (GMU) is the largest R1 (Carnegie Classification of very high research activity) university in Virginia. Mason scientists have been involved in the discovery and development of novel diagnostics and therapeutics in areas as diverse as infectious diseases and cancer. Below are highlights of the efforts being led by Mason researchers in the drug discovery arena. To enable targeted cellular delivery, and non-biomedical applications, Veneziano and colleagues have developed a synthesis strategy that enables the design of self-assembling DNA nanoparticles (DNA origami) with prescribed shape and size in the 10 to 100 nm range. The nanoparticles can be loaded with molecules of interest such as drugs, proteins and peptides, and are a promising new addition to the drug delivery platforms currently in use. The investigators also recently used the DNA origami nanoparticles to fine tune the spatial presentation of immunogens to study the impact on B cell activation. These studies are an important step towards the rational design of vaccines for a variety of infectious agents. To elucidate the parameters for optimizing the delivery efficiency of lipid nanoparticles (LNPs), Buschmann, Paige and colleagues have devised methods for predicting and experimentally validating the pKa of LNPs based on the structure of the ionizable lipids used to formulate the LNPs. These studies may pave the way for the development of new LNP delivery vehicles that have reduced systemic distribution and improved endosomal release of their cargo post administration. To better understand protein-protein interactions and identify potential drug targets that disrupt such interactions, Luchini and colleagues have developed a methodology that identifies contact points between proteins using small molecule dyes. The dye molecules noncovalently bind to the accessible surfaces of a protein complex with very high affinity, but are excluded from contact regions. When the complex is denatured and digested with trypsin, the exposed regions covered by the dye do not get cleaved by the enzyme, whereas the contact points are digested. The resulting fragments can then be identified using mass spectrometry. The data generated can serve as the basis for designing small molecules and peptides that can disrupt the formation of protein complexes involved in disease processes. For example, using peptides based on the interleukin 1 receptor accessory protein (IL-1RAcP), Luchini, Liotta, Paige and colleagues disrupted the formation of IL-1/IL-R/IL-1RAcP complex and demonstrated that the inhibition of complex formation reduced the inflammatory response to IL-1B. Working on the discovery of novel antimicrobial agents, Bishop, van Hoek and colleagues have discovered a number of antimicrobial peptides from reptiles and other species. DRGN-1, is a synthetic peptide based on a histone H1-derived peptide that they had identified from Komodo Dragon plasma. DRGN-1 was shown to disrupt bacterial biofilms and promote wound healing in an animal model. The peptide, along with others, is being developed and tested in preclinical studies. Other research by van Hoek and colleagues focuses on in silico antimicrobial peptide discovery, screening of small molecules for antibacterial properties, as well as assessment of diffusible signal factors (DFS) as future therapeutics. The above examples provide insight into the cutting-edge studies undertaken by GMU scientists to develop novel methodologies and platform technologies important to drug discovery.
59 BASIC BIOLOGICAL SCIENCES↗
Induced Chirality in QDs Using Thermoresponsive Elastin-like Polypeptides
Circular dichroism (CD) spectroscopy has emerged as a potent tool for probing chiral small-molecule ligand exchange on natively achiral quantum dots (QDs). In this study, we report a novel approach to identifying QD–biomolecule interactions by inducing chirality in CdS QDs using thermoresponsive elastin-like polypeptides (ELPs) engineered with C-terminal cysteine residues. Our method is based on a versatile two-step ligand exchange process starting from monodisperse oleate-capped QDs in nonpolar media and proceeding through an easily accessed achiral glycine-capped QD intermediate. Successful conjugation of the ELPs onto the QDs is confirmed by the diagnostic CD response corresponding to the QD electronic transitions in the visible range. The resulting ELP:CdS conjugates demonstrate thermally reversible coacervation, as observed through dynamic light scattering, small-angle X-ray scattering, and electron microscopy. Furthermore, this research provides a foundation for using induced chirality in QD electronic transitions to probe QD conjugation to complex peptides and proteins, opening pathways for designing dynamic, stimuli-responsive hybrid nanomaterials.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Using Multiple Isotope-Labeled Infrared Spectra for the Structural Characterization of an Intrinsically Disordered Peptide
Intrinsically disordered proteins (IDPs) rapidly interconvert between conformers, requiring an ensemble description. This complicates their experimental characterization, and force field limitations pose challenges for their simulation. Here, in this work, we use isotope-labeled and unlabeled infrared (IR) spectra to reweight simulated ensembles of the elastin-like peptide GVGVPGVG, a paradigmatic disordered peptide. By comparing the results obtained with different spectra, we explicitly show that the weights are underdetermined by the ensemble averaged data. We identify which labels and frequency regions maximize structural information while minimizing sensitivity to simulation error and show that these regions report on whether the peptide makes specific interactions. Our work shows the importance of incorporating simulations and simulated spectra at the planning stages of isotope-labeled IR experiments and more generally provides a framework for interpreting IR data for IDPs.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Design and Function of α-Helix-Rich, Heme-Binding Peptide Materials
Peptide materials often employ short peptides that self-assemble into unique nanoscale architectures and have been employed across many fields relevant to medicine and energy. A majority of peptide materials are high in beta-sheet, secondary structure content, including heme-binding peptide materials. To broaden the structural diversity of heme-binding peptide materials, a small series of peptides were synthesized to explore the design criteria required for (1) folding into an alpha-helix structure, (2) assembling into a nanoscale material, (3) binding heme, and (4) demonstrating functions similar to that of heme proteins. One peptide was identified to meet all four criteria, including the heme protein function of CO binding and its microsecond-to-millisecond recombination rates, as measured by transient absorption spectroscopy. In conclusion, implications of new design criteria and peptide material function through heme incorporation are discussed.
59 BASIC BIOLOGICAL SCIENCES↗