A colorimetric competitive displacement assay for the evaluation of catalytic peptides.

An indicator displacement assay has been adapted to detect the diol products of the aldol reaction between 4-nitrobenzaldehyde and hydroxyacetone in crude reaction mixtures. This provides a rapid colorimetric method of detecting product formation and thus evaluating potential catalysts, which is demonstrated using multiple catalytic peptides.

Optimization and expansion of a site-selective N-methylpyridinium-4-carboxaldehyde-mediated transamination for bacterially expressed proteins.

Site-selective bioconjugation methods are valuable because of their ability to confer new properties to proteins by the chemical attachment of specific functional groups. Well-defined bioconjugates obtained through these methods have found utility for the study of protein function and the creation of protein-based materials. We have previously reported a protein modification strategy to modify the N-terminus of peptides and proteins using N-methylpyridinium-4-carboxaldehyde benzenesulfonate (Rapoport's salt, RS) as a transamination reagent, which oxidizes the N-terminal amino group to provide a uniquely reactive aldehyde or ketone. This functional handle can subsequently be modified with an alkoxyamine reagent of choice. Previous work had found glutamate terminal sequences to be highly reactive toward RS-mediated transamination. However, proteins of interest are often recombinantly expressed in E. coli, where the expression of a glutamate-terminal protein is rendered difficult because the N-terminal methionine derived from the start codon is not cleaved when Glu is in the second position. In this work, we describe a way to overcome this difficulty via the insertion of a Factor Xa proteolytic cleavage site to acquire the optimal glutamate residue at the N-terminus. Additionally, we present studies on alternative high-yielding sequences containing N-terminal residues that can be expressed directly. We have used site-directed mutagenesis to validate these findings on a model cellulase enzyme, an endoglucanase from the thermophilic Pyrococcus horikoshii. Activity assays performed with these mutants show that RS transamination and subsequent modification with alkoxyamines have no negative impact on cellulolytic ability.

Hyperpolarized xenon-based molecular sensors for label-free detection of analytes.

Nuclear magnetic resonance (NMR) can reveal the chemical constituents of a complex mixture without resorting to chemical modification, separation, or other perturbation. Recently, we and others have developed magnetic resonance agents that report on the presence of dilute analytes by proportionately altering the response of a more abundant or easily detected species, a form of amplification. One example of such a sensing medium is xenon gas, which is chemically inert and can be optically hyperpolarized, a process that enhances its NMR signal by up to 5 orders of magnitude. Here, we use a combinatorial synthetic approach to produce xenon magnetic resonance sensors that respond to small molecule analytes. The sensor responds to the ligand by producing a small chemical shift change in the Xe NMR spectrum. We demonstrate this technique for the dye, Rhodamine 6G, for which we have an independent optical assay to verify binding. We thus demonstrate that specific binding of a small molecule can produce a xenon chemical shift change, suggesting a general approach to the production of xenon sensors targeted to small molecule analytes for in vitro assays or molecular imaging in vivo.

Relative contractile motion of the rings in a switchable palindromic [3]rotaxane in aqueous solution driven by radical-pairing interactions.

Artificial muscles are an essential component for the development of next-generation prosthetic devices, minimally invasive surgical tools, and robotics. This communication describes the design, synthesis, and characterisation of a mechanically interlocked molecule (MIM), capable of switchable and reversible linear molecular motion in aqueous solution that mimics muscular contraction and extension. Compatibility with aqueous solution was achieved in the doubly bistable palindromic [3]rotaxane design by using radical-based molecular recognition as the driving force to induce switching.

Site-specific protein transamination using N-methylpyridinium-4-carboxaldehyde.

The controlled attachment of synthetic groups to proteins is important for a number of fields, including therapeutics, where antibody-drug conjugates are an emerging area of biologic medicines. We have previously reported a site-specific protein modification method using a transamination reaction that chemoselectively oxidizes the N-terminal amine of a polypeptide chain to a ketone or an aldehyde group. The newly introduced carbonyl can be used for conjugation to a synthetic group in one location through the formation of an oxime or a hydrazone linkage. To expand the scope of this reaction, we have used a combinatorial peptide library screening platform as a method to explore new transamination reagents while simultaneously identifying their optimal N-terminal sequences. N-Methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport's salt, RS) was identified as a highly effective transamination reagent when paired with glutamate-terminal peptides and proteins. This finding establishes RS as a transamination reagent that is particularly well suited for antibody modification. Using a known therapeutic antibody, herceptin, it was demonstrated that RS can be used to modify the heavy chains of the wild-type antibody or to modify both the heavy and the light chains after N-terminal sequence mutation to add additional glutamate residues.

Using synthetically modified proteins to make new materials.

The uniquely diverse structures and functions of proteins offer many exciting opportunities for creating new materials with advanced properties. Exploiting these capabilities requires a set of versatile chemical reactions that can attach nonnatural groups to specific locations on protein surfaces. Over the years, we and others have developed a series of new techniques for protein bioconjugation, with a particular emphasis on achieving high site selectivity and yield. Using these reactions, we have been able to prepare a number of new materials with functions that depend on both the natural and the synthetic components. In this Account, we discuss our progress in protein bioconjugation over the past decade, focusing on three distinct projects. We first consider our work to harness sunlight artificially by mimicking features of the photosynthetic apparatus, with its beautifully integrated system of chromophores, electron transfer groups, and catalytic centers. Central to these photosystems are light-harvesting antennae having hundreds of precisely aligned chromophores with positions that are dictated by the proteins within the arrays. Our approach to generating similar arrangements involves the self-assembly of tobacco mosaic virus coat proteins bearing synthetic chromophore groups. These systems offer efficient light collection, are easy to prepare, and can be used to build complex photocatalytic systems through the modification of multiple sites on the protein surfaces. We then discuss protein-based carriers that can deliver drugs and imaging agents to diseased tissues. The nanoscale agents we have built for this purpose are based on the hollow protein shell of bacteriophage MS2. These 27 nm capsids have 32 pores, which allow the entry of relatively large organic molecules into the protein shell without requiring disassembly. Our group has developed a series of chemical strategies that can install dyes, radiolabels, MRI contrast agents, and anticancer drugs on the inside surface of these capsids. We have also developed methods to decorate the external surfaces with binders for specific proteins on cancer cells. As a third research area, our group has developed protein-polymer hybrid materials for water remediation. To reduce the toxicity of heavy metals in living cells, Nature has evolved metallothioneins, which are sulfur-rich polypeptides that bind mercury, cadmium, and other toxic ions at sub-parts-per-billion concentrations. Unfortunately, these proteins are very difficult to incorporate into polymers, largely because typical protein modification reactions target the very cysteine, lysine, and carboxylate-containing residues that are required for their proper function. To address this challenge, we developed a new way to attach these (and many other) proteins to polymer chains by expressing them as part of an N- and C-terminal modification "cassette". The resulting materials retain their selectivity and can remove trace amounts of toxic metal ions from ocean water. Each of these examples has presented a new set of protein bioconjugation challenges that have been met through the development of new reaction methodology. Future progress in the generation of protein-based materials will require scalable synthetic techniques with improved yields and selectivities, inexpensive purification methods for bioconjugates, and theoretical and dynamical treatments for designing new materials through protein self-assembly.

A randomized controlled trial of a video intervention shows evidence of increasing COVID-19 vaccination intention.

Increasing acceptance of COVID-19 vaccines is imperative for public health. Previous research on educational interventions to overcome vaccine hesitancy have shown mixed effects in increasing vaccination intention, although much of this work has focused on parental attitudes toward childhood vaccination. In this study, we conducted a randomized controlled trial to investigate whether vaccination intention changes after viewing an animated YouTube video explaining how COVID-19 mRNA vaccines work. We exposed participants to one of four interventions-watching the video with a male narrator, watching the same video with a female narrator, reading the text of the transcript of the video, or receiving no information (control group). We found that participants who watched the version of the video with a male narrator expressed statistically significant increased vaccination intention compared to the control group. The video with a female narrator had more variation in results. As a whole, there was a non-significant increased vaccination intention when analyzing all participants who saw the video with a female narrator; however, for politically conservative participants there was decreased vaccination intention for this intervention compared to the control group at a threshold between being currently undecided and expressing probable interest. These results are encouraging for the ability of interventions as simple as YouTube videos to increase vaccination propensity, although the inconsistent response to the video with a female narrator demonstrates the potential for bias to affect how certain groups respond to different messengers.

Investigation of the Effect of Turn Residues on Tetrapeptide Aldol Catalysts with ß-Turn Propensity.

Peptide catalysts for a wide diversity of reaction types contain a common motif-residues that bias the sequence toward ß-turn secondary structure. In this work, we explore what role that secondary structure plays in the catalysis of aldol reactions for primary amine tetrapeptide aldol catalysts. Using a lead tetrapeptide ß-turn catalytic sequence, we varied the i + 1 and i + 2 residues to amino acids that would affect the ß-turn propensity. We then studied the correlation between secondary structure, aldol rate enhancement, and stereoselectivity of the reaction between hydroxyacetone and 4-nitrobenzaldehyde. Using the i + 3 amide chemical shift as a measure of ß-turn character, we found a rough correlation between the peptide structure and reaction kinetics but minimal effect on stereoselectivity. These trends may help aid the design of future catalytic sequences.

The role of ß-hairpin conformation in ester hydrolysis peptide catalysts based on a TrpZip scaffold.

To explore the role of peptide conformation on catalytic activity in the context of ester hydrolysis catalysts, pairs of sequences were designed that contained or lacked ß-hairpin character. For the hydrolysis of para-nitrophenylacetate in aqueous media, we found small but consistent trends wherein His-containing sequences based on a TrpZip scaffold showed higher catalytic activity without ß-hairpin character.

Identification of highly reactive sequences for PLP-mediated bioconjugation using a combinatorial peptide library.

Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We have previously reported a pyridoxal 5'-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine-lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.

Self-assembling peptide coatings designed for highly luminescent suspension of single-walled carbon nanotubes.

A series of self-assembling multidomain peptides have been designed, synthesized, and tested for their ability to individually suspend single-walled carbon nanotubes (SWCNTs) in water while preserving strong near-IR nanotube luminescence. Photometric and spectral measurements on individual SWCNTs revealed that emission in the common biocompatible coating agents Pluronic F127, ss-DNA, and BSA is approximately an order of magnitude weaker than in the bioincompatible ionic surfactant SDBS. By contrast, one of the engineered peptides gave SWCNT emission approximately 40% as intense as in SDBS. A strong inverse correlation was also found between the spectral line widths of coated SWCNTs and the efficiency of their emission. Peptides with rationally designed self-assembly properties appear to be promising coatings that may enable SWCNT optical sensing applications in biological environments.

Oxime ligation on the surface of mesoporous silica nanoparticles.

A versatile surface-functionalization strategy applicable to mesoporous silica nanoparticles, which could potentially serve as drug delivery vehicles, is described that makes use of alkoxyamine tethers on the surface of the nanoparticles. A wide variety of carbonyl compounds can be attached readily to these tethers under the mild conditions of oxime ether formation, simply by incubating the chemically modified mesoporous silica nanoparticles with aldehydes or ketones in water.

Anticancer activity expressed by a library of 2,9-diazaperopyrenium dications.

Polyaromatic compounds are well-known to intercalate DNA. Numerous anticancer chemotherapeutics have been developed upon the basis of this recognition motif. The compounds have been designed such that they interfere with the role of the topoisomerases, which control the topology of DNA during the cell-division cycle. Although many promising chemotherapeutics have been developed upon the basis of polyaromatic DNA intercalating systems, these candidates did not proceed past clinical trials on account of their dose-limiting toxicity. Herein, we discuss an alternative, water-soluble class of polyaromatic compounds, the 2,9-diazaperopyrenium dications, and report in vitro cell studies for a library of these dications. These investigations reveal that a number of 2,9-diazaperopyrenium dications show similar activities as doxorubicin toward a variety of cancer cell lines. Additionally, we report the solid-state structures of these dications, and we relate their tendency to aggregate in solution to their toxicity profiles. The addition of bulky substituents to these polyaromatic dications decreases their tendency to aggregate in solution. The derivative substituted with 2,6-diisopropylphenyl groups proved to be the most cytotoxic against the majority of the cell lines tested. In the solid state, the 2,6-diisopropylphenyl-functionalized derivative does not undergo π···π stacking, while in aqueous solution, dynamic light scattering reveals that this derivative forms very small (50-100 nm) aggregates, in contrast with the larger ones formed by dications with less bulky substituents. Alteration of the aromaticitiy in the terminal heterocycles of selected dications reveals a drastic change in the toxicity of these polyaromatic species toward specific cell lines.

Site-Specific Protein Bioconjugation via a Pyridoxal 5'-Phosphate-Mediated N-Terminal Transamination Reaction.

The covalent attachment of chemical groups to proteins is a critically important tool for the study of protein function and the creation of protein-based materials. Methods of site-specific protein modification are necessary for the generation of well defined bioconjugates possessing a new functional group in a single position in the amino acid sequence. This article describes a pyridoxal 5'-phosphate (PLP)-mediated transamination reaction that is specific for the N-terminus of a protein. The reaction oxidizes the N-terminal amine to a ketone or an aldehyde, which can form a stable oxime linkage with an alkoxyamine reagent of choice. Screening studies have identified the most reactive N-terminal residues, facilitating the use of site-directed mutagenesis to achieve high levels of conversion. Additionally, this reaction has been shown to be effective for a number of targets that are not easily accessed through heterologous expression, such as monoclonal antibodies. Curr. Protoc. Chem. Biol. 2:125-134 © 2010 by John Wiley & Sons, Inc.