Aggregation of Charge Acceptors on Nanocrystal Surfaces Alters Rates of Photoinduced Electron Transfer.

Semiconductor nanocrystals (NCs) interfaced with molecular ligands that function as charge and energy acceptors are an emerging platform for the design of light-harvesting, photon-upconverting, and photocatalytic materials. However, NC systems explored for these applications often feature high concentrations of bound acceptor ligands, which can lead to ligand-ligand interactions that may alter each system's ability to undergo charge and energy transfer. Here, we demonstrate that aggregation of acceptor ligands impacts the rate of photoinduced NC-to-ligand charge transfer between lead(II) sulfide (PbS) NCs and perylenediimide (PDI) electron acceptors. As the concentration of PDI acceptors is increased, we find the average electron transfer rate from PbS to PDI ligands decreases by nearly an order of magnitude. The electron transfer rate slowdown with increasing PDI concentration correlates strongly with the appearance of PDI aggregates in steady-state absorption spectra. Electronic structure calculations and molecular dynamics (MD) simulations suggest PDI aggregation slows the rate of electron transfer by reducing orbital overlap between PbS charge donors and PDI charge acceptors. While we find aggregation slows electron transfer in this system, the computational models we employ predict ligand aggregation could also be used to speed electron transfer by producing delocalized states that exhibit improved NC-molecule electronic coupling and energy alignment with NC conduction band states. Our results demonstrate that ligand aggregation can alter rates of photoinduced electron transfer between NCs and organic acceptor ligands and should be considered when designing hybrid NC:molecule systems for charge separation.

Evaluating the Effect of Dye-Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides.

A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG)10 linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide-alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the single-molecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.

Mechanistic Analysis of Solid-State Colorimetric Switching: Monoalkoxynaphthalene-Naphthalimide Donor-Acceptor Dyads.

There is growing interest in creating solids that are responsive to various stimuli. Herein we report the first molecular-level mechanistic picture of the thermochromic polymorphic transition in a series of MAN-NI dyad crystals that turn from orange to yellow upon heating with minimal changes to the microscopic morphology following the transition. Detailed structural analyses revealed that the dyads assemble to create an alternating bilayer type structure, with horizontal alternating alkyl and stacked aromatic layers in both the orange and yellow forms. The observed dynamic behavior in the solid state moves as a yellow wavefront through the orange crystal. The overall process is critically dependent on a complex interplay between the layered structure of the starting crystal, the thermodynamics of the two differently colored forms, and similar densities of the two polymorphs. Upon heating, the orange form alkyl chain layers become disordered, allowing for some lateral diffusion of dyads within their own layer. Moving to either adjacent stack in the same layer allows a dyad to exchange a head-to-head stacking geometry (orange) for a head-to-tail stacking geometry (yellow). This transition is unique in that it involves a nucleation and growth mechanism that converts to a faster cooperative wavefront mechanism during the transition. The fastest moving of the wavefronts have an approximately 38° angle with respect to the long axis of the crystal, corresponding to a nonconventional C-H···O hydrogen bond network of dyad molecules in adjacent stacks that enables a transition with cooperative character to proceed within layers of orange crystals. The orange-to-yellow transition is triggered at a temperature that is very close to the temperature at which the orange and yellow forms exchange as the more stable, while being lower than the melting temperature of the original orange, or final yellow, solids.

Characterization of aromatic residue-controlled protein retention in the endoplasmic reticulum of Saccharomyces cerevisiae.

An endoplasmic reticulum (ER) retention sequence (ERS) is a characteristic short sequence that mediates protein retention in the ER of eukaryotic cells. However, little is known about the detailed molecular mechanism involved in ERS-mediated protein ER retention. Using a new surface display-based fluorescence technique that effectively quantifies ERS-promoted protein ER retention within Saccharomyces cerevisiae cells, we performed comprehensive ERS analyses. We found that the length, type of amino acid residue, and additional residues at positions -5 and -6 of the C-terminal HDEL motif all determined the retention of ERS in the yeast ER. Moreover, the biochemical results guided by structure simulation revealed that aromatic residues (Phe-54, Trp-56, and other aromatic residues facing the ER lumen) in both the ERS (at positions -6 and -4) and its receptor, Erd2, jointly determined their interaction with each other. Our studies also revealed that this aromatic residue interaction might lead to the discriminative recognition of HDEL or KDEL as ERS in yeast or human cells, respectively. Our findings expand the understanding of ERS-mediated residence of proteins in the ER and may guide future research into protein folding, modification, and translocation affected by ER retention.

Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries.

Myriad new applications of proteases would be enabled by an ability to fine-tune substrate specificity and activity. Herein we present a general strategy for engineering protease selectivity and activity by capitalizing on sequestration of the protease to be engineered within the yeast endoplasmic reticulum (ER). A substrate fusion protein composed of yeast adhesion receptor subunit Aga2, selection and counterselection substrate sequences, multiple intervening epitope tag sequences, and a C-terminal ER retention sequence is coexpressed with a protease library. Cleavage of the substrate fusion protein by the protease eliminates the ER retention sequence, facilitating transport to the yeast surface. Yeast cells that display Aga2 fusions in which only the selection substrate is cleaved are isolated by multicolor FACS with fluorescently labeled antiepitope tag antibodies. Using this system, the Tobacco Etch Virus protease (TEV-P), which strongly prefers Gln at P1 of its canonical ENLYFQ↓S substrate, was engineered to recognize selectively Glu or His at P1. Kinetic analysis indicated an overall 5,000-fold and 1,100-fold change in selectivity, respectively, for the Glu- and His-specific TEV variants, both of which retained high catalytic turnover. Human granzyme K and the hepatitis C virus protease were also shown to be amenable to this unique approach. Further, by adjusting the signaling strategy to identify phosphorylated as opposed to cleaved sequences, this unique system was shown to be compatible with the human Abelson tyrosine kinase.

NDI and DAN DNA: nucleic acid-directed assembly of NDI and DAN.

Two novel DNA base surrogate phosphoramidites 1 and 2, based upon relatively electron-rich 1,5-dialkoxynaphthalene (DAN) and relatively electron-deficient 1,4,5,8-naphthalenetetracarboxylic diimide (NDI), respectively, were designed, synthesized, and incorporated into DNA oligonucleotide strands. The DAN and NDI artificial DNA bases were inserted within a three-base-pair region within the interior of a 12-mer oligonucleotide duplex in various sequential arrangements and investigated with CD spectroscopy and UV melting curve analysis. The CD spectra of the modified duplexes indicated B-form DNA topology. Melting curve analyses revealed trends in DNA duplex stability that correlate with the known association of DAN and NDI moieties in aqueous solution as well as the known favorable interactions between NDI and natural DNA base pairs. This demonstrates that DNA duplex stability and specificity can be driven by the electrostatic complementarity between DAN and NDI. In the most favorable case, an NDI-DAN-NDI arrangement in the middle of the DNA duplex was found to be approximately as stabilizing as three A-T base pairs.

Therapeutic enzyme deimmunization by combinatorial T-cell epitope removal using neutral drift.

A number of heterologous enzymes have been investigated for cancer treatment and other therapeutic applications; however, immunogenicity issues have limited their clinical utility. Here, a new approach has been created for heterologous enzyme deimmunization whereby combinatorial saturation mutagenesis is coupled with a screening strategy that capitalizes on the evolutionary biology concept of neutral drift, and combined with iterative computational prediction of T-cell epitopes to achieve extensive reengineering of a protein sequence for reduced MHC-II binding propensity without affecting catalytic and pharmacological properties. Escherichia coli L-asparaginase II (EcAII), the only nonhuman enzyme approved for repeated administration, is critical in treatment of childhood acute lymphoblastic leukemia (ALL), but elicits adverse antibody responses in a significant fraction of patients. The neutral drift screening of combinatorial saturation mutagenesis libraries at a total of 12 positions was used to isolate an EcAII variant containing eight amino acid substitutions within computationally predicted T-cell epitopes--of which four were nonconservative--while still exhibiting k(cat)/K(M) = 10(6) M(-1) s(-1) for L-Asn hydrolysis. Further, immunization of HLA-transgenic mice expressing the ALL-associated DRB1*0401 allele with the engineered variant resulted in significantly reduced T-cell responses and a 10-fold reduction in anti-EcAII IgG titers relative to the existing therapeutic. This significant reduction in the immunogenicity of EcAII may be clinically relevant for ALL treatment and illustrates the potential of employing neutral drift screens to achieve large jumps in sequence space as may be required for the deimmunization of heterologous proteins.

High-Resolution Substrate Specificity Profiling of SARS-CoV-2 Mpro; Comparison to SARS-CoV Mpro.

The SARS-CoV-2 Mpro protease from COVID-19 cleaves the pp1a and pp2b polyproteins at 11 sites during viral maturation and is the target of Nirmatrelvir, one of the two components of the frontline treatment sold as Paxlovid. We used the YESS 2.0 platform, combining protease and substrate expression in the yeast endoplasmic reticulum with fluorescence-activated cell sorting and next-generation sequencing, to carry out the high-resolution substrate specificity profiling of SARS-CoV-2 Mpro as well as the related SARS-CoV Mpro from SARS 2003. Even at such a high level of resolution, the substrate specificity profiles of both enzymes are essentially identical. The population of cleaved substrates isolated in our sorts is so deep, the relative catalytic efficiencies of the different cleavage sites on the SARS-CoV-2 polyproteins pp1a and pp2b are qualitatively predicted. These results not only demonstrated the precise and reproducible nature of the YESS 2.0/NGS approach to protease substrate specificity profiling but also should be useful in the design of next generation SARS-CoV-2 Mpro inhibitors, and by analogy, SARS-CoV Mpro inhibitors as well.

Chemical and linguistic considerations for encoding Chinese characters: an embodiment using chain-end degradable sequence-defined oligourethanes created by consecutive solid phase click chemistry.

Sequence-defined polymers (SDPs) are currently being investigated for use as information storage media. As the number of monomers in the SDPs increases, with a corresponding increase in mathematical base, the use of tandem-MS for de novo sequencing becomes more challenging. In contrast, chain-end degradation routines are truly de novo, potentially allowing very large mathematical bases for encoding. While alphabetic scripts have a few dozen symbols, logographic scripts, such as Chinese, can have several thousand symbols. Using a new in situ consecutive click reaction approach on an oligourethane backbone for writing, and a previously reported chain-end degradation routine for reading, we encoded/decoded a confucius proverb written in Chinese characters using two encoding schemes: Unicode and Zhèng Ma. Unicode is an internationally standardized arbitrary string of hexadecimal (base-16) symbols which efficiently encodes uniquely identifiable symbols but requires complete fidelity of transmission, or context-based inferential strategies to be interpreted. The Zhèng Ma approach encodes with a base-26 system using the visual characteristics and internal composition of Chinese characters themselves, which leads to greater ambiguity of encoded strings, but more robust retrievability of information from partial or corrupted encodings. The application of information-encoded oligourethanes to two different encoding systems allowed us to establish their flexibility and versatility for data storage. We found the oligourethanes immensely adaptable to both encoding schemes for Chinese characters, and we highlight the expected tradeoff between the efficiency and uniqueness of Unicode encoding on the one hand, and the fidelity to a scripts' particular visual characteristics on the other.

Threading polyintercalators with extremely slow dissociation rates and extended DNA binding sites.

The development of small molecules that bind DNA sequence specifically has the potential to modulate gene expression in a general way. One mode of DNA binding is intercalation, or the insertion of molecules between DNA base pairs. We have developed a modular polyintercalation system in which intercalating naphthalene diimide (NDI) units are connected by flexible linkers that alternate between the minor and major grooves of DNA when bound. We recently reported a threading tetraintercalator with a dissociation half-life of 16 days, the longest reported to date, from its preferred 14 bp binding site. Herein, three new tetraintercalator derivatives were synthesized with one, two, and three additional methylene units in the central major groove-binding linker. These molecules displayed dissociation half-lives of 57, 27, and 18 days, respectively, from the 14 bp site. The optimal major groove-binding linker was used in the design of an NDI hexaintercalator that was analyzed by gel-shift assays, DNase I footprinting, and UV-vis spectroscopy. The hexaintercalator bound its entire 22 bp binding site, the longest reported specific binding site for a synthetic, non-nucleic acid-based DNA binding molecule, but with a significantly faster dissociation rate compared to the tetraintercalators.

More than meets the eye: conformational switching of a stacked dialkoxynaphthalene-naphthalenetetracarboxylic diimide (DAN-NDI) foldamer to an NDI-NDI fibril aggregate.

The thermally induced conformational switching of a stacked dialkxoynaphthalene-naphthalenetetracarboxylic diimide (DAN-NDI) amphiphilic foldamer to an NDI-NDI fibril aggregate is described. The aggregated fibril structures were explored by UV/Vis, circular dichroism (CD), atomic-force microscopy (AFM), and TEM techniques. Our findings indicate that the aromatic DAN-NDI interactions of the original foldamer undergoes transformation to a fibrillar assembly with aromatic NDI-NDI stacked interactions. These structural insights could help inform new molecular designs and increase our understanding of fibrillar assembly and aggregation process in aqueous solution.

Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways.

The first and highly conserved step in glutathione (GSH) biosynthesis is formation of γ-glutamyl cysteine by the enzyme glutamate-cysteine ligase (GshA). However, bioinformatic analysis revealed that many prokaryotic species that encode GSH-dependent proteins lack the gene for this enzyme. To understand how bacteria cope without gshA, we isolated Escherichia coli ΔgshA multigenic suppressors that accumulated physiological levels of GSH. Mutations in both proB and proA, the first two genes in L-proline biosynthesis, provided a new pathway for γ-glutamyl cysteine formation via the selective interception of ProB-bound γ-glutamyl phosphate by amino acid thiols, likely through an S-to-N acyl shift mechanism. Bioinformatic analysis suggested that the L-proline biosynthetic pathway may have a second role in γ-glutamyl cysteine formation in prokaryotes. Also, we showed that this mechanism could be exploited to generate cytoplasmic redox buffers bioorthogonal to GSH.

Subtle recognition of 14-base pair DNA sequences via threading polyintercalation.

Small molecules that bind DNA in a sequence-specific manner could act as antibiotic, antiviral, or anticancer agents because of their potential ability to manipulate gene expression. Our laboratory has developed threading polyintercalators based on 1,4,5,8-naphthalene diimide (NDI) units connected in a head-to-tail fashion by flexible peptide linkers. Previously, a threading tetraintercalator composed of alternating minor-major-minor groove-binding modules was shown to bind specifically to a 14 bp DNA sequence with a dissociation half-life of 16 days [Holman, G. G., et al. (2011) Nat. Chem. 3, 875-881]. Herein are described new NDI-based tetraintercalators with a different major groove-binding module and a reversed N to C directionality of one of the minor groove-binding modules. DNase I footprinting and kinetic analyses revealed that these new tetraintercalators are able to discriminate, by as much as 30-fold, 14 bp DNA binding sites that differ by 1 or 2 bp. Relative affinities were found to correlate strongly with dissociation rates, while overall C(2) symmetry in the DNA-binding molecule appeared to contribute to enhanced association rates.

Using Spectator Ligands to Enhance Nanocrystal-to-Molecule Electron Transfer.

Semiconductor nanocrystals (NCs) have emerged as promising photocatalysts. However, NCs are often functionalized with complex ligand shells that contain not only charge acceptors but also other "spectator ligands" that control NC solubility and affinity for target reactants. Here, we show that spectator ligands are not passive observers of photoinduced charge transfer but rather play an active role in this process. We find the rate of electron transfer from quantum-confined PbS NCs to perylenediimide acceptors can be varied by over a factor of 4 simply by coordinating cinnamate ligands with distinct dipole moments to NC surfaces. Theoretical calculations indicate this rate variation stems from both ligand-induced changes in the free energy for charge transfer and electrostatic interactions that alter perylenediimide electron acceptor orientation on NC surfaces. Our work shows NC-to-molecule charge transfer can be fine-tuned through ligand shell design, giving researchers an additional handle for enhancing NC photocatalysis.

YESS 2.0, a Tunable Platform for Enzyme Evolution, Yields Highly Active TEV Protease Variants.

Here we describe YESS 2.0, a highly versatile version of the yeast endoplasmic sequestration screening (YESS) system suitable for engineering and characterizing protein/peptide modifying enzymes such as proteases with desired new activities. By incorporating features that modulate gene transcription as well as substrate and enzyme spatial sequestration, YESS 2.0 achieves a significantly higher operational and dynamic range compared with the original YESS. To showcase the new advantages of YESS 2.0, we improved an already efficient TEV protease variant (TEV-EAV) to obtain a variant (eTEV) with a 2.25-fold higher catalytic efficiency, derived almost entirely from an increase in turnover rate (kcat). In our analysis, eTEV specifically digests a fusion protein in 2 h at a low 1:200 enzyme to substrate ratio. Structural modeling indicates that the increase in catalytic efficiency of eTEV is likely due to an enhanced interaction between the catalytic Cys151 with the P1 substrate residue (Gln). Furthermore, the modeling showed that the ENLYFQS peptide substrate is buried to a larger extent in the active site of eTEV compared with WT TEV. The new eTEV variant is functionally the fastest TEV variant reported to date and could potentially improve efficiency in any TEV application.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes.

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.

Highly active and selective endopeptidases with programmed substrate specificities.

A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (kcat/KM > 10(40 M(- 1) s(- 1)). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including GluArg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic.

A systematic study of thermochromic aromatic donor-acceptor materials.

Molar mixtures (1:1) of electron-rich dialkoxynapthalene (Dan) and electron-deficient 1,4,5,8-napthalenetetracarboxylic diimide (Ndi) derivatives form highly tunable, columnar mesophases with a dark red color due to a charge transfer absorbance derived from alternating face-centered stacking. Certain Dan-Ndi mixtures undergo a dramatic color change from dark red to an almost colorless material upon crystallizing from the mesophase. Macroscopic morphology of the solid is not changed during this process. In order to investigate the origins of this interesting thermochromic behavior, Dan and Ndi side chains were systematically altered and their 1:1 mixtures were studied. We have previously speculated that the presence or absence of steric interactions due to side chain branching on the aromatic units controlled the level of color change associated with crystallization. Results from the present study further refine this conclusion including a key crystal structure that provides a structural rationale for the observed results.

Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli.

We describe facile isolation of full-length IgG antibodies from combinatorial libraries expressed in E. coli. Full-length heavy and light chains are secreted into the periplasm, where they assemble into aglycosylated IgGs that are captured by an Fc-binding protein that is tethered to the inner membrane. After permeabilizing the outer membrane, spheroplast clones expressing so-called E-clonal antibodies, which specifically recognize fluorescently labeled antigen, are selected using flow cytometry. Screening of a library constructed from an immunized animal yielded several antibodies with nanomolar affinities toward the protective antigen of Bacillus anthracis.

Quantitative Analysis of the Substrate Specificity of Human Rhinovirus 3C Protease and Exploration of Its Substrate Recognition Mechanisms.

Human rhinovirus 3C protease (HRV 3C-P) is a high-value commercial cysteine protease that could specifically recognize the short peptide sequence of LEVLFQ↓GP. In here, a strategy based on our previous Yeast Endoplasmic Reticulum Sequestration Screening (YESS) approach was developed in Saccharomyces cerevisiae, a model microorganism, to fully characterize the substrate specificity of a typical human virus protease, HRV 3C-P, in a quantitative and fast manner. Our results demonstrated that HRV 3C-P had very high specificity at P1 and P1' positions, only recognizing Gln/Glu at the P1 position and Gly/Ala/Cys/Ser at the P1' position, respectively. Comparably, it exhibited efficient recognition of most residues at the P2' position, except Trp. Further biochemical characterization through site mutagenesis, enzyme structural modeling, and comparison with other 3C proteases indicated that the S1 pocket of HRV 3C-P was constituted by neutral and basic amino acids, in which His160 and Thr141 specifically interacted with Gln or Glu residues at the substrate P1 position. Additionally, the stringent S1' pocket determined its unique property of only accommodating residues without or with short side chains. Based on our characterization, LEVLFQ↓GM was identified as a more favorable substrate than the original LEVLFQ↓GP at high temperature, which might be caused by the conversion of random coils to ß-turns in HRV 3C-P along with the temperature increase. Our studies prompted a further understanding of the substrate specificity and recognition mechanism of HRV 3C-P. Besides, the YESS-PSSC combined with the enzyme modeling strategy in this study provides a general strategy for deciphering the substrate specificities of proteases.