SEARCH · Search NASA

Results for “complementarity”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7

All sources NASA NTRS DOE OSTI

DOE OSTI2024

Vaccination induces broadly neutralizing antibody precursors to HIV gp41

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

CRISPR–Cas9 bends and twists DNA to read its sequence

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9–RNA–DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 ‘reads’ snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single-domain camelid antibody

Transporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

A native IgE in complex with profilin provides insights into allergen recognition and cross-reactivity

Allergies have become a rising health problem, where plentiful substances can trigger IgE-mediated allergies in humans. While profilins are considered minor allergens, these ubiquitous proteins are primary molecules involved in cross-reactivity and pollen-food allergy syndrome. Here we report the first crystal structures of murine Fab/IgE, with its chains naturally paired, in complex with the allergen profilin from Hevea brasiliensis (Hev b 8). The crystallographic models revealed that the IgE’s six complementarity-determining regions (CDRs) interact with the allergen, comprising a rigid paratope-epitope surface of 926 Å 2 , which includes an extensive network of interactions. Interestingly, we also observed previously unreported flexibility at Fab/IgE’s elbow angle, which did not influence the shape of the paratope. The Fab/IgE exhibits a high affinity for Hev b 8, even when using 1 M NaCl in BLI experiments. Finally, based on the encouraging cross-reactivity assays using two mutants of the maize profilin (Zea m 12), this antibody could be a promising tool in IgE engineering for diagnosis and research applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Global tuning of hadronic interaction models with accelerator-based and astroparticle data

In high-energy and astroparticle physics, event generators play an essential role, even in the simplest data analyses. As analysis techniques become more sophisticated, e.g. based on deep neural networks, their correct description of the observed event characteristics becomes even more important. Physical processes occurring in hadronic collisions are simulated within a Monte Carlo framework. A major challenge is the modeling of hadron dynamics at low momentum transfer, which includes the initial and final phases of every hadronic collision. QCD-inspired phenomenological models used for these phases cannot guarantee completeness or correctness over the full phase space. These models usually include parameters which must be tuned to suitable experimental data. Until now, event generators have been developed and tuned mainly on the basis of data from high-energy physics experiments at accelerators. The wealth of data available from the latest generation of astroparticle experiments has not yet been fully exploited, and in many cases is not satisfactorily described. Both kinds of data sets are complementary as astroparticle experiments provide sensitivity especially to hadrons produced nearly parallel to the collision axis and cover center-of-mass energies up to several hundred TeV, well beyond those reached at colliders so far. In this report, we provide an overview of state-of-the-art event generators and their tuning, including the most relevant inputs from high-energy accelerator and astroparticle experiments. We present a road map that shows, for the first time, how the unified tuning of event generators with accelerator-based and astroparticle data can be performed.

Albrecht, J. [Ruhr U., Bochum, RAPP Ctr.; Ruhr U.,↗

DOE OSTI2025

VHH antibody loop guides design of a synthetic macrocyclic peptide that potently blocks influenza virus membrane fusion

Abstract Miniaturizing biologically complex structural motifs to produce synthetic functional mimetics holds significant promise for development of new therapeutic modalities. Here, we demonstrate a unique approach using the key binding loop of the single variable domain of a heavy chain (V H H) llama antibody as a starting point for peptide design. V H H antibodies of camelids and sharks generally have longer, but more ligand-efficient complementarity determining region 3 (CDR3) loops and are relatively stable structures. We harnessed these attributes as templates for design of a series of synthetic macrocyclic peptides. The designed peptides exhibit nanomolar binding to influenza hemagglutinin (HA) and heterosubtypic in vitro neutralization breadth against influenza A viruses by inhibiting the low pH mediated HA conformational changes that lead to membrane fusion. X-ray structures of peptide-HA complexes reveal high structural mimicry with the parent V H H antibody. One such macrocycle peptide candidate is promising for further development of broad protection against influenza A group 1 viruses.

Kadam, Rameshwar U.↗

DOE OSTI2021

Identification and quantification of lignin monomers and oligomers from reductive catalytic fractionation of pine wood with GC × GC – FID/MS

Thorough lignin characterization is vital to understand the physicochemical properties of lignin and to evaluate lignocellulose biorefinery processes. In this study, an in-depth characterization of lignin oil, obtained from reductive catalytic fractionation (RCF) of pine wood, was performed with quantitative GC × GC – FID analysis and qualitative GC × GC – MS. By utilizing high-temperature resistant column sets in the GC × GC system and by applying a derivatization step, unambiguous detection of lignin monomers, dimers, and trimers is enabled. In addition to confirm the identity of eleven monomers, corresponding to 34 wt% of the RCF lignin oil, thirty-six dimers (16 wt%) and twenty-one trimers (7 wt%) were comprehensively identified by analysis of their mass spectra and quantified by a FID, encompassing the identity of an additional 23 wt% of the RCF lignin oil. The proposed structures reveal the interlinkages present in the dimeric and trimeric oligomers, containing β-5, β-1, β–β, 5–5, and a minor fraction of β-O-4 and 4-O-5 bonds. Furthermore, aliphatic end-units in the dimeric and trimeric molecules were identified, consisting of various substituents at the C4 position, that have been previously observed in the RCF-derived lignin monomers. To reduce complexity for analysis, the RCF oil was separated into six fractions, prior to analysis. The structural motifs (inter-unit linkages and end-units) that are found in the different fractions vary significantly, such that the lignin fractions extracted in more polar solvents contained higher molecular weight fragments and more hydroxyl containing structural motifs. Additionally, the identified structures of individual dimer and trimer molecules by GC × GC align well with and further complement the recent findings from 1 H– 13 C HSQC NMR spectroscopy, demonstrating complementarity between both 2D techniques to obtain a holistic view on both the molecular structures and the distribution of bonds and end-units in RCF oil. The combination of these two techniques provides a powerful tool for future RCF and other lignin depolymerization research.

09 BIOMASS FUELS↗

DOE OSTI2025

Understanding the structural mechanics of ligated DNA crystals via molecular dynamics simulation

DNA self-assembly is a highly programmable method to construct arbitrary architectures based on sequence complementarity. Among various constructs, DNA crystals are macroscopic crystalline materials formed by assembling motifs via sticky end association. Due to their high structural integrity and size ranging from tens to hundreds of micrometers, DNA crystals offer unique opportunities to study the structural properties and deformation behaviors of DNA assemblies. For example, enzymatic ligation of sticky ends can selectively seal nicks resulting in more robust structures with enhanced mechanical properties. However, the research efforts have been mostly on experiments involving different motif designs, structural optimization, or new synthesis methods, while their mechanics are not yet fully understood. The complex properties of DNA crystals are difficult to study via experiments alone, and numerical simulation can complement and aid the experiments. The coarse-grained molecular dynamics (MD) simulation is a powerful tool that can probe the mechanics of DNA assemblies. Here, we investigate DNA crystals made of four different motif lengths with various ligation patterns (full ligation, major directions, connectors, and in-plane) using oxDNA, an open-source, coarse-grained MD platform. We found that several distinct deformation stages emerge in response to mechanical loading and that the number and the location of ligated nucleotides can significantly modulate structural behaviors. These findings should be useful for predicting crystal properties and thus improving the design.

DNA crystal↗

DOE OSTI2021

Clustering of CODEX clusters

The clustering of galaxy clusters links the spatial nonuniformity of dark matter halos to the growth of the primordial spectrum of perturbations. The amplitude of the clustering signal is widely used to estimate the halo mass of astrophysical objects. The advent of cluster mass calibrations enables using clustering in cosmological studies. Aims. We analyze the autocorrelation function of a large contiguous sample of galaxy clusters, the Constrain Dark Energy with X-ray (CODEX) sample, in which we take particular care of cluster definition. These clusters were X-ray selected using the ROentgen SATellite All-Sky Survey and then identified as galaxy clusters using the code redMaPPer run on the photometry of the Sloan Digital Sky Survey. We develop methods for precisely accounting for the sample selection effects on the clustering and demonstrate their robustness using numerical simulations. Methods. Using the clean CODEX sample, which was obtained by applying a redshift-dependent richness selection, we computed the two-point autocorrelation function of galaxy clusters in the 0.1 < z < 0.3 and 0.3 < z < 0.5 redshift bins. We compared the bias in the measured correlation function with values obtained in numerical simulations using a similar cluster mass range. Results. By fitting a power law, we measured a correlation length r 0 = 18.7 ± 1.1 and slope γ = 1.98 ± 0.14 for the correlation function in the full redshift range. By fixing the other cosmological parameters to their nine-year Wilkinson Microwave Anisotropy Probe values, we reproduced the observed shape of the correlation function under the following cosmological conditions: Ω m0 = $0.22_{-0.03}^{+0.04}$ and S 8 = σ 8 (Ω m0 /0.3)0.5 = $0.85_{-0.08}^{+0.10}$ with estimated additional systematic errors of σ Ω m0 = 0.02 and σS 8 = 0.20. We illustrate the complementarity of clustering constraints by combining them with CODEX cosmological constraints based on the X-ray luminosity function, deriving Ωm = 0.25 ± 0.01 and σ 8 = $0.81_{-0.02}^{+0.01}$ with an estimated additional systematic error of σ Ω m0 = 0.07 and σ σ8 = 0.04. The mass calibration and statistical quality of the mass tracers are the dominant source of uncertainty.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2023

Simultaneous measurements of volume, pressure, optical images, and crystal structure with a dynamic diamond anvil cell: A real-time event monitoring system

The dynamic diamond anvil cell (dDAC) technique has attracted great interest because it possibly provides a bridge between static and dynamic compression studies with fast, repeatable, and controllable compression rates. The dDAC can be a particularly useful tool to study the pathways and kinetics of phase transitions under dynamic pressurization if simultaneous measurements of physical quantities are possible as a function of time. We report the development of a real-time event monitoring (RTEM) system with dDAC, which can simultaneously record the volume, pressure, optical image, and structure of materials during dynamic compression runs. In particular, the volume measurement using both Fabry–Pérot interferogram and optical images facilitates the construction of an equation of state (EoS) using the dDAC in a home-laboratory. We also developed an in-line ruby pressure measurement (IRPM) system to be deployed at a synchrotron x-ray facility. This system provides simultaneous measurements of pressure and x-ray diffraction in low and narrow pressure ranges. The EoSs of ice VI obtained from the RTEM and the x-ray diffraction data with the IRPM are consistent with each other. The complementarity of both RTEM and IRPM systems will provide a great opportunity to scrutinize the detailed kinetic pathways of phase transitions using dDAC.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

DOE OSTI2020

Photo-printing of faceted DNA patchy particles

Patchy particles with shape complementarity can serve as building blocks for assembling colloidal superstructures. Alternatively, encoding information on patches using DNA can direct assembly into a variety of crystalline or other preprogrammed structures. Here, we present a tool where DNA is used both to engineer shape and to encode information on colloidal particles. Two reactive oil emulsions with different but complementary DNA (cDNA) brushes are assembled into CsCl-like crystalline lattices. The DNA brushes are recruited to and ultimately localized at the junctions between neighboring droplets, which gives rise to DNA-encoded faceted patches. The emulsions are then solidified by ultraviolet (UV) polymerization, producing faceted patchy particles. The facet size and DNA distribution are determined by the balance between the DNA binding energy and the elastic deformation energy of droplets. This method leads to a variety of new patchy particles with directional interactions in scalable quantities.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Discovery of Marburg virus neutralizing antibodies from virus-naïve human antibody repertoires using large-scale structural predictions

Marburg virus (MARV) disease is lethal, with fatality rates up to 90%. Neutralizing antibodies (Abs) are promising drug candidates to prevent or treat the disease. Current efforts are focused in part on vaccine development to induce such MARV-neutralizing Abs. We analyzed the antibody repertoire from healthy unexposed and previously MARV-infected individuals to assess if naïve repertoires contain suitable precursor antibodies that could become neutralizing with a limited set of somatic mutations. Here, we computationally searched the human Ab variable gene repertoire for predicted structural homologs of the neutralizing Ab MR78 that is specific to the receptor binding site (RBS) of MARV glycoprotein (GP). Eight Ab heavy-chain complementarity determining region 3 (HCDR3) loops from MARV-naïve individuals and one from a previously MARV-infected individual were selected for testing as HCDR3 loop chimeras on the MR78 Ab framework. Three of these chimerized antibodies bound to MARV GP. We then tested a full-length native Ab heavy chain encoding the same 17-residue-long HCDR3 loop that bound to the MARV GP the best among the chimeric Abs tested. Despite only 57% amino acid sequence identity, the Ab from a MARV-naïve donor recognized MARV GP and possessed neutralizing activity against the virus. Crystallization of both chimeric and full-length native heavy chain-containing Abs provided structural insights into the mechanism of binding for these types of Abs. Our work suggests that the MARV GP RBS is a promising candidate for epitope-focused vaccine design to induce neutralizing Abs against MARV.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Structural basis for differential recognition of phosphohistidine-containing peptides by 1-pHis and 3-pHis monoclonal antibodies

In 2015, monoclonal antibodies (mAbs) that selectively recognize the 1-pHis or 3-pHis isoforms of phosphohistidine were developed by immunizing rabbits with degenerate Ala/Gly peptides containing the nonhydrolyzable phosphohistidine (pHis) analog- phosphotriazolylalanine (pTza). Here, we report structures of five rabbit mAbs bound to cognate pTza peptides: SC1-1 and SC50-3 that recognize 1-pHis, and their 3-pHis–specific counterparts, SC39-4, SC44-8, and SC56-2. These cocrystal structures provide insights into the binding modes of the pTza phosphate group that are distinct for the 1- and 3-pHis mAbs with the selectivity arising from specific contacts with the phosphate group and triazolyl ring. The mode of phosphate recognition in the 3-pHis mAbs recapitulates the Walker A motif, as present in kinases. The complementarity-determining regions (CDRs) of four of the Fabs interact with the peptide backbone rather than peptide side chains, thus conferring sequence independence, whereas SC44-8 shows a proclivity for binding a GpHAGA motif mediated by a sterically complementary CDRL3 loop. Specific hydrogen bonding with the triazolyl ring precludes recognition of pTyr and other phosphoamino acids by these mAbs. Kinetic binding experiments reveal that the affinity of pHis mAbs for pHis and pTza peptides is submicromolar. Bound pHis mAbs also shield the pHis peptides from rapid dephosphorylation. The epitope–paratope interactions illustrate how these anti-pHis antibodies are useful for a wide range of research techniques and this structural information can be utilized to improve the specificity and affinity of these antibodies toward a variety of pHis substrates to understand the role of histidine phosphorylation in healthy and diseased states.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

A conserved epitope III on hepatitis C virus E2 protein has alternate conformations facilitating cell binding or virus neutralization

Epitope III, a highly conserved amino acid motif of 524 APTYSW 529 on the hepatitis C virus (HCV) E2 glycoprotein, resides in the critical loop that binds to the host receptor CD81, thus making it one of the most important antibody targets for blocking HCV infections. Here, we have determined the X-ray crystal structure of epitope III at a 2.0-Å resolution when it was captured by a site-specific neutralizing antibody, monoclonal antibody 1H8 (mAb1H8). The snapshot of this complex revealed that epitope III has a relatively rigid structure when confined in the binding grooves of mAb1H8, which confers the residue specificity at both ends of the epitope. Such a high shape complementarity is reminiscent of the “lock and key” mode of action, which is reinforced by the incompatibility of an antibody binding with an epitope bearing specific mutations. By subtly positioning the side chains on the three residues of Tyr 527 , Ser 528 , and Trp 529 while preserving the spatial rigidity of the rest, epitope III in this cocrystal complex adopts a unique conformation that is different from previously described E2 structures. With further analyses of molecular docking and phage display–based peptide interactions, we recognized that it is the arrangements of two separate sets of residues within epitope III that create these discrete conformations for the epitope to interact selectively with either mAb1H8 or CD81. These observations thus raise the possibility that local epitope III conformational dynamics, in conjunction with sequence variations, may act as a regulatory mechanism to coordinate “mAb1H8-like” antibody-mediated immune defenses with CD81-initiated HCV infections.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This “knob” domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC 50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

PE5–PPE4–EspG 3 heterotrimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems

Mycobacterium tuberculosis has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. ESX-1, ESX-3, and ESX-5 systems have been shown to each secrete a distinct set of substrates, including PE and PPE families of proteins, named for conserved Pro-Glu and Pro-Pro-Glu motifs in their N termini. Proper secretion of the PE–PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems, and how each EspG recognizes its subset of PE–PPE proteins is currently unknown. The only current structural characterization of PE–PPE–EspG heterotrimers is from the ESX-5 system. Here we present the crystal structure of the PE5 mt –PPE4 mt –EspG 3mm heterotrimer from the ESX-3 system. Our heterotrimer reveals that EspG 3mm interacts exclusively with PPE4 mt in a similar manner to EspG 5 , shielding the hydrophobic tip of PPE4 mt from solvent. The C-terminal helical domain of EspG 3mm is dynamic, alternating between “open” and “closed” forms, and this movement is likely functionally relevant in the unloading of PE–PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 heterotrimers, the PE–PPE heterodimer of our ESX-3 heterotrimer is interacting with its chaperone at a drastically different angle and presents different faces of the PPE protein to the chaperone. We conclude that the PPE–EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate among noncognate PE–PPE pairs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Interdependence in active mobility adoption: Joint modeling and motivational spillover in walking, cycling and bike-sharing

Active mobility offers an array of physical, emotional, and social well-being benefits. However, with the proliferation of the sharing economy, new nonmotorized means of transport are entering the fold, complementing some existing mobility options while competing with others. The purpose of this research study is to investigate the adoption of three active travel modes—namely walking, cycling, and bikesharing—in a joint modeling framework. Here, the analysis is based on an adaptation of the stages of change framework, which originates from the health behavior sciences. Multivariate ordered probit modeling drawing on U.S. survey data provides well-needed insights into individuals’ preparedness to adopt multiple active modes as a function of personal, neighborhood, and psychosocial factors. The research suggests three important findings. (1) The joint model structure confirms interdependence among different active mobility choices. The strongest complementarity is found for walking and cycling adoption. (2) Each mode has a distinctive adoption path with either three or four separate stages. We discuss the implications of derived stage-thresholds and plot adoption contours for selected scenarios. (3) Psychological and neighborhood variables generate more coupling among active modes than individual and household factors. Specifically, identifying strongly with active mobility aspirations, experiences with multimodal travel, possessing better navigational skills, along with supportive local community norms are the factors that appear to drive the joint adoption decisions. This study contributes to the understanding of how decisions within the same functional domain are related and help to design policies that promote active mobility by identifying positive spillovers and joint determinants.

42 ENGINEERING↗

DOE OSTI2023

Molecular recognition requires dimerization of a VHH antibody

Camelid heavy-chain-only antibodies are a unique class of antibody that possesses only a single variable domain (termed VHH) for antigen recognition. Despite their apparent canonical mechanism of target recognition, where a single VHH domain binds a single target, an anti-caffeine VHH has been observed to possess 2:1 stoichiometry. Here, the structure of the anti-caffeine VHH/caffeine complex enabled the generation and biophysical analysis of variants that were used to better understand the role of VHH homodimerization in caffeine recognition. VHH interface mutants and caffeine analogs, which were examined to probe the mechanism of caffeine binding, suggested caffeine recognition is only possible with the VHH dimer species. Correspondingly, in the absence of caffeine, the anti-caffeine VHH was found to form a dimer with a dimerization constant comparable to that observed with VH:VL domains in conventional antibody systems, which was most stable near physiological temperature. While the VHH:VHH dimer structure (at 1.13 Å resolution) is reminiscent of conventional VH:VL heterodimers, the homodimeric VHH possesses a smaller angle of domain interaction, as well as a larger amount of apolar surface area burial. To test the general hypothesis that the short complementarity-determining region-3 (CDR3) may help drive VHH:VHH homodimerization, an anti-picloram VHH domain containing a short CDR3 was generated and characterized, which revealed it also existed as dimer species in solution. These results suggest homodimer-driven recognition may represent a more common method of VHH ligand recognition, opening opportunities for novel VHH homodimer affinity reagents and helping to guide their use in chemically induced dimerization applications.

60 APPLIED LIFE SCIENCES↗