Solution NMR Research Articles

Unraveling the Facet-Dependent Surface Chemistry at Molecular Scale: Photoassisted Oxidation of InP Nanocrystals.

The facet-dependent surface chemistry of nanocrystals (NCs) provides fundamental insights into chemical reactivities, which are critical for obtaining precise control over the NC surface. In this study, by obtaining InP NCs with well-defined {111} and {110}/{-1-1-1} facets (tetrahedrons and tetrapods, respectively) capped with chloride-oleylamine ligands, the previously underinvestigated facet-dependent surface chemistry of III-V materials is explored. Solid-state and solution NMR analyses show that InP tetrahedrons, with their smaller surface heterogeneity (single facet composition and lesser edge/vertex contribution) and stronger Lewis acidity, exhibit narrow 31P and 115In resonances as well as deshielded 13C signals of α-carbon adjacent to the NH2 group of oleylamine. As a result, InP tetrahedra exhibit strong ligand binding and a notable presence of less-mobile oleylamine ligands on the surface, leading to the blocking of access to external species. This is also consistent with the minimal blue shift of the first excitonic peak in absorption spectra and the strong resistance to photoassisted surface oxidation of InP tetrahedrons. Our findings, supported by solid-state/solution NMR, FT-IR, and XPS analyses, highlight the significance of facet-dependent reactivities to surface ligands and, thus, atmospheric moieties, enhancing the potential of III-V NCs in various optoelectronic applications.

Structures and NMR Solution Dynamics of Diorganotin Complexes with Bulky Quadridentate Salen‐ and Salphen‐Type Schiff Bases (N2O2) Incorporating Diazenylpyridyl Side Arms

AbstractA series of six complexes [n‐Bu2Sn(L1)]⋅S (S=Benzene/toluene, mixed/partial) (1), [Ph2Sn(L1)]n⋅S (S=Benzene) (2), [Bz2Sn(L1)] (3), [n‐Bu2Sn(L2)]n (4), [Ph2Sn(L2)] (5) and [Bz2Sn(L2)] (6) were synthesized using diorganotin(IV) oxide precursors and two new bis‐Schiff bases containing N2O2 donor atoms. The Schiff bases, H2L1 and H2L2, were prepared by reacting ethane‐1,2‐diamine or benzene‐1,2‐diamine with 2‐hydroxy‐5‐(pyridin‐3‐yldiazenyl)benzaldehyde in absolute ethanol. Compounds 1–6 were characterized by IR and NMR spectroscopies, as well as by solid‐state single‐crystal X‐ray diffraction (except for 3 and 6). In all the complexes the bis‐Schiff bases occupy the equatorial positions and the R groups are in axial sites. However, while 1 and 5 are discrete molecules where the tin atoms adopt distorted octahedral geometries, 2 and 4 are monoperiodic polymers with the metal cations in pentagonal bipyramidal environments. Although solid state crystallographic results revealed asymmetrical molecular configurations, solution NMR studies proved symmetric arrangements for all the compounds. Tin NMR spectroscopy confirmed that all complexes are six‐coordinate in solution, despite variations in solid‐state coordination. Further analysis using Hirshfeld surface and fingerprint plots provided insights into intermolecular interactions in compounds 1, 2, 4, and 5. This comprehensive study underscores the structural diversity and solution‐state dynamics of organotin(IV) complexes synthesized using novel Schiff bases.

Insights into the Structure and Dynamics of Proteins from 19F Solution NMR Spectroscopy.

19F NMR spectroscopy has recently witnessed a resurgence as an attractive analytical tool for the study of the structure and dynamics of biomolecules in vitro and in cells, despite reports of its applications in biomolecular NMR since the 1970s. The high gyromagnetic ratio, large chemical shift dispersion, and complete absence of the spin 1/2 19F nucleus from biomolecules results in background-free, high-resolution 19F NMR spectra. The introduction of 19F probes in a few selected locations in biomolecules reduces spectral crowding despite its increased line width in comparison to typical 1H NMR line widths and allows rapid site-specific measurements from simple 1D spectra alone. The design and synthesis of novel 19F probes with reduced line widths and increased chemical shift sensitivity to the surrounding environment, together with advances in labeling techniques, NMR methodology, and hardware, have overcome several drawbacks of 19F NMR spectroscopy. The increased interest and widespread use of 19F NMR spectroscopy of biomolecules is gradually establishing it as a sensitive and high-resolution probe of biomolecular structure and dynamics, supplementing traditional 13C/15N-based methods. This Review focuses on the advances in 19F solution NMR spectroscopy of proteins in the past 5 years, with an emphasis on novel 19F tags and labeling techniques, NMR experiments to probe protein structure and conformational dynamics in vitro, and in-cell NMR applications.

On the Properties of Styrene-Maleic Acid Copolymer-Lipid Nanoparticles: A Solution NMR Perspective.

The production of functionally active membrane proteins (MPs) in an adequate membrane environment is a key step in structural biology. Polymer-lipid particles based on styrene and maleic acid (SMA) represent a promising type of membrane mimic, as they can extract properly folded MPs directly from their native lipid environment. However, the original SMA polymer is sensitive to acidic pH levels, which has led to the development of several modifications: SMA-EA, SMA-QA, and others. Here, we introduce a novel SMA derivative with a negatively charged taurine moiety, SMA-tau, and investigate the formation and characteristics of lipid-SMA-EA and lipid-SMA-tau membrane-mimicking particles. Our findings demonstrate that both polymers can form nanodiscs with a patch of lipid bilayer that can undergo phase transitions at temperatures close to those of the lipid bilayer membranes. Finally, we discuss the potential applications of these SMAs for NMR spectroscopy.

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa/CD16a structure.

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa/CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues, we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step, with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating that the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa/CD16a structure

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa/CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues, we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step, with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating that the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.

Dissecting the Conformational Heterogeneity of Stem-Loop Substructures of the Fifth Element in the 5'-Untranslated Region of SARS-CoV-2.

Throughout the family of coronaviruses, structured RNA elements within the 5' region of the genome are highly conserved. The fifth stem-loop element from SARS-CoV-2 (5_SL5) represents an example of an RNA structural element, repeatedly occurring in coronaviruses. It contains a conserved, repetitive fold within its substructures SL5a and SL5b. We herein report the detailed characterization of the structure and dynamics of elements SL5a and SL5b that are located immediately upstream of the SARS-CoV-2 ORF1a/b start codon. Exploiting the unique ability of solution NMR methods, we show that the structures of both apical loops are modulated by structural differences in the remote parts located in their stem regions. We further integrated our high-resolution models of SL5a/b into the context of full-length 5_SL5 structures by combining different structural biology methods. Finally, we evaluated the impact of the two most common VoC mutations within 5_SL5 with respect to individual base-pair stability.

The new pincer-type NHCs obtained by synthesizing Ag(I)-NHC complexes with various tails containing hydroxyl or acetate derivatives: Structural properties and in vitro antibacterial activities

This article discusses the synthesis, chemistry, and uses of transition metal complexes containing functionalized N-Heterocyclic carbenes (NHCs). NHCs were obtained by coupling functional group compounds containing ethyl acetate, propyl acetate, and ethanol to the 5,6-dimethylbenzimidazole compound. Thermogravimetric analysis (TGA), FT-IR, and solution (1H and 13C) NMR spectroscopy were used to characterize the elements in these compounds, and Rietveld analysis using X-ray powder diffraction (XPD) and ICP (Inductively Coupled Plasma) data was used to determine their structures. Major uses include catalyst modification and immobilization using a functional group to immobilize catalysts on a 5,6-dimethylbenzimidazole derivative support. The effects of the drug ampicillin were compared with the antimicrobial activities of 3a-c and 4a-c determined by the minimum inhibitory concentration (MIC; μg/ml). The MIC value of the compound bis(1,3-bis(3-ethoxy-3-oxopropyl)-5,6-dimethyl-1H-3λ4-benzo[d]imidazol-2-ylidene)silver(I)hexafluorophosphate (4b) was measured as 0.25 ± 2.05 μg/mL. This value indicates that this complex has strong antibacterial abilities.

The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19F-NMR.

Endogenous phospholipids influence the conformational equilibria of G protein-coupled receptors, regulating their ability to bind drugs and form signaling complexes. However, most studies of GPCR-lipid interactions have been carried out in mixed micelles or lipid nanodiscs. Though useful, these membrane mimetics do not fully replicate the physical properties of native cellular membranes associated with large assemblies of lipids. We investigated the conformational equilibria of the human A2A adenosine receptor (A2AAR) in phospholipid vesicles using 19F solid-state magic angle spinning NMR (SSNMR). By applying an optimized sample preparation workflow and experimental conditions, we were able to obtain 19F-SSNMR spectra for both antagonist- and agonist-bound complexes with sensitivity and linewidths closely comparable to those achieved using solution NMR. This facilitated a direct comparison of the A2AAR conformational equilibria across detergent micelle, lipid nanodisc, and lipid vesicle preparations. While antagonist-bound A2AAR showed a similar conformational equilibria across all membrane and membrane mimetic systems, the conformational equilibria of agonist-bound A2AAR exhibited differences among different environments. This suggests that the conformational equilibria of GPCRs may be influenced not only by specific receptor-lipid interactions but also by the membrane properties found in larger lipid assemblies.

Multimode Masers of Thermally Polarized Nuclear Spins in Solution NMR.

We present experimental single and multimode sustained ^{1}H NMR masers in solution on thermally polarized spins at room temperature and 9.4T achieved through the electronic control of radiation feedback (radiation damping). Our observations illustrate the breakdown of the usual three-dimensional Maxwell-Bloch equations for radiation feedback and a simple toy model of few coupled classical moments is used to interpret these experiments. This Letter represents a significant step to bring the spontaneous radiation damping based NMR masers in various contexts to the next stage of feedback-controlled and reproducible experiments.

Molecular Interactions between Tau Protein and TIA1: Distinguishing Physiological Condensates from Pathological Fibrils.

The interaction of tau protein with other key proteins essential for stress granule formation determines their functional and pathological impact. In a biological framework, the synergy between Alzheimer's associated tau protein and the stress granule core protein TIA1 is widely recognized. However, the molecular details of this association remain unclear. In this study, we throw light on the importance of the state in which the TIA1 exists in mediating its association with the tau protein. Investigations were carried out on the three repeat constructs of tau (K19) and different structures formed by TIA1. Specifically, the condensate formed by TIA1 full-length (TIA1-FL) protein as well as fibril formed by low complexity domain of TIA1 (TIA1-LCD). The dynamics of K19 inside TIA1-FL condensates and the aggregation kinetics of K19 in the presence of TIA1-LCD fibrils were examined using various biophysical techniques. Relaxation-based solution NMR spectroscopic investigations suggest a weak interaction with TIA1 condensates and indicated a reduction in the dynamics of K19 within these TIA1 condensates. In contrast, a significant interaction was observed between K19, and TIA1-LCD fibrils primarily mediated through 321KCGS324 and 306VQIVYKPVDLSKV317. Our findings emphasize that the interaction between Tau and TIA1 varies depending on whether TIA1 is in its physiological condensate form or its pathological fibril state.

Slow Conformational Exchange between Partially Folded and Near-Native States of Ubiquitin: Evidence for a Multistate Folding Model.

The mechanism by which small proteins fold, i.e., via intermediates or via a two-state mechanism, is a subject of intense investigation. Intermediate states in the folding pathways of these proteins are sparsely populated due to transient lifetimes under normal conditions rendering them transparent to a majority of the biophysical methods employed for structural, thermodynamic, and kinetic characterization, which attributes are essential for understanding the cooperative folding/unfolding of such proteins. Dynamic NMR spectroscopy has enabled the characterization of folding intermediates of ubiquitin that exist in equilibrium under conditions of low pH and denaturants. At low pH, an unlocked state defined as N' is in fast exchange with an invisible state, U″, as observed by CEST NMR. Addition of urea to ubiquitin at pH 2 creates two new states F' and U', which are in slow exchange (kF'→U' = 0.14 and kU'→F' = 0.28 s-1) as indicated by longitudinal ZZ-magnetization exchange spectroscopy. High-resolution solution NMR structures of F' show it to be in an "unlocked" conformation with measurable changes in rotational diffusion, translational diffusion, and rotational correlational times. U' is characterized by the presence of just the highly conserved N-terminal β1-β2 hairpin. The folding of ubiquitin is cooperative and is nucleated by the formation of an N-terminal β-hairpin followed by significant hydrophobic collapse of the protein core resulting in the formation of bulk of the secondary structural elements stabilized by extensive tertiary contacts. U' and F' may thus be described as early and late folding intermediates in the ubiquitin folding pathway.

Advanced Methods for Characterizing Battery Interfaces: Towards a Comprehensive Understanding of Interfacial Evolution in Modern Batteries

Batteries are complex systems operating far from equilibrium, relying on intricate reactions at interfaces for performance. Understanding and optimizing these interfaces is crucial, but challenges arise due to the diverse factors influencing their development, making comprehensive characterization essential despite experimental difficulties. Recent advancements in characterization tools offer new opportunities to explore interfacial evolution, particularly in the solid electrolyte interphase (SEI).In this perspective article, leading experts in physical-chemical characterization techniques for electrochemical systems discuss the current state-of-the-art and emerging approaches to study interfaces and their evolution in batteries. The focus here is on the capabilities, technical challenges, limitations, and requirements that these techniques must meet to advance our understanding of battery interfacial evolution. The emphasis is placed on techniques that enable probing interfaces under realistic conditions, close to commercial battery systems, and on the integration of multiple approaches within a single measurement (multimodal) to minimise variable effects.This article focuses on the most promising techniques for characterizing all phases relevant to interfacial processes, as well as their integration with correlative analyses and computational modelling. We discuss solid phase characterization with X-ray spectroscopies and microscopies (XPS, XAS, STXM, X-PEEM & XCT), Raman spectroscopies (SERS, TERS & SHINERS), solid-state NMR and electron microscopies and spectroscopies (STEM, EDSX, EELS & 4D-STEM). The liquid phase characterization is discussed in terms of solution NMR spectroscopy, TEM and optical spectroscopies, while the gas phase can be characterized using OEMS, Pressure monitoring and GCMS. Computational modelling and simulation (DFT, ReaxFF & MLIP) are also discussed

Rapid Quantification of Protein Secondary Structure Composition from a Single Unassigned 1D 13C Nuclear Magnetic Resonance Spectrum.

The function of a protein is predicated upon its three-dimensional fold. Representing its complex structure as a series of repeating secondary structural elements is one of the most useful ways by which we study, characterize, and visualize a protein. Consequently, experimental methods that quantify the secondary structure content allow us to connect a protein's structure to its function. Here, we introduce an automated gradient descent-based method we refer to as secondary-structure distribution by NMR that allows for rapid quantification of the protein secondary structure composition of a protein from a single, 1D 13C NMR spectrum without chemical shift assignments. The analysis of nearly 900 proteins with known structure and chemical shifts demonstrates the capabilities of our approach. We show that these results rival alternative techniques such as FT-IR and circular dichroism that are commonly used to estimate secondary structure compositions. The resulting method requires only the primary sequence of the protein and its referenced 13C NMR spectrum. Each residue is modeled in an ensemble of secondary structures with percentage contributions from random coil, α-helix, and β-sheet secondary structures obtained by minimizing the difference between a simulated and experimental 1D 13C NMR spectrum. The capabilities of the method are demonstrated as applied to samples at natural abundance or enriched in 13C, acquired by either solution or solid-state NMR, and even on low magnetic field benchtop NMR spectrometers. This approach allows for rapid characterization of protein secondary structure across traditionally challenging to characterize states including liquid-liquid phase-separated, membrane-bound, or aggregated states.

Backbone assignment of CcdB_G100T toxin from E.coli in complex with the toxin binding C-terminal domain of its cognate antitoxin CcdA.

The CcdAB system expressed in the E.coli cells is a prototypical example of the bacterial toxin-antitoxin (TA) systems that ensure the survival of the bacterial population under adverse environmental conditions. The solution and crystal structures of CcdA, CcdB and of CcdB in complex with the toxin-binding C-terminal domain of CcdA have been reported. Our interest lies in the dynamics of CcdB-CcdA complex formation. Solution NMR studies have shown that CcdB_G100T, in presence of saturating concentrations of CcdA-c, a truncated C-terminal fragment of CcdA exists in equilibrium between two major populations. Sequence specific backbone resonance assignments of both equilibrium forms of the ~ 27kDa complex, have been obtained from a suite of triple resonance NMR experiments acquired on 2H, 13C, 15N enriched samples of CcdB_G100T. Analysis of 1H, 13Cα, 13Cβ secondary chemical shifts, shows that both equilibrium forms of CcdB_G100T have five beta-strands and one alpha-helix as the major secondary structural elements in the tertiary structure. The results of these studies are presented below.

Study on hydrolytic, oxidative and photolytic degradation behaviour of anticancer drug sonidegib: Characterization of the products by UHPLC-Q-TOF-MS/MS, 2D NMR and in silico toxicity assessment

Sonidegib (SDG) is a USFDA-approved anticancer drug developed for treating basal cell carcinoma. The presented study is focused on forced degradation studies of SDG with special emphasis on oxidative and photolytic degradation. Stress testing was performed as per ICH guidelines in hydrolytic, oxidative, and photolytic stress, revealing the formation of 15 transformation products. The results demonstrated the sensitivity of SDG to photolytic exposure under UV-A light, as well as to oxidative stress conditions involving hydrogen peroxide and a radical initiator. The observed DPs are separated and identified using RP-LC-PDA using a linear gradient program equipped with C18 (4.6 ×250 mm, 5 µm) column. All the DPs with >0.1 % concentration are characterized using tandem mass spectrometry and DPs with complex, unstable and isomeric nature are enriched and studied using solution NMR. Enrichment studies unveiled the formation of DP-6, DP-11, DP-12 and DP-13 as major transformation products. Interpretation of characteristic fragments from MS/MS spectra, NMR chemical shifts, and NOEs are utilized to solve the structure of the DPs. Further elucidated structures are employed to propose the involved mechanisms in degradation pathway. In addition, the DPs are studied for toxicity in silico and the potential/plausible carcinogenic, genotoxic and mutagenic nature of the DPs are illustrated.

Enantioselective Friedel-Crafts alkylation of 1,3,5-trimethoxybenzene with trans-β-nitrostyrenes catalysed by [Cp*Rh{(R)-Prophos}(H2O)][SbF6]2 (Prophos = propane-1,2-diyl-bis(diphenylphosphane)

The aqua complex (SRh,RC)-[Cp*Rh{(R)-Prophos}(H2O)][SbF6]2 (Prophos = propane-1,2-diyl-bis(diphenylphosphane) catalyses the asymmetric Friedel-Crafts (FC) alkylation of 1,3,5-trimethoxybenzene with trans-β-nitrostyrenes. Only the monoalkylated adduct was obtained in all cases with enantioselectivities of up to 93/7 e.r. Solution NMR measurements have allowed us to detect and characterise catalytic intermediates such as metal-trans-β-nitrostyrene and metal‑aci-nitro complexes, as well as free aci‑nitro compound. Based on these studies, a Michael-type Friedel-Crafts mechanism is proposed.

Solution NMR backbone resonance assignment of the full-length resistance-related calcium-binding protein Sorcin.

Sorcin is a penta-EF hand calcium-binding protein that confers multidrug resistance in cancer cells. It regulates cellular Ca2+ homeostasis by interacting with calcium channels such as Ryanodine receptor 2 and Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase in a calcium-dependent manner. The crystal structure of the Sorcin has been determined in both calcium-free and calcium-bound states to understand calcium-binding induced conformational change. However, due to its flexibility, most of the N-terminal domain is invisible in these crystal structures. Here we report the 1H, 13C, and 15N backbone resonance assignments of full-length Sorcin in the calcium-free state using solution NMR. The protein secondary structure was predicted based on the assigned backbone chemical shifts using TALOS+ and CSI 3.0. Our backbone resonance assignment of the full-length Sorcin provides a foundation for future NMR spectroscopic studies to uncover the mechanism of Ca2+ sensing by Sorcin.

Solution NMR backbone assignment of the N-terminal tandem Zα1-Zα2 domains of Z-DNA binding protein 1.

The detection of nucleic acids that are present in atypical conformations is a crucial trigger of the innate immune response. Human Z-DNA binding protein 1 (ZBP1) is a pattern recognition receptor that harbors two Zα domains that recognize Z-DNA and Z-RNA. ZBP1 detects this alternate nucleic acid conformation as foreign, and upon stabilization of these substrates, it triggers activation of an immune response. Here, we present the backbone chemical shift assignment of a construct encompassing the Zα1 and Zα2 domains as well as the interconnecting linker of ZBP1. These assignments can be directly transferred to the isolated Zα1 and Zα2 domains, thereby demonstrating that these domains maintain virtually identical structures in the tandem context.

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa / CD16a structure.

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa / CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.