Multidimensional Nuclear Magnetic Resonance Research Articles

Transferring principles of solid-state and Laplace NMR to the field of in vivo brain MRI.

Magnetic resonance imaging (MRI) is the primary method for noninvasive investigations of the human brain in health, disease, and development but yields data that are difficult to interpret whenever the millimeter-scale voxels contain multiple microscopic tissue environments with different chemical and structural properties. We propose a novel MRI framework to quantify the microscopic heterogeneity of the living human brain as spatially resolved five-dimensional relaxation-diffusion distributions by augmenting a conventional diffusion-weighted imaging sequence with signal encoding principles from multidimensional solid-state nuclear magnetic resonance (NMR) spectroscopy, relaxation-diffusion correlation methods from Laplace NMR of porous media, and Monte Carlo data inversion. The high dimensionality of the distribution space allows resolution of multiple microscopic environments within each heterogeneous voxel as well as their individual characterization with novel statistical measures that combine the chemical sensitivity of the relaxation rates with the link between microstructure and the anisotropic diffusivity of tissue water. The proposed framework is demonstrated on a healthy volunteer using both exhaustive and clinically viable acquisition protocols.

COLMAR Lipids Web Server and Ultrahigh-Resolution Methods for Two-Dimensional Nuclear Magnetic Resonance- and Mass Spectrometry-Based Lipidomics

Accurate identification of lipids in biological samples is a key step in lipidomics studies. Multidimensional nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for this purpose as it provides comprehensive structural information on lipid composition at atomic resolution. However, the interpretation of NMR spectra of complex lipid mixtures is currently hampered by limited spectral resolution and the absence of a customized lipid NMR database along with user-friendly spectral analysis tools. We introduce a new two-dimensional (2D) NMR metabolite database "COLMAR Lipids" that was specifically curated for hydrophobic metabolites presently containing 501 compounds with accurate experimental 2D 13C-1H heteronuclear single quantum coherence (HSQC) chemical shift data measured in CDCl3. A new module in the public COLMAR suite of NMR web servers was developed for the (semi)automated analysis of complex lipidomics mixtures (http://spin.ccic.osu.edu/index.php/colmarm/index2). To obtain 2D HSQC spectra with the necessary high spectral resolution along both 13C and 1H dimensions, nonuniform sampling in combination with pure shift spectroscopy was applied allowing the extraction of an abundance of unique cross-peaks belonging to hydrophobic compounds in complex lipidomics mixtures. As shown here, this information is critical for the unambiguous identification of underlying lipid molecules by means of the new COLMAR Lipids web server, also in combination with mass spectrometry, as is demonstrated for Caco-2 cell and lung tissue cell extracts.

Unlocking the architecture of native plant cell walls via solid-state nuclear magnetic resonance.

Understanding the biosynthesis and architecture of the plant cell wall is key to predictably engineering plants as a sustainable bioenergy source. Multi-dimensional solid-state nuclear magnetic resonance (ssNMR) is a promising technique to investigate the three-dimensional networks formed between cell wall components in their native state, and develop models of cell wall architecture. To do this, it is necessary to produce plant material that is highly enriched in the carbon-13 isotope (13C), since carbon-12 (12C) is inactive in NMR. Here, we present a cost-effective way to generate 13C-enriched mature plant tissue and to determine the degree of 13C incorporation. We describe a series of multi-dimensional ssNMR experiments that have been used in recent studies of cell wall architecture, and provide an introduction to interpreting the resulting ssNMR spectra.

Realtime optimization of multidimensional NMR spectroscopy on embedded sensing devices

The increasingly ubiquitous use of embedded devices calls for autonomous optimizations of sensor performance with meager computing resources. Due to the heavy computing needs, such optimization is rarely performed, and almost never carried out on-the-fly, resulting in a vast underutilization of deployed assets. Aiming at improving the measurement efficiency, we show an OED (Optimal Experimental Design) routine where quantities of interest of probable samples are partitioned into distinctive classes, with the corresponding sensor signals learned by supervised learning models. The trained models, digesting the compressed live data, are subsequently executed at the constrained device for continuous classification and optimization of measurements. We demonstrate the closed-loop method with multidimensional NMR (Nuclear Magnetic Resonance) relaxometry, an analytical technique seeing a substantial growth of field applications in recent years, on a wide range of complex fluids. The realtime portion of the procedure demands minimal computing load, and is ideally suited for instruments that are widely used in remote sensing and IoT networks.

Three-dimensional structure of Megabalanus rosa Cement Protein 20 revealed by multi-dimensional NMR and molecular dynamics simulations.

Barnacles employ a protein-based cement to firmly attach to immersed substrates. The cement proteins (CPs) have previously been identified and sequenced. However, the molecular mechanisms of adhesion are not well understood, in particular, because the three-dimensional molecular structure of CPs remained unknown to date. Here, we conducted multi-dimensional nuclear magnetic resonance (NMR) studies and molecular dynamics (MD) simulations of recombinant Megabalanus rosa Cement Protein 20 (rMrCP20). Our NMR results show that rMrCP20 contains three main folded domain regions intervened by two dynamic loops, resulting in multiple protein conformations that exist in equilibrium. We found that 12 out of 32 Cys in the sequence engage in disulfide bonds that stabilize the β-sheet domains owing to their placement at the extremities of β-strands. Another feature unveiled by NMR is the location of basic residues in turn regions that are exposed to the solvent, playing an important role for intermolecular contact with negatively charged surfaces. MD simulations highlight a highly stable and conserved β-motif (β7-β8), which may function as nuclei for amyloid-like nanofibrils previously observed in the cured adhesive cement. To the best of our knowledge, this is the first report describing the tertiary structure of an extracellular biological adhesive protein at the molecular level. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

A miniaturized spectrometer for NMR relaxometry under extreme conditions

With the advent of integrated electronics, microfabrication and novel chemistry, NMR (Nuclear Magnetic Resonance) methods, embodied in miniaturized spectrometers, have found profound uses in recent years that are beyond their conventional niche. In this work, we extend NMR relaxometry on a minute sample below 20 μL to challenging environment of 150 °C in temperature and 900 bar in pressure. Combined with a single-board NMR spectrometer, we further demonstrate multidimensional NMR relaxometries capable of resolving compositions of complex fluids. The confluence of HTHP (high-pressure high-temperature) capability, minimal sample volume, and reduced sensor envelop and power budget creates a new class of mobile NMR platforms, bringing the powerful analytical toolkit in a miniaturized footprint to extreme operating conditions.

Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct 19F Labels.

To probe ligand-receptor binding at the atomic-level, a frequent approach involves multidimensional nuclear magnetic resonance (NMR) spectroscopy experiments relying on 13C- and/or 15N-enrichment alongside 1H. Alternatively, the lack of fluorine in biomolecules may be exploited through specific incorporation of 19F nuclei into a sample. The 19F nucleus is highly sensitive to environmental changes and allows for one-dimensional NMR spectroscopic study, with perturbation to chemical shift and spin dynamics diagnostic of structural change, ligand binding, and modified conformational sampling. This was applied to the apelinergic system, which comprises a rhodopsin-like G protein-coupled receptor (the apelin receptor (AR)/APJ) and two families of cognate ligands, the apelin and apela (ELABELA/toddler) peptides. Specifically, AR fragments consisting of either the N-terminal tail and first transmembrane (TM) α-helix (AR55) or the first three transmembrane α-helices (TM1-3) were prepared with biosynthetic fluorotryptophan incorporation. Interactions of each AR fragment with a high-affinity, 2,4,5-trifluorophenylalanine labeled apelin analogue were compared by 19F NMR. Distinct ranges of 19F chemical shifts for ligand and receptor provide unambiguous tracking of both species, with distinct binding behaviour observed for each AR fragment implying that AR55 is not sufficient to recapitulate the physiological binding event. Site-specific perturbation was also apparent for the apelin analogue as a function of substitution site, indicating an orientational binding preference. As a whole, this strategy of distinctive 19F labelling for ligand and receptor provides a relatively fast (i.e., employing 1D NMR experiments) and highly sensitive method to simultaneously and definitively track binding in both species.

PEA-15 C-Terminal Tail Allosterically Modulates Death-Effector Domain Conformation and Facilitates Protein-Protein Interactions.

Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) exerts its regulatory roles on several critical cellular pathways through protein–protein interactions depending on its phosphorylation states. It can either inhibit the extracellular signal-regulated kinase (ERK) activities when it is dephosphorylated or block the assembly of death-inducing signaling complex (DISC) and the subsequent activation of apoptotic initiator, caspase-8, when it is phosphorylated. Due to the important roles of PEA-15 in regulating these pathways that lead to opposite cellular outcomes (cell proliferation vs. cell death), we proposed a phosphostasis (phosphorylation homeostasis) model, in which the phosphorylation states of the protein are vigorously controlled and regulated to maintain a delicate balance. The phosphostasis gives rise to the protective cellular functions of PEA-15 to preserve optimum cellular conditions. In this article, using advanced multidimensional nuclear magnetic resonance (NMR) techniques combined with a novel chemical shift (CS)-Rosetta algorithm for de novo protein structural determination, we report a novel conformation of PEA-15 death-effector domain (DED) upon interacting with ERK2. This new conformation is modulated by the irregularly structured C-terminal tail when it first recognizes and binds to ERK2 at the d-peptide recruitment site (DRS) in an allosteric manner, and is facilitated by the rearrangement of the surface electrostatic and hydrogen-bonding interactions on the DED. In this ERK2-bound conformation, three of the six helices (α2, α3, and α4) comprising the DED reorient substantially in comparison to the free-form structure, exposing key residues on the other three helices that directly interact with ERK2 at the DEF-docking site (docking site for ERK, FxF) and the activation loop. Additionally, we provide evidence that the phosphorylation of the C-terminal tail leads to a distinct conformation of DED, allowing efficient interactions with Fas-associated death domain (FADD) protein at the DISC. Our results substantiate the allosteric regulatory roles of the C-terminal tail in modulating DED conformation and facilitating protein–protein interactions of PEA-15.

Resolution enhancement and proton proximity probed by 3D TQ/DQ/SQ proton NMR spectroscopy under ultrafast magic-angle-spinning beyond 70 kHz.

Proton nuclear magnetic resonance (NMR) in solid state has gained significant attention in recent years due to the remarkable resolution and sensitivity enhancement afforded by ultrafast magic-angle-spinning (MAS). In spite of the substantial suppression of 1H-1H dipolar couplings, the proton spectral resolution is still poor compared to that of 13C or 15N NMR, rendering it challenging for the structural and conformational analysis of complex chemicals or biological solids. Herein, by utilizing the benefits of double-quantum (DQ) and triple-quantum (TQ) coherences, we propose a 3D single-channel pulse sequence that correlates proton triple-quantum/double-quantum/single-quantum (TQ/DQ/SQ) chemical shifts. In addition to the two-spin proximity information, this 3D TQ/DQ/SQ pulse sequence enables more reliable extraction of three-spin proximity information compared to the regular 2D TQ/SQ correlation experiment, which could aid in revealing the proton network in solids. Furthermore, the TQ/DQ slice taken at a specific SQ chemical shift only reveals the local correlations to the corresponding SQ chemical shift, and thus it enables accurate assignments of the proton peaks along the TQ and DQ dimensions and simplifies the interpretation of proton spectra especially for dense proton networks. The high performance of this 3D pulse sequence is well demonstrated on small compounds, L-alanine and a tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (MLF). We expect that this new methodology can inspire the development of multidimensional solid-state NMR pulse sequences using the merits of TQ and DQ coherences and enable high-throughput investigations of complex solids using abundant protons.

Frequency selective coherence transfer NMR spectroscopy to study the structural dynamics of high molecular weight proteins.

Multidimensional nuclear magnetic resonance (NMR) spectroscopy has enabled detailed characterizations of protein structures and dynamics that are closely linked to functions. However, it leads to a large sensitivity loss in applications to high molecular weight proteins, which is caused by spin relaxation during the frequency discrimination period in the indirect dimension. Here, we describe a selective coherence transfer scheme, which enables us to selectively observe 1H nuclei bonded to 15N or 13C nuclei with specified resonance frequencies. By utilizing this scheme, we achieved a 2.5- to 6-fold increase in signal height per unit of time with this scheme by avoiding the relaxation loss in the indirect dimension, as compared to the conventional two-dimensional heteronuclear correlation spectroscopy. We also demonstrated the effectiveness of this approach with applications to the membrane protein KirBac1.1, and characterized the functionally relevant conformational exchange process in both detergent micelles and a reconstituted membrane environment, corresponding to the apparent molecular masses of 220 kDa and 300 kDa, respectively.

Bacterial Analogs of Plant Tetrahydropyridine Alkaloids Mediate Microbial Interactions in a Rhizosphere Model System.

Plants expend significant resources to select and maintain rhizosphere communities that benefit their growth and protect them from pathogens. A better understanding of assembly and function of rhizosphere microbial communities will provide new avenues for improving crop production. Secretion of antibiotics is one means by which bacteria interact with neighboring microbes and sometimes change community composition. In our analysis of a taxonomically diverse consortium from the soybean rhizosphere, we found that Pseudomonas koreensis selectively inhibits growth of Flavobacterium johnsoniae and other members of the Bacteroidetes grown in soybean root exudate. A genetic screen in P. koreensis identified a previously uncharacterized biosynthetic gene cluster responsible for the inhibitory activity. Metabolites were isolated based on biological activity and were characterized using tandem mass spectrometry, multidimensional nuclear magnetic resonance, and Mosher ester analysis, leading to the discovery of a new family of bacterial tetrahydropyridine alkaloids, koreenceine A to D (metabolites 1 to 4). Three of these metabolites are analogs of the plant alkaloid γ-coniceine. Comparative analysis of the koreenceine cluster with the γ-coniceine pathway revealed distinct polyketide synthase routes to the defining tetrahydropyridine scaffold, suggesting convergent evolution. Koreenceine-type pathways are widely distributed among Pseudomonas species, and koreenceine C was detected in another Pseudomonas species from a distantly related cluster. This work suggests that Pseudomonas and plants convergently evolved the ability to produce similar alkaloid metabolites that can mediate interbacterial competition in the rhizosphere.IMPORTANCE The microbiomes of plants are critical to host physiology and development. Microbes are attracted to the rhizosphere due to massive secretion of plant photosynthates from roots. Microorganisms that successfully join the rhizosphere community from bulk soil have access to more abundant and diverse molecules, producing a highly competitive and selective environment. In the rhizosphere, as in other microbiomes, little is known about the genetic basis for individual species' behaviors within the community. In this study, we characterized competition between Pseudomonas koreensis and Flavobacterium johnsoniae, two common rhizosphere inhabitants. We identified a widespread gene cluster in several Pseudomonas spp. that is necessary for the production of a novel family of tetrahydropyridine alkaloids that are structural analogs of plant alkaloids. We expand the known repertoire of antibiotics produced by Pseudomonas in the rhizosphere and demonstrate the role of the metabolites in interactions with other rhizosphere bacteria.

Optimization of band-selective homonuclear dipolar recoupling in solid-state NMR by a numerical phase search.

Spin polarization transfers among aliphatic 13C nuclei, especially 13Cα-13Cβ transfers, permit correlations of their nuclear magnetic resonance (NMR) frequencies that are essential for signal assignments in multidimensional solid-state NMR of proteins. We derive and demonstrate a new radio-frequency (RF) excitation sequence for homonuclear dipolar recoupling that enhances spin polarization transfers among aliphatic 13C nuclei at moderate magic-angle spinning (MAS) frequencies. The phase-optimized recoupling sequence with five π pulses per MAS rotation period (denoted as PR5) is derived initially from systematic numerical simulations in which only the RF phases are varied. Subsequent theoretical analysis by average Hamiltonian theory explains the favorable properties of numerically optimized phase schemes. The high efficiency of spin polarization transfers in simulations is preserved in experiments, in part because the RF field amplitude in PR5 is only 2.5 times the MAS frequency so that relatively low 1H decoupling powers are required. Experiments on a microcrystalline sample of the β1 immunoglobulin binding domain of protein G demonstrate an average enhancement factor of 1.6 for 13Cα → 13Cβ polarization transfers, compared to the standard 13C-13C spin-diffusion method, implying a two-fold time saving in relevant 2D and 3D experiments.

A finite element approach to forward modeling of nuclear magnetic resonance measurements in coupled pore systems.

Porous media characterized by a hierarchy of length scales are ubiquitous in industry and nature, and include carbonate rocks, cements, heterogeneous catalysts, and biological cells. Nuclear magnetic resonance (NMR) is a popular tool for studying liquid-saturated porous materials, where the spin relaxation rate is generally considered proportional to pore size. However, in porous granular media, the relaxation rate is modified by diffusion between the intraparticle and interparticle pores. The observed relaxation rates do not reflect the pore size under such conditions. Deconvolving the various contributions of surface relaxation, geometry, and diffusion is nontrivial, and forward models are a powerful technique for elucidating the underlying pore structure. Various forward models have been proposed previously, including analytic solutions and random walk simulations. Here, a finite element method is adopted to simulate the diffusion of nuclear magnetization in a coupled pore geometry. We validate our model against existing solutions and use the simulations to determine the surface relaxivity of powdered silica by matching experimental results. The finite element approach is more versatile than other modeling methods, allowing direct visualization of the diffusing magnetization and being trivially extensible to multidimensional NMR exchange experiments.

Duet of Acetate and Water at the Defects of Metal-Organic Frameworks.

Metal-organic frameworks (MOFs) are porous crystalline materials with promising applications in molecular adsorption, separation, and catalysis. It has been discovered recently that structural defects introduced unintentionally or by design could have a significant impact on their properties. However, the exact chemical composition and structural evolution under different conditions at the defects are still under debate. In this study, we performed multidimensional solid-state nuclear magnetic resonance (SSNMR) coupled with computer simulations to elucidate an important scenario of MOF defects, uncovering the dynamic interplay between residual acetate and water. Acetate, as a defect modulator, and water, as a byproduct, are prevalent defect-associated species, which are among the key factors determining the reactivity and stability of defects. We discovered that acetate molecules coordinate to a single metal site monodentately and pair with water at the neighboring position. The acetates are highly flexible, which undergo fast libration as well as a slow kinetic exchange with water through dynamic hydrogen bonds. The dynamic processes under variable temperatures and different hydration levels have been quantitatively analyzed across a broad time scale from microseconds to seconds. The integration of SSNMR and computer simulations allows a precision probe into defective MOF structures with intrinsic dynamics and disorder.

Expression and Purification of Chemokine MIP-3α (CCL20) through a Calmodulin-Fusion Protein System.

Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. Moreover, the MIP-3α protein has been shown to display direct antimicrobial, antiviral and antiprotozoal activities. Because of the potential therapeutic uses of this protein, the efficient production of MIP-3α is of great interest. However, bacterial recombinant production of the MIP-3α protein has been limited by the toxicity of this extremely basic protein (pI 9.7) toward prokaryotic cells, and by solubility problems during expression and purification. In an attempt to overcome these issues, we have investigated the bacterial recombinant expression of MIP-3α by using several common expression and fusion tags, including 6× histidine (His), small ubiquitin modifier protein (SUMO), thioredoxin (TRX), ketosteroid isomerase (KSI), and maltose binding protein (MBP). We have also evaluated a recently introduced calmodulin (CaM)-tag that has been used for the effective expression of many basic antimicrobial peptides (AMPs). Here, we show that the CaM fusion tag system effectively expressed soluble MIP-3α in the cytoplasm of Escherichia coli with good yields. Rapid purification was facilitated by the His-tag that was integrated in the CaM-fusion protein system. Multidimensional nuclear magnetic resonance (NMR) studies demonstrated that the recombinant protein was properly folded, with the correct formation of disulfide bonds. In addition, the recombinant MIP-3α had antibacterial activity, and was shown to inhibit the formation of Pseudomonas aeruginosa biofilms.

Metabolic Profiling Using In Vivo High Field Flow NMR.

In vivo NMR (nuclear magnetic resonance) has the potential to monitor and record metabolic flux in close to real time, which is essential for better understanding the toxic mode of action of a contaminant and deciphering complex interconnected stress-induced pathways impacted inside an organism. Here, we describe how to construct and use a simple flow system to keep small aquatic organisms alive inside the NMR spectrometer. In living organisms, magnetic susceptibility distortions lead to severe broadening in conventional NMR. Two main approaches can be employed to overcome this issue: (1) use a pulse sequence to reduce the distortions, or (2) employ multidimensional NMR in combination with isotopic enrichment to provide the spectral dispersion required to separate peaks from overlapping resonances. Both approaches are discussed, and protocols for both approaches are provided here in the context of small aquatic organisms.

Novel protein science enabled by total chemical synthesis.

Chemical synthesis is a well-established method for the preparation in the research laboratory of multiple-tens-of-milligram amounts of correctly folded, high purity protein molecules. Chemically synthesized proteins enable a broad spectrum of novel protein science. Racemic mixtures consisting of d-protein and l-protein enantiomers facilitate crystallization and determination of protein structures by X-ray diffraction. d-Proteins enable the systematic development of unnatural mirror image protein molecules that bind with high affinity to natural protein targets. The d-protein form of a therapeutic target can also be used to screen natural product libraries to identify novel small molecule leads for drug development. Proteins with novel polypeptide chain topologies including branched, circular, linear-loop, and interpenetrating polypeptide chains can be constructed by chemical synthesis. Medicinal chemistry can be applied to optimize the properties of therapeutic protein molecules. Chemical synthesis has been used to redesign glycoproteins and for the a priori design and construction of covalently constrained novel protein scaffolds not found in nature. Versatile and precise labeling of protein molecules by chemical synthesis facilitates effective application of advanced physical methods including multidimensional nuclear magnetic resonance and time-resolved FTIR for the elucidation of protein structure-activity relationships. The chemistries used for total synthesis of proteins have been adapted to making artificial molecular devices and protein-inspired nanomolecular constructs. Research to develop mirror image life in the laboratory is in its very earliest stages, based on the total chemical synthesis of d-protein forms of polymerase enzymes.

Tri-comb spectroscopy

Multi-dimensional coherent spectroscopy (MDCS)1,2 is a powerful method for optical spectroscopy that has become an important tool for studying ultrafast dynamics in a wide range of systems. It is an optical analog of multidimensional nuclear magnetic resonance spectroscopy that enables the measurement of homogeneous linewidths in inhomogeneously broadened systems, many-body interactions, and coupling between excited resonances, all of which are not simultaneously accessible by any other linear or non-linear method. Current implementations of MDCS require a bulky apparatus and suffer from resolution and acquisition speed limitations that constrain their applications outside the laboratory3-5. Here we propose and demonstrate an approach to nonlinear coherent spectroscopy that utilizes three frequency combs with slightly different repetition rates. Unlike traditional nonlinear methods, tri-comb spectroscopy uses only a single photodetector and no mechanical moving elements to enable faster acquisition times, while also providing comb resolution. As a proof of concept, a multidimensional coherent spectrum with comb cross-diagonal resolution is generated using only 365 ms of data. These improvements make multidimensional coherent spectroscopy relevant for systems with narrow resonances (especially cold atomic and molecular systems). In addition the method has the potential to be field deployable for chemical sensing applications.

Theoretical investigation of heterogeneous wettability in porous media using NMR

It is highly important to understand the heterogeneous wettability properties of porous media for enhanced oil recovery (EOR). However, wettability measurements are still challenging in directly investigating the wettability of porous media. In this paper, we propose a multidimensional nuclear magnetic resonance (NMR) method and the concept of apparent contact angles to characterize the heterogeneous wettability of porous media. The apparent contact angle, which is determined by both the wetting surface coverage and the local wettability (wetting contact angles of each homogeneous wetting regions or wetting patches), is first introduced as an indicator of the heterogeneous wettability of porous media using the NMR method. For homogeneously wetting patches, the relaxation time ratio T1/T2 is employed to probe the local wettabiity of wetting patches. The T2 − D is introduced to obtain the wetting surface coverage using the effective relaxivity. Numerical simulations are conducted to validate this method.

Comparative study of atmospheric water-soluble organic aerosols composition in contrasting suburban environments in the Iberian Peninsula Coast

This study investigates the structural composition and major sources of water-soluble organic matter (WSOM) from PM2.5 collected, in parallel, during summer and winter, in two contrasting suburban sites at Iberian Peninsula Coast: Aveiro (Portugal) and Coruña (Spain). PM10 samples were also collected at Coruña for comparison. Ambient concentrations of PM2.5, total nitrogen (TN), and WSOM were higher in Aveiro than in Coruña, with the highest levels found in winter at both locations. In Coruña, concentrations of PM10, TN, and WSOM were higher than those from PM2.5. Regardless of the season, stable isotopic δ13C and δ15N in PM2.5 suggested important contributions of anthropogenic fresh organic aerosols (OAs) at Aveiro. In Coruña, δ13C and δ15N of PM2.5 and PM10 suggests decreased anthropogenic input during summer. Although excitation-emission fluorescence profiles were similar for all WSOM samples, multi-dimensional nuclear magnetic resonance (NMR) spectroscopy confirmed differences in their structural composition, reflecting differences in aging processes and/or local sources between the two locations. In PM2.5 WSOM in Aveiro, the relative distribution of non-exchangeable proton functional groups was in the order: HC (40–43%) > HCC (31–39%) > HCO (12–15%) > Ar-H (5.0–13%). However, in PM2.5 and PM10 WSOM in Coruña, the relative contribution of HCO groups (24–30% and 23–29%, respectively) equals and/or surpasses that of HCC (25–26% and 25–29%, respectively), being also higher than those of Aveiro. In both locations, the highest aromatic contents were observed during winter due to biomass burning emissions. The structural composition of PM2.5 and PM10 WSOM in Coruña is dominated by oxygenated aliphatic compounds, reflecting the contribution of secondary OAs from biogenic, soil dust, and minor influence of anthropogenic emissions. In contrast, the composition of PM2.5 WSOM in Aveiro appears to be significantly impacted by fresh and secondary anthropogenic OAs. Marine and biomass burning OAs are important contributors, common to both sites.