NIR-II emissive donor-acceptor-donor fluorophores for dual fluorescence bioimaging and photothermal therapy applications.

Fluorescence bioimaging with near-infrared II (NIR-II) emissive organic fluorophores has proven to be a viable noninvasive diagnostic technique. However, there is still the need for the development of fluorophores that possess increased stability as well as functionalities that impart stimuli responsiveness. Through strategic design, we can synthesize fluorophores that possess not only NIR-II optical profiles but also pH-sensitivity and the ability to generate heat upon irradiation. In this work, we employ a donor-acceptor-donor (D-A-D) design to synthesize a series of NIR-II fluorophores. Here we use thienothiadiazole (TTD) as the acceptor, 3-hexylthiophene (HexT) as the π-spacer and vary the alkyl amine donor units: N,N-dimethylaniline (DMA), phenylpiperidine (Pip), and phenylmorpholine (Morp). Spectroscopic analysis shows that all three derivatives exhibit emission in the NIR-II region with λemimax ranging from 1030 to 1075 nm. Upon irradiation, the fluorophores exhibited noticeable heat generation through non-radiative processes. The ability to generate heat indicates that these fluorophores will act as theranostic (combination therapeutic and diagnostic) agents in which simultaneous visualization and treatment can be performed. Additionally, biosensing capabilities were supported by changes in the absorbance properties while under acidic conditions as a result of protonation of the alkyl amine donor units. The fluorophores also show minimal toxicity in a human mammary cell line and with murine red blood cells. Overall, initial results indicate viable NIR-II materials for multiple biomedical applications.

Exploring Residue-Level Interactions between the Biofilm-Driving R2ab Protein and Polystyrene Nanoparticles.

In biological systems, proteins can bind to nanoparticles to form a "corona" of adsorbed molecules. The nanoparticle corona is of significant interest because it impacts an organism's response to a nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. A residue-level mapping of protein behavior on nanoparticle surfaces is needed, but this mapping is difficult to obtain with traditional approaches. Here, we have investigated the interaction between R2ab and polystyrene nanoparticles (PSNPs) at the level of individual residues. R2ab is a bacterial surface protein from Staphylococcus epidermidis and is known to interact strongly with polystyrene, leading to biofilm formation. We have used mass spectrometry after lysine methylation and hydrogen-deuterium exchange (HDX) NMR spectroscopy to understand how the R2ab protein interacts with PSNPs of different sizes. Lysine methylation experiments reveal subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX rates become slower overall in the presence of PSNPs. However, some regions of the R2ab protein exhibit faster than average exchange rates in the presence of PSNPs, while others are slower than the average behavior, suggesting conformational changes upon binding. HDX rates and methylation ratios support a recently proposed "adsorbotope" model for PSNPs, wherein adsorbed proteins consist of unfolded anchor points interspersed with partially structured regions. Our data also highlight the challenges of characterizing complex protein-nanoparticle interactions using these techniques, such as fast exchange rates. While providing insights into how R2ab adsorbs onto PSNP surfaces, this research emphasizes the need for advanced methods to comprehend residue-level interactions in the nanoparticle corona.

Protein-Functionalized Gold Nanospheres with Tunable Photothermal Efficiency for the Near-Infrared Photothermal Ablation of Biofilms.

Temperature-responsive nanostructures with high antimicrobial efficacy are attractive for therapeutic applications against multidrug-resistant bacteria. Here, we report temperature-responsive nanospheres (TRNs) engineered to undergo self-association and agglomeration above a tunable transition temperature (Tt). The temperature-responsive behavior of the nanoparticles is obtained by functionalizing citrate-capped spherical gold nanoparticles (AuNPs) with elastin-like polypeptides (ELPs). Using protein design principles, we achieve a broad range of attainable Tt values and photothermal conversion efficiencies (η). Two approaches were used to adjust this range: First, by altering the position of the cysteine residue used to attach ELP to the AuNP, we attained a Tt range from 34 to 42 °C. Then, by functionalizing the AuNP with an additional small globular protein, we could extend this range to 34-50 °C. Under near-infrared (NIR) light exposure, all TRNs exhibited reversible agglomeration. Moreover, they showed an enhanced photothermal conversion efficiency in their agglomerated state relative to the dispersed state. Despite their spherical shape, TRNs have a photothermal conversion efficiency approaching that of gold nanorods (η = 68 ± 6%), yet unlike nanorods, the synthesis of TRNs requires no cytotoxic compounds. Finally, we tested TRNs for the photothermal ablation of biofilms. Above Tt, NIR irradiation of TRNs resulted in a 10,000-fold improvement in killing efficiency compared to untreated controls (p < 0.0001). Below Tt, no enhanced antibiofilm effect was observed. In conclusion, engineering the interactions between proteins and nanoparticles enables the tunable control of TRNs, resulting in a novel antibiofilm nanomaterial with low cytotoxicity.

Protein Binding Leads to Reduced Stability and Solvated Disorder in the Polystyrene Nanoparticle Corona.

Understanding the conformation of proteins in the nanoparticle corona has important implications in how organisms respond to nanoparticle-based drugs. These proteins coat the nanoparticle surface, and their properties will influence the nanoparticle's interaction with cell targets and the immune system. While some coronas are thought to be disordered, two key unanswered questions are the degree of disorder and solvent accessibility. Here, a model is developed for protein corona disorder in polystyrene nanoparticles of varying size. For two different proteins, it is found that binding affinity decreases as nanoparticle size increases. The stoichiometry of binding, along with changes in the hydrodynamic size, supports a highly solvated, disordered protein corona anchored at a small number of attachment sites. The scaling of the stoichiometry versus nanoparticle size is consistent with disordered polymer dimensions. Moreover, it is found that proteins are destabilized less in the presence of larger nanoparticles, and hydrophobic exposure decreases at lower curvatures. The observations hold for proteins on flat polystyrene surfaces, which have the lowest hydrophobic exposure. The model provides an explanation for previous observations of increased amyloid fibrillation rates in the presence of larger nanoparticles, and it may rationalize how cell receptors can recognize protein disorder in therapeutic nanoparticles.

What is allosteric regulation? Exploring the exceptions that prove the rule!

"Allosteric" was first introduced to mean the other site (i.e., a site distinct from the active or orthosteric site), an adjective for "regulation" to imply a regulatory outcome resulting from ligand binding at another site. That original idea outlines a system with two ligand-binding events at two distinct locations on a macromolecule (originally a protein system), which defines a four-state energy cycle. An allosteric energy cycle provides a quantifiable allosteric coupling constant and focuses our attention on the unique properties of the four equilibrated protein complexes that constitute the energy cycle. Because many observed phenomena have been referenced as "allosteric regulation" in the literature, the goal of this work is to use literature examples to explore which systems are and are not consistent with the two-ligand thermodynamic energy cycle-based definition of allosteric regulation. We emphasize the need for consistent language so comparisons can be made among the ever-increasing number of allosteric systems. Building on the mutually exclusive natures of an energy cycle definition of allosteric regulation versus classic two-state models, we conclude our discussion by outlining how the often-proposed Rube-Goldberg-like mechanisms are likely inconsistent with an energy cycle definition of allosteric regulation.

Exploring the Residue-Level Interactions between the R2ab Protein and Polystyrene Nanoparticles.

In biological systems, proteins can bind to nanoparticles to form a "corona" of adsorbed molecules. The nanoparticle corona is of high interest because it impacts the organism's response to the nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. Ultimately, a residue-level mapping of protein behavior on nanoparticle surfaces is needed, but this mapping is difficult to obtain with traditional approaches. Here, we have investigated the interaction between R2ab and polystyrene nanoparticles (PSNPs) at the level of individual residues. R2ab is a bacterial surface protein from Staphylococcus epidermidis and is known to interact strongly with polystyrene, leading to biofilm formation. We have used mass spectrometry after lysine methylation and hydrogen-deuterium exchange (HDX) NMR spectroscopy to understand how the R2ab protein interacts with PSNPs of different sizes. Through lysine methylation, we observe subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX measurements reveal that certain regions of the R2ab protein undergo faster exchange rates in the presence of PSNPs, suggesting conformational changes upon binding. Both results support a recently proposed "adsorbotope" model, wherein adsorbed proteins consist of unfolded anchor points interspersed with regions of partial structure. Our data also highlight the challenges of characterizing complex protein-nanoparticle interactions using these techniques, such as fast exchange rates. While providing insights into how proteins respond to nanoparticle surfaces, this research emphasizes the need for advanced methods to comprehend these intricate interactions fully at the residue level.

Near-Infrared Photothermal Ablation of Biofilms using ProteinFunctionalized Gold Nanospheres with a Tunable Temperature Response.

Temperature-responsive nanostructures with high antimicrobial efficacy are attractive for therapeutic applications against multi-drug-resistant bacteria. Here, we report temperature-responsive nanospheres (TRNs) that are engineered to undergo self-association and agglomeration above a tunable transition temperature (Tt). Temperature-responsive behavior of the nanoparticles is obtained by functionalizing citrate-capped, spherical gold nanoparticles (AuNPs) with elastin-like polypeptides (ELPs). Using protein design principles, we achieve a broad range of attainable Tt values and photothermal conversion efficiencies (η). Two approaches were used to adjust this range: First, by altering the position of the cysteine residue used to attach ELP to the AuNP, we attained a Tt range from 34-42 °C. Then, functionalizing the AuNP with an additional small globular protein, we were able to extend this range to 34-50 °C. Under near-infrared (NIR) light exposure, all TRNs exhibited reversible agglomeration. Moreover, they showed enhanced photothermal conversion efficiency in their agglomerated state relative to the dispersed state. Despite their spherical shape, TRNs have a photothermal conversion efficiency approaching that of gold nanorods (η = 68±6%), yet unlike nanorods, the synthesis of TRNs requires no cytotoxic compounds. Finally, we tested TRNs for photothermal ablation of biofilms. Above Tt, NIR irradiation of TRNs resulted in a 10,000-fold improvement in killing efficiency compared to untreated controls (p < 0.0001). Below Tt, no enhanced anti-biofilm effect was observed. In conclusion, engineering the interactions between proteins and nanoparticles enables the tunable control of TRNs, resulting in a novel, anti-biofilm nanomaterial with low cytotoxicity.

ParSe 2.0: A web tool to identify drivers of protein phase separation at the proteome level.

We have developed an algorithm, ParSe, which accurately identifies from the primary sequence those protein regions likely to exhibit physiological phase separation behavior. Originally, ParSe was designed to test the hypothesis that, for flexible proteins, phase separation potential is correlated to hydrodynamic size. While our results were consistent with that idea, we also found that many different descriptors could successfully differentiate between three classes of protein regions: folded, intrinsically disordered, and phase-separating intrinsically disordered. Consequently, numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. Built from that finding, ParSe 2.0 uses an optimal set of property scales to predict domain-level organization and compute a sequence-based prediction of phase separation potential. The algorithm is fast enough to scan the whole of the human proteome in minutes on a single computer and is equally or more accurate than other published predictors in identifying proteins and regions within proteins that drive phase separation. Here, we describe a web application for ParSe 2.0 that may be accessed through a browser by visiting https://stevewhitten.github.io/Parse_v2_FASTA to quickly identify phase-separating proteins within large sequence sets, or by visiting https://stevewhitten.github.io/Parse_v2_web to evaluate individual protein sequences.

Selective Near-Infrared Blood Detection Driven by Ionic Liquid-Dye-Albumin Nanointeractions.

Due to its abundance in blood, a great deal of research has been undertaken to develop efficient biosensors for serum albumin and provide insight into the interactions that take place between these biosensing molecules and the protein. Near-infrared (NIR, >700 nm) organic dyes have been shown to be effective biosensors of serum albumin, but their effectiveness is diminished in whole blood. Herein, it is shown that an NIR sulfonate indolizine-donor-based squaraine dye, SO3SQ, can be strengthened as a biosensor of albumin through the addition of biocompatible ionic liquids (ILs). Specifically, the IL choline glycolate (1:1), at a concentration of 160 mM, results in the enhanced fluorescence emission ("switch-on") of the dye in the presence of blood. The origin of the fluorescence enhancement was investigated via methods, including DLS, ITC, and molecular dynamics. Further, fluorescence measurements were conducted to see the impact the dye-IL system had on the fluorescence of the tryptophan residue of human serum albumin (HSA), as well as to determine its apparent association constants in relation to albumin. Circular dichroism (CD) spectroscopy was used to provide evidence that the dye-IL system does not alter the secondary structures of albumin or DNA. Our results suggest that the enhanced fluorescence of the dye in the presence of IL and blood is due to diversification of binding sites in albumin, controlled by the interaction of the IL-dye-albumin complex.

Protein Binding Leads to Reduced Stability and Solvated Disorder in the Polystyrene Nanoparticle Corona.

Understanding the conformation of proteins in the nanoparticle corona has important implications in how organisms respond to nanoparticle-based drugs. These proteins coat the nanoparticle surface, and their properties will influence the nanoparticle's interaction with cell targets and the immune system. While some coronas are thought to be disordered, two key unanswered questions are the degree of disorder and solvent accessibility. Here, using a comprehensive thermodynamic approach, along with supporting spectroscopic experiments, we develop a model for protein corona disorder in polystyrene nanoparticles of varying size. For two different proteins, we find that binding affinity decreases as nanoparticle size increases. The stoichiometry of binding, along with changes in the hydrodynamic size, support a highly solvated, disordered protein corona anchored at a small number of enthalpically-driven attachment sites. The scaling of the stoichiometry vs. nanoparticle size is consistent disordered polymer dimensions. Moreover, we find that proteins are destabilized less severely in the presence of larger nanoparticles, and this is supported by measurements of hydrophobic exposure, which becomes less pronounced at lower curvatures. Our observations hold for flat polystyrene surfaces, which, when controlled for total surface area, have the lowest hydrophobic exposure of all systems. Our model provides an explanation for previous observations of increased amyloid fibrillation rates in the presence of larger nanoparticles, and it may rationalize how cell receptors can recognize protein disorder in therapeutic nanoparticles.

Human disease-associated calmodulin mutations alter calcineurin function through multiple mechanisms.

Calmodulin (CaM) is a ubiquitous, calcium-sensing protein that regulates a multitude of processes throughout the body. In response to changes in [Ca2+], CaM modifies, activates, and deactivates enzymes and ion channels, as well as many other cellular processes. The importance of CaM is highlighted by the conservation of an identical amino acid sequence in all mammals. Alterations to CaM amino acid sequence were once thought to be incompatible with life. During the last decade modifications to the CaM protein sequence have been observed in patients suffering from life-threatening heart disease (calmodulinopathy). Thus far, inadequate or untimely interaction between mutant CaM and several proteins (LTCC, RyR2, and CaMKII) have been identified as mechanisms underlying calmodulinopathy. Given the extensive number of CaM interactions in the body, there are likely many consequences for altering CaM protein sequence. Here, we demonstrate that disease-associated CaM mutations alter the sensitivity and activity of the Ca2+-CaM-enhanced serine/threonine phosphatase calcineurin (CaN). Biophysical characterization by circular dichroism, solution NMR spectroscopy, stopped-flow kinetic measurements, and MD simulations provide mechanistic insight into mutation dysfunction as well as highlight important aspects of CaM Ca2+ signal transduction. We find that individual CaM point mutations (N53I, F89L, D129G, and F141L) impair CaN function, however, the mechanisms are not the same. Specifically, individual point mutations can influence or modify the following properties: CaM binding, Ca2+ binding, and/or Ca2+kinetics. Moreover, structural aspects of the CaNCaM complex can be altered in manners that indicate changes to allosteric transmission of CaM binding to the enzyme active site. Given that loss of CaN function can be fatal, as well as evidence that CaN modifies ion channels already associated with calmodulinopathy, our results raise the possibility that altered CaN function contributes to calmodulinopathy.

1H, 15N, and 13C chemical shift backbone resonance NMR assignment of the accumulation-associated protein (Aap) lectin domain from Staphylococcus epidermidis.

Staphylococcus epidermidis is the leading causative agent for hospital-acquired infections, especially device-related infections, due to its ability to form biofilms. The accumulation-associated protein (Aap) of S. epidermidis is primarily responsible for biofilm formation and consists of two domains, A and B. It was found that the A domain is responsible for the attachment to the abiotic/biotic surface, whereas the B domain is responsible for the accumulation of bacteria during biofilm formation. One of the parts of the A domain is the Aap lectin, which is a carbohydrate-binding domain having 222 amino acids in its structure. Here we report the near complete backbone chemical shift assignments for the lectin domain, as well as its predicted secondary structure. This data will provide a platform for future NMR studies to explore the role of lectin in biofilm formation.

Mechanistic basis of staphylococcal interspecies competition for skin colonization.

Staphylococci, whether beneficial commensals or pathogens, often colonize human skin, potentially leading to competition for the same niche. In this multidisciplinary study we investigate the structure, binding specificity, and mechanism of adhesion of the Aap lectin domain required for Staphylococcus epidermidis skin colonization and compare its characteristics to the lectin domain from the orthologous Staphylococcus aureus adhesin SasG. The Aap structure reveals a legume lectin-like fold with atypical architecture, showing specificity for N-acetyllactosamine and sialyllactosamine. Bacterial adhesion assays using human corneocytes confirmed the biological relevance of these Aap-glycan interactions. Single-cell force spectroscopy experiments measured individual binding events between Aap and corneocytes, revealing an extraordinarily tight adhesion force of nearly 900 nN and a high density of receptors at the corneocyte surface. The SasG lectin domain shares similar structural features, glycan specificity, and corneocyte adhesion behavior. We observe cross-inhibition of Aap-and SasG-mediated staphylococcal adhesion to corneocytes. Together, these data provide insights into staphylococcal interspecies competition for skin colonization and suggest potential avenues for inhibition of S. aureus colonization.

NMR-based metabolomics of plasma from dairy calves infected with two primary causal agents of bovine respiratory disease (BRD).

Each year, bovine respiratory disease (BRD) results in significant economic loss in the cattle sector, and novel metabolic profiling for early diagnosis represents a promising tool for developing effective measures for disease management. Here, 1H-nuclear magnetic resonance (1H-NMR) spectra were used to characterize metabolites from blood plasma collected from male dairy calves (n = 10) intentionally infected with two of the main BRD causal agents, bovine respiratory syncytial virus (BRSV) and Mannheimia haemolytica (MH), to generate a well-defined metabolomic profile under controlled conditions. In response to infection, 46 metabolites (BRSV = 32, MH = 33) changed in concentration compared to the uninfected state. Fuel substrates and products exhibited a particularly strong effect, reflecting imbalances that occur during the immune response. Furthermore, 1H-NMR spectra from samples from the uninfected and infected stages were discriminated with an accuracy, sensitivity, and specificity ≥ 95% using chemometrics to model the changes associated with disease, suggesting that metabolic profiles can be used for further development, understanding, and validation of novel diagnostic tools.

Intrinsically disordered regions that drive phase separation form a robustly distinct protein class.

Protein phase separation is thought to be a primary driving force for the formation of membrane-less organelles, which control a wide range of biological functions from stress response to ribosome biogenesis. Among phase-separating (PS) proteins, many have intrinsically disordered regions (IDRs) that are needed for phase separation to occur. Accurate identification of IDRs that drive phase separation is important for testing the underlying mechanisms of phase separation, identifying biological processes that rely on phase separation, and designing sequences that modulate phase separation. To identify IDRs that drive phase separation, we first curated datasets of folded, ID, and PS ID sequences. We then used these sequence sets to examine how broadly existing amino acid property scales can be used to distinguish between the three classes of protein regions. We found that there are robust property differences between the classes and, consequently, that numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. This result indicates that multiple, redundant mechanisms contribute to the formation of phase-separated droplets from IDRs. The top-performing scales were used to further optimize our previously developed predictor of PS IDRs, ParSe. We then modified ParSe to account for interactions between amino acids and obtained reasonable predictive power for mutations that have been designed to test the role of amino acid interactions in driving protein phase separation. Collectively, our findings provide further insight into the classification of IDRs and the elements involved in protein phase separation.

Predicting protein function and orientation on a gold nanoparticle surface using a residue-based affinity scale.

The orientation adopted by proteins on nanoparticle surfaces determines the nanoparticle's bioactivity and its interactions with living systems. Here, we present a residue-based affinity scale for predicting protein orientation on citrate-gold nanoparticles (AuNPs). Competitive binding between protein variants accounts for thermodynamic and kinetic aspects of adsorption in this scale. For hydrophobic residues, the steric considerations dominate, whereas electrostatic interactions are critical for hydrophilic residues. The scale rationalizes the well-defined binding orientation of the small GB3 protein, and it subsequently predicts the orientation and active site accessibility of two enzymes on AuNPs. Additionally, our approach accounts for the AuNP-bound activity of five out of six additional enzymes from the literature. The model developed here enables high-throughput predictions of protein behavior on nanoparticles, and it enhances our understanding of protein orientation in the biomolecular corona, which should greatly enhance the performance and safety of nanomedicines used in vivo.

Metal Ion Binding Induces Local Protein Unfolding and Destabilizes Human Carbonic Anhydrase II.

Human carbonic anhydrase II (HCA) is a robust metalloprotein and an excellent biological model system to study the thermodynamics of metal ion coordination. Apo-HCA binds one zinc ion or two copper ions. We studied these binding processes at five temperatures (15-35 °C) using isothermal titration calorimetry, yielding thermodynamic parameters corrected for pH and buffer effects. We then sought to identify binding-induced structural changes. Our data suggest that binding at the active site organizes 6-8 residues; however, copper binding near the N-terminus results in a net unfolding of 6-7 residues. This surprising destabilization was confirmed using circular dichroism and protein stability measurements. Metal binding induced unfolding may represent an important regulatory mechanism, but it may be easily missed by NMR and X-ray crystallography. Thus, in addition to highlighting a highly novel binding-induced unfolding event, we demonstrate the value of calorimetry for studying the structural implications of metal binding.

Understanding the Adsorption of Peptides and Proteins onto PEGylated Gold Nanoparticles.

Polyethylene glycol (PEG) surface conjugations are widely employed to render passivating properties to nanoparticles in biological applications. The benefits of surface passivation by PEG are reduced protein adsorption, diminished non-specific interactions, and improvement in pharmacokinetics. However, the limitations of PEG passivation remain an active area of research, and recent examples from the literature demonstrate how PEG passivation can fail. Here, we study the adsorption amount of biomolecules to PEGylated gold nanoparticles (AuNPs), focusing on how different protein properties influence binding. The AuNPs are PEGylated with three different sizes of conjugated PEG chains, and we examine interactions with proteins of different sizes, charges, and surface cysteine content. The experiments are carried out in vitro at physiologically relevant timescales to obtain the adsorption amounts and rates of each biomolecule on AuNP-PEGs of varying compositions. Our findings are relevant in understanding how protein size and the surface cysteine content affect binding, and our work reveals that cysteine residues can dramatically increase adsorption rates on PEGylated AuNPs. Moreover, shorter chain PEG molecules passivate the AuNP surface more effectively against all protein types.

Beta turn propensity and a model polymer scaling exponent identify intrinsically disordered phase-separating proteins.

The complex cellular milieu can spontaneously demix, or phase separate, in a process controlled in part by intrinsically disordered (ID) proteins. A protein's propensity to phase separate is thought to be driven by a preference for protein-protein over protein-solvent interactions. The hydrodynamic size of monomeric proteins, as quantified by the polymer scaling exponent (v), is driven by a similar balance. We hypothesized that mean v, as predicted by protein sequence, would be smaller for proteins with a strong propensity to phase separate. To test this hypothesis, we analyzed protein databases containing subsets of proteins that are folded, disordered, or disordered and known to spontaneously phase separate. We find that the phase-separating disordered proteins, on average, had lower calculated values of v compared with their non-phase-separating counterparts. Moreover, these proteins had a higher sequence-predicted propensity for ß-turns. Using a simple, surface area-based model, we propose a physical mechanism for this difference: transient ß-turn structures reduce the desolvation penalty of forming a protein-rich phase and increase exposure of atoms involved in π/sp2 valence electron interactions. By this mechanism, ß-turns could act as energetically favored nucleation points, which may explain the increased propensity for turns in ID regions (IDRs) utilized biologically for phase separation. Phase-separating IDRs, non-phase-separating IDRs, and folded regions could be distinguished by combining v and ß-turn propensity. Finally, we propose a new algorithm, ParSe (partition sequence), for predicting phase-separating protein regions, and which is able to accurately identify folded, disordered, and phase-separating protein regions based on the primary sequence.

Solution NMR of Nanoparticles in Serum: Protein Competition Influences Binding Thermodynamics and Kinetics.

The spontaneous formation of a protein corona on a nanoparticle surface influences the physiological success or failure of the synthetic nanoparticle as a drug carrier or imaging agent used in vivo. A quantitative understanding of protein-nanoparticle interactions is therefore critical for the development of nanoparticle-based therapeutics. In this perspective, we briefly discuss the challenges and limitations of current approaches used for studying protein-nanoparticle binding in a realistic biological medium. Subsequently, we demonstrate that solution nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to monitor protein competitive binding in a complex serum medium in situ. Importantly, when many serum proteins are competing for a gold nanoparticle (AuNP) surface, solution NMR is able to detect differences in binding thermodynamics, and kinetics of a tagged protein. Combined with other experimental approaches, solution NMR is an invaluable tool to understand protein behavior in the nanoparticle corona.