Biology subjects

Fitzkee, N. C.

Publications and source records attributed to Fitzkee, N. C..

13 recordsLinked to original sources

bioRxiv2026

Mapping the vRNA Interaction with HIV-1 Integrase

A series of critical interactions within the viral core between the viral RNA (vRNA) and HIV-1 Integrase (IN) has previously been reported. In these studies, contact points between vRNA and IN were identified using RNA-seq and MS-based protein foot-printing approaches. Several IN amino acids located in its C-terminal domain (CTD) were found to be essential for vRNA binding and their alanine substitution severely impacted the correct morphogenesis of the matured viral core. Here, we have extended these studies by performing a comprehensive mapping of the IN-vRNA interaction by deploying RNA crosslinking and NMR methodologies. Together, these approaches were able to identify additional contacts points between the vRNA and IN. Our results reveal several new basic amino acids located in the IN CTD critical for the vRNA-IN interaction, viral replication and correct morphology of the matured viral core.

biochemistry↗

bioRxiv2025

Precision control of nanoparticle delivery with engineered biomimetic protein coronas

Nanoparticle delivery to tumors remains inefficient, with current nanomedicines achieving only 0.7% injected dose per gram (ID/g) of tumor tissue due to uncontrolled protein corona formation that redirects nanoparticles away from target sites. We engineered biomimetic protein coronas to control nanoparticle-protein interactions and enhance tumor targeting. Competitive binding studies using NMR spectroscopy revealed that transferrin (Tf) and fibronectin (Fn) outcompete albumin (BSA) and immunoglobulin G (IgG) for 15 nm gold nanoparticle surfaces, establishing a binding hierarchy that enables predictable corona composition. Pre-coating nanoparticles with a four-protein combination (BSA+Tf+Fn+IgG) created coronas that selectively enhanced cancer cell uptake while reducing macrophage recognition in vitro. When administered to tumor-bearing mice, these engineered coronas achieved 13 ppm/g tumor accumulation--equivalent to 4% ID/g--representing 6.5-fold improvement over bare nanoparticles and 2.6-fold improvement over PEGylated formulations. Proteomics analysis of secondary coronas formed in human serum revealed that engineered nanoparticles selectively recruit transport and adhesion proteins while limiting immune recognition signatures. The pre-formed coronas maintained targeting protein retention and reduced complement binding compared to controls. Circular dichroism confirmed minimal protein structural perturbation, preserving receptor-binding functionality for active targeting. The strategy harnesses natural protein adsorption processes to create "smart" biological interfaces that simultaneously evade immune clearance and promote tumor cell recognition through multiple receptor pathways. This approach demonstrates the feasibility of treating the coron as a programmable interface, addressing delivery limitations that have hindered clinical translation of cancer nanomedicines. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/693224v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@5dd106org.highwire.dtl.DTLVardef@1460e3corg.highwire.dtl.DTLVardef@50c251org.highwire.dtl.DTLVardef@5604f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Thermodynamic Analysis of Protein-Nanoparticle Interactions Links Binding Affinity and Structural Stability

When nanoparticles and nanoplastics enter biological fluids, their surfaces are rapidly coated with proteins, forming a corona that governs biological responses. However, understanding protein- surface interaction energetics remains a significant challenge. Here, we examine how protein charge distribution affects adsorption to polystyrene nanoparticles (PSNPs) by generating a series of lysine-to-alanine variants of the GB3 protein. Using isothermal titration calorimetry (ITC), we found that the K19A variant binds most strongly to both non-functionalized and carboxylate- functionalized PSNPs. ITC thermograms indicate that K19A forms a stable monolayer, while other variants exhibit multilayer adsorption. We hypothesize that removing lysine at position 19 creates a flatter, more neutral interaction surface that promotes efficient initial binding. Fluorescence denaturation experiments show that PSNPs destabilize GB3 protein variants, and binding correlates strongly with protein unfolding (r = 0.82, p < 0.01 for COOH-PSNPs and r = 0.76, p < 0.03 for non-functionalized PSNPs). These results reveal how protein stability and charge distribution shape adsorption thermodynamics, offering a framework for predicting protein-surface interactions. TOC Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/671591v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1741baforg.highwire.dtl.DTLVardef@1c5be81org.highwire.dtl.DTLVardef@15e3118org.highwire.dtl.DTLVardef@e92583_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Using a Bacterial Protein to Selectively Target Bacterial Biofilms: Treatment of S. epidermidis Biofilms with Targeted Photothermal Gold Nanoparticles

Biofilm-related infections are associated with high mortality and morbidity combined with increased treatment costs. Traditional antibiotics are becoming less effective due to the emergence of drug-resistant bacterial strains. The need to treat biofilms on medical implants is particularly acute, and one persistent challenge is selectively directing nanoparticles to the biofilm site. Here, we present a protein-based functionalization strategy that targets the extracellular matrix of biofilms. The protein, derived from the extracellular Staphylococcus epidermidis autolysin, directs nanoparticles to S. epidermidis cell wall components, which are not expected to be present in mammalian tissues. This functionalization is applied to a gold nanoparticle (AuNP) core, along with elastin-like polypeptides (ELPs), which generate a robust photothermal response. In addition to biofilm targeting, the particles exhibit low protein binding, and the photothermal conversion can be modulated by changing the ELP transition temperature. These functionalized AuNPs strongly interact with biofilms under static and flow conditions but exhibit weak interactions with serum-coated surfaces. Near-infrared laser irradiation resulted in a 10,000-fold improvement in killing efficiency compared to untreated controls (p < 0.0001). The targeting strategy utilized here represents a versatile approach to targeting drug-resistant infections and could be readily expanded to other anti-biofilm nanoparticle platforms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/610983v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@a7bf65org.highwire.dtl.DTLVardef@106855aorg.highwire.dtl.DTLVardef@11cdd5dorg.highwire.dtl.DTLVardef@9325f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

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 organisms 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. TOC Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/554951v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@19b8ac5org.highwire.dtl.DTLVardef@b3948aorg.highwire.dtl.DTLVardef@1dd2ef5org.highwire.dtl.DTLVardef@35e938_HPS_FORMAT_FIGEXP M_FIG C_FIG Lysine methylation and hydrogen-deuterium exchange can reveal useful structural details about protein adsorption to nanoparticle surfaces.

biophysics↗

bioRxiv2023

Near-Infrared Photothermal Ablation of Biofilms using Protein-Functionalized 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 ({eta}). 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 {degrees}C. Then, functionalizing the AuNP with an additional small globular protein, we were able to extend this range to 34-50 {degrees}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 ({eta} = 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. TOC Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/553096v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@ffb632org.highwire.dtl.DTLVardef@12cbb4org.highwire.dtl.DTLVardef@1fbac00org.highwire.dtl.DTLVardef@1d950b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

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 nanoparticles 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. TOC Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/548033v1_ufig1.gif" ALT="Figure 1"> View larger version (90K): org.highwire.dtl.DTLVardef@ff243corg.highwire.dtl.DTLVardef@a3f010org.highwire.dtl.DTLVardef@e5d2c5org.highwire.dtl.DTLVardef@70bb5d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

ParSe 2.0: A web application that enables proteome-scale searches for sequences that drive protein-mediated phase separation

We have developed an algorithm, ParSe, that 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.

biophysics↗

bioRxiv2023

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.

microbiology↗

bioRxiv2022

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

Liquid-liquid phase separation (LLPS) of proteins is thought to be a primary driving force for the formation of membraneless organelles, which control a wide range of biological functions from stress response to ribosome biogenesis. LLPS of proteins in cells is primarily, though not exclusively, driven by intrinsically disordered (ID) domains. Accurate identification of ID regions (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 phase-separating (PS) ID sequences. We then used these sequence sets to examine how broadly existing amino acids 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 LLPS. 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 LLPS.

biophysics↗

bioRxiv2022

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 nanoparticles 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.

biophysics↗

bioRxiv2020

Beta Turn Propensity and Polymer Scaling Exponent Identify Intrinsically Disordered Proteins that Phase Separate

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

biophysics↗

bioRxiv2018

SP1433-1438 operon of Streptococcuspneumoniae regulates metal homeostasis and cellular metabolism during zinc-stress

Streptococcus pneumoniae colonizes the mucosa of the human nasopharynx and is a leading cause of community-acquired pneumonia, acute otitis media, and bacterial meningitis. Metal ion homeostasis is vital to the survival of this pathogen and contributes significantly to both colonization and invasive disease. Microarray and qRT-PCR analysis revealed an upregulation of an uncharacterized operon (SP1433-1438) in pneumococci subjected to metal-chelation by N,N,N,N-tetrakis-(2-Pyridylmethyl)ethylenediamine (TPEN). Supplementation of either zinc or cobalt following TPEN treatment drastically abrogated induction. BLAST analysis predicted this operon to encode two ABC-transporters, sharing homology to a multidrug resistance system (SP1434-1435) and an energy-coupling factor (ECF) transport system (SP1436-1438). Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated changes in intracellular concentrations of iron, zinc, and manganese ions in a {Delta}1434-8 strain compared to parental T4R. Analysis of the secreted metabolomic profile of the T4R and {Delta}1434-8 strains identified significant changes in pneumococcal glycolytic pathways, indicating a shift towards increased production of acetate. Additionally, proteomic analysis revealed 41 differentially expressed proteins in the {Delta}1434-8 strain, with roughly 20% of them regulated by the global catabolite repressor, CcpA. Based on these findings, we propose that the SP1433-1438 operon is largely involved in the central metabolism of S. pneumoniae during zinc-limitation.\n\nImportanceMetal sequestration is a common strategy utilized by the host immune response as well as antibiotics such as vancomycin to kill invading bacterial pathogens (1). However, pneumococcus is still able to thrive under zinc-limiting conditions. This study describes a previously uncharacterized operon encoding two ABC transport systems that are strongly induced during zinc-limiting conditions. This operon was found to be regulated by a zinc-dependent regulator (SP1433) that functions independently of the overarching AdcR regulon. We have additionally utilized a 2D-NMR approach to analyze the secreted metabolome and have employed proteomic analysis to identify a role for these systems in the maintenance of cellular metabolism. This study provides new information on how Streptococcus pneumoniae responds and adapts to zinc-limiting conditions.

microbiology↗