Extrinsic Probe Research Articles

Biophysical Studies of Amyloid-Binding Fluorophores to Tau AD Core Fibrils Formed without Cofactors.

Tau is an intrinsically disordered protein involved in several neurodegenerative diseases where a common hallmark is the appearance of tau aggregates in the brain. One common approach to elucidate the mechanisms behind the aggregation of tau has been to recapitulate in vitro the self-assembly process in a fast and reproducible manner. While the seeding of tau aggregation is prompted by negatively charged cofactors, the obtained fibrils are morphologically distinct from those found in vivo. The Tau AD core fragment (TADC, tau 306-378) has emerged as a new model and potential solution for the cofactor-free in vitro aggregation of tau. Here, we use TADC to further study this process combining multiple amyloid-detecting fluorophores and fibril bioimaging. We confirmed by transmission electron microscopy that this fragment forms fibrils after quiescent incubation at 37 °C. We then employed a panel of eight amyloid-binding fluorophores to query the formed species by acquiring their emission spectra. The results obtained showed that nearly all dyes detect TADC self-assembled species. However, the successful monitoring of TADC aggregation kinetics was limited to three fluorophores (X-34, Bis-ANS, and pFTAA) which yielded sigmoidal curves but different aggregation half-times, hinting to different species being detected. Altogether, this study highlights the potential of using multiple extrinsic fluorescent probes, alone or in combination, as tools to further clarify mechanisms behind the aggregation of amyloidogenic proteins.

Unveiling the Structural and Dynamic Characteristics of Concentrated LiNO3 Aqueous Solutions through Ultrafast Infrared Spectroscopy and Molecular Dynamics Simulations.

Highly concentrated aqueous electrolytes have attracted a significant amount of attention for their potential applications in lithium-ion batteries. Nevertheless, a comprehensive understanding of the Li+ solvation structure and its migration within electrolyte solutions remains elusive. This study employs linear vibrational spectroscopy, ultrafast infrared spectroscopy, and molecular dynamics (MD) simulations to elucidate the structural dynamics in LiNO3 solutions by using intrinsic and extrinsic vibrational probes. The N-O stretching vibrations of NO3- exhibit a distinct spectral splitting, attributed to its asymmetric interaction with the surrounding solvation structure. Analysis of the vibrational relaxation dynamics of intrinsic and extrinsic probes, in combination with MD simulations, reveals cage-like networks formed through electrostatic interactions between Li+ and NO3-. This microscopic heterogeneity is reflected in the intertwined arrangement of ions and water molecules. Furthermore, both vehicular transport and structural diffusion assisted by solvent rearrangement for Li+ were analyzed, which are closely linked with the bulk concentration.

Inhibition kinetics and mechanism of genistein against α‐glucosidase

AbstractSeveral studies have documented the effective inhibition of genistein, a component in soybeans against α‐glucosidase. However, the details of the inhibition mechanism and kinetics remain unfulfilled. Here, the authors aim to investigate the anti‐diabetic potential of genistein through IC50 value, fluorescence quenching and inhibition kinetics. Genistein was found to exhibit an inhibition activity on α‐glucosidase with an IC50 value of 7.79 ± 0.36 µm. Analysis of fluorescence spectra indicated that genistein quenched enzyme emission through a static mechanism with a bimolecular quenching constant, kq = 4.04 × 1013 m−1 s−1. In addition, it was found that genistein was bound to α‐glucosidase with a high affinity of binding constant, Ka = 3.38 × 105 m−1 and a 1:1 stoichiometric ratio (n = 1.06). In order to suggest a possible contact involved, 8‐anilino‐1‐naphthalenesulfonic acid (ANS) was added as an extrinsic fluorescence probe to enzyme solution and analyzed fluorescence spectra of the α‐glucosidase‐ANS complexes. When treated with genistein, fluorescence intensities of the complexes were reduced remarkably, indicating that genistein interacts with the enzyme via a hydrophobic domain. Finally, the inhibition constant and inhibition mode were studied by inhibition kinetics. The results revealed that genistein bound easily to α‐glucosidase with a Ki value of 25.61 × 10−6 m through a non‐competitive type.

Dynamics of Formamide-Water Mixtures Investigated by Linear and Nonlinear Infrared Spectroscopy.

Formamide (FA) exhibits complete miscibility with water, offering a simplified model for exploring the solvation dynamics of peptide linkages in biophysical processes. Its liquid state demonstrates a three-dimensional hydrogen bonding network akin to water, reflecting solvent-like behavior. Analyzing the microscopic structure and dynamics of FA-water mixtures is expected to provide crucial insights into hydrogen bonding dynamics─a key aspect of various biophysical phenomena. This study is focused on the dynamics of FA-water mixtures using linear and femtosecond infrared spectroscopies. By using the intrinsic OD stretch and extrinsic probe SCN-, the local vibrational behaviors across various FA-water compositions were systematically investigated. The vibrational relaxation of OD stretch revealed a negligible impact of FA addition on the vibrational lifetime of water molecules, underscoring the mixture's water-like behavior. However, the reorientational dynamics of OD stretch slowed with increasing FA mole fraction (XFA), plateauing beyond XFA > 0.5. This suggests a correlation between OD's reorientational time and the strength of the hydrogen bond network, likely tied to the solution's changing dielectric constant. Conversely, the vibrational relaxation dynamics of SCN- was strongly correlated with XFA, highlighting a competition between water and FA molecules in solvating SCN-. Moreover, a linear relationship between rising viscosity and the prolonged correlation time of SCN-'s slow dynamics indicates that the solution's macroscopic viscosity is dictated by the extended structures formed between FA and water molecules. The relation between the reorientation dynamics of the SCN- and the macroscopic viscosity in aqueous FA-water mixture solutions was analyzed by using the Stokes-Einstein-Debye equations. The direct viscosity-diffusion coupling is observed, which can be attributed to the homogeneous dynamics feature in FA-water mixture solutions. The inclusion of these intrinsic and extrinsic probes not only enhances the comprehensiveness of our analysis but also provides valuable insights into various aspects of the dynamics within the FA-water system. This investigation sheds light on the fundamental dynamics of FA-water mixtures, emphasizing their molecular-level homogeneity in this binary mixture solution.

Phase separation in model lipid membranes investigated with cryogenic electron microscopy.

We describe a method for investigating lateral membrane heterogeneity using cryogenic electron microscopy (cryo-EM) images of liposomes. The method takes advantage of differences in the thickness and molecular density of ordered and disordered phases that are resolvable in phase contrast cryo-EM. Compared to biophysical techniques like FRET or neutron scattering that yield ensemble-averaged information, cryo-EM provides direct visualization of individual vesicles and can therefore reveal variability that would otherwise be obscured by averaging. Moreover, because the contrast mechanism involves inherent properties of the lipid phases themselves, no extrinsic probes are required. We explain and discuss various complementary analyses of spatially resolved thickness and intensity measurements that enable an assessment of the membrane's phase state. The method opens a window to nanodomain structure in synthetic and biological membranes that should lead to an improved understanding of lipid raft phenomena.

Fluorometric Measurement of Calmodulin-Dependent Peptide-Protein Interactions Using Dansylated Calmodulin.

The assessment of peptide-protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides. In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring. In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan. Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes. Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance. DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time. In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein. This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions. Key features • Dynamic fluorescence and real-time monitoring: dansyl-modified CaM enables sensitive, real-time fluorescence, providing valuable insights into the dynamics of molecular interactions and ligand binding. • Selective interaction and stable fluorescent adducts: DNSC selectively interacts with primary amino groups, ensuring specific detection and forming stable fluorescent sulfonamide adducts. • Versatility in research and ease of identification: D-CaM is a versatile tool in biological research, facilitating identification, precise quantification, and drug assessment for therapeutic development. • Sensitivity to surrounding alterations: D-CaM exhibits sensitivity to its surroundings, particularly ligand-induced changes, offering subtle insights into molecular interactions and environmental influences.

Chain-like Structures Facilitate Li+ Transport in Concentrated Aqueous Electrolytes: Insights from Ultrafast Infrared Spectroscopy and Molecular Dynamics Simulations.

Highly concentrated aqueous electrolytes have attracted attention due to their unique applications in lithium ion batteries (LIBs). However, the solvation structure and transport mechanism of Li+ cations at concentrated concentrations remain largely unexplored. To address this gap in knowledge, we employ ultrafast infrared spectroscopy and molecular dynamics (MD) simulations to reveal the dynamic and spatial structural heterogeneity in aqueous lithium chloride (LiCl) solutions. The coupling between the reorientation dynamics of the extrinsic probe and the macroscopic viscosity in aqueous LiCl solutions was analyzed using the Stokes-Einstein-Debye (SED) equations. MD simulations reveal that the Cl- and Li+ form chain-like structures through electrostatic interactions, supporting the vehicular migration of Li+ through the chain-like structure. The concentration dependent conductivity of the LiCl solution is well reproduced, where Li(H2O)2+ and Li(H2O)3+ are the dominant species that contribute to the conduction of Li+. This study is expected to establish correlations between ion pair structures and macroscopic properties.

Allosteric and substrate site effects of relevant amino acids on structural stability and flexibility of glutamate dehydrogenase-1 (GDH-1).

Mammalian GDH-1, which converts glutamate to alpha-ketoglutarate and ammonia while reducing NAD(P), is constitutively expressed in virtually all cells of the body. The extensive literature on GDH-1 suggests that functionally relevant GDH-1 structures may be distributed over a conformational flexibility landscape: polyhexamer, hexamer, trimer, monomer, monomer (unfolded), monomer (misfolded), polymer (disassembled). We have used both intrinsic (tryptophan) and extrinsic (ANS) fluorescent probes to study the dependence of the reversible hexamer to trimer dissociation on the concentrations of the denaturing agents urea and guanidine hydrochloride (GdnHCl). Dissociation at each denaturant concentration increases with increase in concentration and/or temperature, and these dependencies are shown to be affected by amino acids which bind to allosteric and/or substrate binding sites. Dissociation of the hexamer is accompanied by a large increase in the fluorescence of ANS due to binding to increase in exposed hydrophobic microdomains on the protein. In contrast, tryptophan fluorescence decreases due to increased exposure of tryptopan in the protein to the aqueous environment. Measurement were made of the influence of several known substrates and allosteric modifiers (i.e. a selection physiologically relevant monocarboxylic amino acids) on the GDH-1 hexamer to trimer transition. The observed effect of adding each amino acid is consistent with independent measurements of its binding affinity. From the temperature dependence of the fluorescence intensity changes for ANS we obtained activation parameters for the hexamer-trimer transitions. Our data are consistent with the hexamer/trimer transition in GDH-1 having significant regulatory function in vivo, perhaps contribution to/complementing allosteric regulation of the enzyme activity.

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu.

Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4nm-10nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

Syntheses of APTMS-Coated ZnO: An Investigation towards Penconazole Detection.

Extrinsic chemiluminescence can be an efficient tool for determining pesticides and fungicides, which do not possess any intrinsic fluorescent signal. On this basis, (3-aminopropyl) trimethoxysilane (APTMS)-coated ZnO (APTMS@ZnO) was synthesized and tested as an extrinsic probe for the fungicide penconazole. Several synthetic routes were probed using either a one-pot or two-steps method, in order to ensure both a green synthetic pathway and a good signal variation for the penconazole concentration. The synthesized samples were characterized using X-ray diffraction (XRD), infrared (IR), Raman and ultraviolet-visible (UV-Vis) spectroscopy, scanning electron microscopy (SEM) imaging and associated energy-dispersive X-ray (EDX) analysis. The average size of the synthesized ZnO nanoparticles (NPs) is 54 ± 10 nm, in line with previous preparations. Of all the samples, those synthesized in two steps, at temperatures ranging from room temperature (RT) to a maximum of 40 °C, using water solvent (G-APTMG@ZnO), appeared to be composed of nanoparticles, homogeneously coated with APTMS. Chemiluminescence tests of G-APTMG@ZnO, in the penconazole concentration range 0.7-1.7 ppm resulted in a quenching of the native signal between 6% and 19% with a good linear response, thus indicating a green pathway for detecting the contaminant. The estimated detection limit (LOD) is 0.1 ± 0.01 ppm.

Distinct Hydrogen Bonding Dynamics Underlies the Microheterogeneity in DMF-Water Mixtures.

The hydrogen bonding interaction between the amide functional group and water is fundamental to understanding the liquid-liquid heterogeneity in biological systems. Herein, the structure and dynamics of the N,N-dimethylformamide (DMF)-water mixtures have been investigated by linear and nonlinear IR spectroscopies, using the hydroxyl stretch and extrinsic probe of thiocyanate as local vibrational reporters. According to vibrational relaxation dynamics measurements, the orientational dynamics of water is not directly tied to those of DMF molecules. Wobbling-in-a-cone analysis demonstrates that the water molecules have varying degrees of angular restriction depending on their composition due to the formation of specific water-DMF networks. Because of the preferential solvation by DMF molecules, the rotational dynamics of the extrinsic probe is slowed significantly, and its rotational time constants are correlated to the change of solution viscosity. The unique structural dynamics observed in the DMF-water mixtures is expected to provide important insights into the underlying mechanism of microscopic heterogeneity in binary mixtures.

Carvacrol protects against carbonyl osmolyte-induced structural modifications and aggregation to serum albumin: Insights from physicochemical and molecular interaction studies

The robust use of osmolytes (i.e., polyols and sugars) in the key therapeutic regimens/formulations has questioned their impact beyond the stability of therapeutic proteins as these osmolytes trigger structural alterations into proteins including misfolding and subsequent aggregation into amyloid fibrils. Therefore, the current study is the first to delineate the inhibitory effect of carvacrol (CRV) on the carbonyl osmolyte-induced aggregation as well as structural alterations to the bovine serum albumin (BSA) via a set of physicochemical as well as artificial intelligence (AI)-based molecular docking studies. Our initial findings from physicochemical investigations revealed that CRV exhibits substantial protection to BSA under carbonyl osmolyte stress as evident by the compromised hyperchromicity, Schiff's bases, carbonyl and hydroxymethyl furfural content, reduced fluorescent signals, low Rayleigh scattering and prevention of covalent modifications at Lys and Arg residues. The protection against aggregate formation by CRV was further confirmed through the reduced amyloid-specific congo red absorbance as well as fluorescent signals recorded after adding the fibril-specific extrinsic fluorophore probes (i.e., ThT and ANS). The AI-based molecular docking analysis further revealed that CRV (ΔG: −4.96 kcal/mol) competes with d-fructose (ΔG: −4.40 kcal/mol) to mask the Lys and Arg residues to restrict the osmolyte-mediated protein modifications. In conclusion, CRV exhibits substantial protective impact against carbonyl osmolyte-induced structural alterations and protein misfolding and aggregation.

Fluorescence Studies of Nicotinic Acetylcholine Receptor and Its Associated Lipid Milieu: The Influence of Erwin London's Methodological Approaches.

Erwin London dedicated considerable effort to understanding lipid interactions with membrane-resident proteins and how these interactions shaped the formation and maintenance of lipid phases and domains. In this endeavor, he developed ad hoc techniques that greatly contributed to advancements in the field. We have employed and/or modified/extended some of his methodological approaches and applied them to investigate lipid interaction with the nicotinic acetylcholine receptor (nAChR) protein, the paradigm member of the superfamily of rapid pentameric ligand-gated ion channels (pLGIC). Our experimental systems ranged from purified receptor protein reconstituted into synthetic lipid membranes having known effects on receptor function, to cellular systems subjected to modification of their lipid content, e.g., varying cholesterol levels. We have often employed fluorescence techniques, including fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and of nAChR intrinsic fluorescence by nitroxide spin-labeled phospholipids, DPH anisotropy, excimer formation of pyrene-phosphatidylcholine, and Förster resonance energy transfer (FRET) from the protein moiety to the extrinsic probes Laurdan, DPH, or pyrene-phospholipid to characterize various biophysical properties of lipid-receptor interactions. Some of these strategies are revisited in this review. Special attention is devoted to the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the nAChR. The receptor protein was shown to organize its PA-containing immediate microenvironment into microdomains with high lateral packing density and rigidity. PA and cholesterol appear to compete for the same binding sites on the nAChR protein.

Recent advancements toward non-invasive imaging of retinal amyloid-beta for early detection of Alzheimer's disease.

Recent advancements toward non-invasive imaging of retinal amyloid-beta for early detection of Alzheimer's disease.

Characterization of Acetonitrile Isotopologues as Vibrational Probes of Electrolytes.

Acetonitrile has emerged as a solvent candidate for novel electrolyte formulations in metal-ion batteries and supercapacitors. It features a bright local C≡N stretch vibrational mode whose infrared (IR) signature is sensitive to battery-relevant cations (Li+, Mg2+, Zn2+, Ca2+) both in pure form and in the presence of water admixture across a full possible range of concentrations from the dilute to the superconcentrated regime. Stationary and time-resolved IR spectroscopy thus emerges as a natural tool to study site-specific intermolecular interactions from the solvent perspective without introducing an extrinsic probe that perturbs solution morphology and may not represent the intrinsic dynamics in these electrolytes. The metal-coordinated acetonitrile, water-separated metal–acetonitrile pair, and free solvent each have a distinct vibrational signature that allows their unambiguous differentiation. The IR band frequency of the metal-coordinated acetonitrile depends on the ion charge density. To study the ion transport dynamics, it is necessary to differentiate energy-transfer processes from structural interconversions in these electrolytes. Isotope labeling the solvent is a necessary prerequisite to separate these processes. We discuss the design principles and choice of the CD313CN label and characterize its vibrational spectroscopy in these electrolytes. The Fermi resonance between 13C≡N and C–D stretches complicates the spectral response but does not prevent its effective utilization. Time-resolved two-dimensional (2D) IR spectroscopy can be performed on a mixture of acetonitrile isotopologues and much can be learned about the structural dynamics of various species in these formulations.

In vivo observation of amyloid-like fibrils produced under stress

The participation of amyloids in neurodegenerative diseases and functional processes has triggered the quest for methods allowing their direct detection in vivo. Despite the plethora of data, those methods are still lacking. The autofluorescence from the extended β-sheets of amyloids is here used to track fibrillation of S. cerevisiae Golgi Reassembly and Stacking Protein (Grh1). Grh1 has been implicated in starvation-triggered unconventional protein secretion (UPS), and here its participation also in heat shock response (HSR) is suggested. Fluorescence Lifetime Imaging (FLIM) is used to detect fibril autofluorescence in cells (E. coli and yeast) under stress (starvation and higher temperature). The formation of Grh1 large complexes under stress is further supported by size exclusion chromatography and ultracentrifugation. The data show for the first time in vivo detection of amyloids without the use of extrinsic probes as well as bring new perspectives on the participation of Grh1 in UPS and HSR.

Thermodynamic Affinity and Nature of Forces Defining Glycosaminoglycan-Protein Systems Using Fluorescence Spectroscopy.

Among the biophysical techniques used to study glycosaminoglycan (GAG)-protein interactions, fluorescence spectroscopy is a quantitative tool that has been extensively used to provide structural and dynamical information. Its advantages include high sensitivity, relative ease of applicability, and wide range of available fluorescence labels and probes. A large majority of protein-GAG systems have been studied using either intrinsic (e.g., Trp) or extrinsic (e.g., a noncovalent fluorophore) probes. It forms the basis for measurement of dissociation constant and stoichiometry of GAG-protein complexes. We describe step-by-step procedures to measure the affinity of GAG-protein complexes, parse the ionic and non-ionic components of the free energy of binding, and identify the site of GAG binding through competitive binding experiments.

Purification, Characterization, and Assessment of Antimicrobial Activity and Toxicity of Portulaca elatior Leaf Lectin (PeLL).

Lectins are carbohydrate-binding proteins with several bioactivities, including antimicrobial properties. Portulaca elatior is a species found at Brazilian Caatinga and data on the biochemical composition of this plant are scarce. The present work describes the purification of P. elatior leaf lectin (PeLL) as well as the assessment of its antimicrobial activity and toxicity. PeLL, isolated by chromatography on a chitin column, had native liquid charge and subunit composition evaluated by electrophoresis. Hemagglutinating activity (HA) of PeLL was determined in the presence of carbohydrates or divalent cations, as well as after heating and incubation at different pH values. Changes in the lectin conformation were monitored by evaluating intrinsic tryptophan fluorescence and using the extrinsic probe bis-ANS. Antimicrobial activity was evaluated against Pectobacterium strains and Candida species. The minimal inhibitory (MIC), bactericidal (MBC), and fungicidal (MFC) concentrations were determined. Finally, PeLL was evaluated for in vitro hemolytic activity in human erythrocytes and in vivo acute toxicity in mice (5 and 10mg/kg b.w. per os). PeLL (pI 5.4; 20kDa) had its HA was inhibited by mannose, galactose, Ca2+, Mg2+, and Mn2+. PeLL HA was resistant to heating at 100°C, although conformational changes were detected. PeLL was more active in the acidic pH range, in which no conformational changes were observed. The lectin presented MIC and MBC of 0.185 and 0.74μg/mL for all Pectobacterium strains, respectively; MIC of 1.48μg/mL for C. albicans, C. tropicalis, and C. krusei; MIC and MFC of 0.74 and 2.96μg/mL for C. parapsilosis. No hemolytic activity or signs of acute toxicity were observed in the mice. In conclusion, a new, low-toxic, and thermostable lectin was isolated from P. elatior leaves, being the first plant compound to show antibacterial activity against Pectobacterium.

Confined Effects on Structural Isomers in the MCM-41-SIL Matrix as Seen by Extrinsic Probes via PALS and ESR: n-Butanol vs tert-Butanol

Confined Effects on Structural Isomers in the MCM-41-SIL Matrix as Seen by Extrinsic Probes via PALS and ESR: <i>n</i>-Butanol vs <i>tert</i>-Butanol

Candida albicans exhibit two classes of cell surface binding sites for serum albumin defined by their affinity, abundance and prospective role in interkingdom signalling.

Serum albumin binding to the yeast form of Candida albicans is described. Two populations of binding site are identified using two complementary spectroscopic techniques: an extrinsic fluorescent probe, 3-hexa-decanoyl-7-hydrocoumarin ([HEXCO) added to the C. albicans yeast cell surface that records the electrostatic surface potential and so responds to the surface binding of serum albumin and secondly a light scattering technique that reveals how albumin modulates aggregation of the yeast population. The albumin binding sites are found to possess different binding affinities and relative abundance leading to different total binding capacities. These are characterized as a receptor population with high affinity binding (Kd ~ 17 μM) but relatively low abundance and a separate population with high abundance but much lower affinity (Kd ~ 364 μM). The low-affinity binding sites are shown to be associated with the yeast cell aggregation. These values are found be dependent on the C. albicans strain and the nature of the culture media; some examples of these effects are explored. The possible physiological consequences of the presence of these sites are speculated in terms of evading the host’s immune response, biofilm formation and possible interkingdom signaling processes.