Yiyan Fei

Effect of Fluorescently Labeling Protein Probes on Kinetics of Protein-Ligand Reactions

We study the kinetic effect of extrinsic fluorescent labeling agents on protein-ligand binding affinity and find that the kinetics is related to the loss or change of protein function when proteins are fluorescent-labeled.

A novel high-throughput scanning microscope for label-free detection of protein and small-molecule chemical microarrays

We describe a novel scanning optical microscope based on a polarization-modulated nulling ellipsometry. The new microscope employs a combination of scanning mirror and sample translation and thus enables high-throughput label-free detection of biomolecular microarrays with more than 10 000 protein or small-molecule targets. For illustration, we show the image of a 2760-spot protein microarray on a functionalized glass slide obtained with such a microscope. The new scanning microscope is also capable of determining, in parallel, the real-time binding kinetics of multiple molecular species under aqueous conditions.

Simultaneous measurement of 10,000 protein-ligand affinity constants using microarray-based kinetic constant assays.

Fluorescence-based endpoint detection of microarrays with 10,000 or more molecular targets is a most useful tool for high-throughput profiling of biomolecular interactions, including screening large molecular libraries for novel protein ligands. However, endpoint fluorescence data such as images of reacted microarrays contain little information on kinetic rate constants, and the reliability of endpoint data as measures of binding affinity depends on reaction conditions and postreaction processing. We here report a simultaneous measurement of binding curves of a protein probe with 10,000 molecular targets in a microarray with an ellipsometry-based (label-free) optical scanner. The reaction rate constants extracted from these curves (k(on), k(off), and k(a)=k(on)/k(off)) are used to characterize the probe-target interactions instead of the endpoints. This work advances the microarray technology to a new milestone, namely, from an endpoint assay to a kinetic constant assay platform. The throughput of this binding curve assay platform is comparable to those at the National Institutes of Health Molecular Library Screening Centers, making it a practical method in screening compound libraries for novel ligands and for system-wide affinity profiling of proteins, viruses, or whole cells against diverse molecular targets.

Macromolecular scaffolds for immobilizing small molecule microarrays in label-free detection of protein-ligand interactions on solid support

We explored two macromolecular scaffolds, bovine serum albumin (BSA) and polyvinyl alcohol (PVA), as chemically complementary platforms for immobilizing small molecule compounds on functionalized glass slides. We conjugated biotin molecules to BSA and amine-derivatized PVA and subsequently immobilized the conjugates on epoxy-functionalized glass slides through reaction of free amine residues on BSA and PVA with surface-bound epoxy groups. We studied binding reactions of such immobilized small molecule targets with solution-phase protein probes using an oblique-incidence reflectivity difference scanning optical microscope. The results showed that both BSA and amine-derivatized PVA were effective and efficient as carriers of small molecules with NHS residues and fluoric residues and for immobilization on epoxy-coated solid surfaces. A significant fraction of the conjugated small molecules retain their innate chemical activity.

Fluorescent labeling agents change binding profiles of glycan-binding proteins.

Interactions of glycan-binding proteins (GBPs) with glycans are essential in cell adhesion, bacterial/viral infection, and cellular signaling pathways. Experimental characterization of these interactions based on glycan microarrays typically involves (1) labeling GBPs directly with fluorescent reagents before incubation with the microarrays, or (2) labeling GBPs with biotin before the incubation and detecting the captured GBPs after the incubation using fluorescently labeled streptavidin, or (3) detecting the captured GBPs after the incubation using fluorescently labeled antibodies raised against the GBPs. The fluorescent signal is mostly measured ex situ after excess fluorescent materials are washed off. In this study, by using a label-free optical scanner for glycan microarray detection, we measured binding curves of 7 plant lectins to 24 glycans: four β1-4-linked galactosides, three β1-3-linked galactosides, one β-linked galactoside, one α-linked N-acetylgalactosaminide, eight α2-3-linked sialosides, and seven α2-6-linked sialosides. From association and dissociation constants deduced by global-fitting the binding curves, we found that (1) labeling lectins directly with fluorescent agents change binding profiles of lectins, in some cases by orders of magnitude; (2) those lectin-glycan binding reactions characterized with large dissociation rates, though biologically relevant, are easily missed or deemed insignificant in ex situ fluorescence-based assays as most captured lectins are washed off before detection. This study highlights the importance of label-free real-time detection of protein-ligand interactions and the potential pitfall in interpreting fluorescence-based assays for characterization of protein-glycan interactions.

Use of real-time, label-free analysis in revealing low-affinity binding to blood group antigens by Helicobacter pylori

Infectious diseases are often initiated by microbial adherence that is mediated by the binding of attachment molecules, termed adhesins, to cell surface receptors on host cells. We present an experimental system, oblique-incidence reflectivity difference (OI-RD) microscopy, which allows the detection of novel, low-affinity microbial attachment mechanisms that may be essential for infectious processes. OI-RD microscopy was used to analyze direct binding of the oncopathogen, Helicobacter pylori ( H. pylori ) to immobilized glycoconjugates in real time with no need for labeling tags. The results suggest the presence of additional Lewis b blood group antigen (Le(b)) binding adhesins that have not been detected previously. OI-RD microscopy also confirmed the high-affinity binding of H. pylori outer-membrane protein BabA to Le(b). The OI-RD microscopy method is broadly applicable to real-time characterization of intact microbial binding to host receptors and offers new strategies to elucidate the molecular interactions of infectious agents with human host cells.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.

Discovering Small Molecule Ligands of Vascular Endothelial Growth Factor That Block VEGF–KDR Binding Using Label-Free Microarray-Based Assays

We present here a label-free microarray-based assay platform that we used to identify inhibitors of vascular endothelial growth factor (VEGF)-kinase-insertion domain receptor (KDR) binding. Supported by a combination of special ellipsometry-based optical detection and small molecule microarrays (SMM), this platform consists of three assays: (1) the first assay detects binding of a target protein with SMM and identifies ligands to the protein as inhibitor candidates; (2) the second assay detects binding of a receptor protein with identical SMM and subsequent binding of the target protein (a sandwich assay) to identify the ligands to the receptor protein that do not interfere with the target-receptor binding; (3) the third assay detects binding of the target protein to the receptor protein in the presence of the ligands of the target protein identified from the first assay, with the receptor protein immobilized to a solid surface through the ligands identified in the second assay, to yield dose-response curves. Using this platform, we screened 7,961 compounds from the National Cancer Institute and found 12 inhibitors to VEGF-KDR (VEGFR2) interactions with IC₅₀ ranging from 0.3 to 60 μM. The inhibitory potency of these inhibitors found in the microarray-based assay was confirmed by their inhibition of VEGF-induced VEGFR2 phosphorylation in a cell-based assay.

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Developing an Efficient and General Strategy for Immobilization of Small Molecules onto Microarrays Using Isocyanate Chemistry

Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%.