Jongseong Kim

A mechanically stabilized receptor-ligand flex-bond important in the vasculature

Haemostasis in the arteriolar circulation mediated by von Willebrand factor (VWF) binding to platelets is an example of an adhesive interaction that must withstand strong hydrodynamic forces acting on cells. VWF is a concatenated, multifunctional protein that has binding sites for platelets as well as subendothelial collagen. Binding of the A1 domain in VWF to the glycoprotein Ib α subunit (GPIbα) on the surface of platelets mediates crosslinking of platelets to one another and the formation of a platelet plug for arterioles. The importance of VWF is illustrated by its mutation in von Willebrand disease, a bleeding diathesis. Here, we describe a novel mechanochemical specialization of the A1–GPIbα bond for force-resistance. We have developed a method that enables, for the first time, repeated measurements of the binding and unbinding of a receptor and ligand in a single molecule (ReaLiSM). We demonstrate two states of the receptor–ligand bond, that is, a flex-bond. One state is seen at low force; a second state begins to engage at 10 pN with a ∼20-fold longer lifetime and greater force resistance. The lifetimes of the two states, how force exponentiates lifetime, and the kinetics of switching between the two states are all measured. For the first time, single-molecule measurements on this system are in agreement with bulk phase measurements. The results have important implications not only for how platelets bound to VWF are able to resist force to plug arterioles, but also how increased flow activates platelet plug formation.

Molecular Fibers Based on the Honeycomb-Like Self-Assembly of an α-Helical Polypeptide

We have succeeded in fabricating well-grown molecular fibers of a polypeptide on substrates by using a conventional solution spin-coating and drying process. These molecular fibers were found to consist of a honeycomb-like molecular assembly formed via the hexagonal close packing of the polypeptide chains in the alpha-helix conformation.

INO80 exchanges H2A.Z for H2A by translocating on DNA proximal to histone dimers

ATP-dependent chromatin remodellers modulate nucleosome dynamics by mobilizing or disassembling nucleosomes, as well as altering nucleosome composition. These chromatin remodellers generally function by translocating along nucleosomal DNA at the H3–H4 interface of nucleosomes. Here we show that, unlike other remodellers, INO80 translocates along DNA at the H2A–H2B interface of nucleosomes and persistently displaces DNA from the surface of H2A–H2B. DNA translocation and DNA torsional strain created near the entry site of nucleosomes by INO80 promotes both the mobilization of nucleosomes and the selective exchange of H2A.Z–H2B dimers out of nucleosomes and replacement by H2A–H2B dimers without any additional histone chaperones. We find that INO80 translocates and mobilizes H2A.Z-containing nucleosomes more efficiently than those containing H2A, partially accounting for the preference of INO80 to replace H2A.Z with H2A. Our data suggest that INO80 has a mechanism for dimer exchange that is distinct from other chromatin remodellers including its paralogue SWR1.

Direct Thrombus Imaging in Stroke.

There is an emergent need for imaging methods to better triage patients with acute stroke for tissue-plasminogen activator (tPA)-mediated thrombolysis or endovascular clot retrieval by directly visualizing the size and distribution of cerebral thromboemboli. Currently, magnetic resonance (MR) or computed tomography (CT) angiography visualizes the obstruction of blood flow within the vessel lumen rather than the thrombus itself. The present visualization method, which relies on observation of the dense artery sign (the appearance of cerebral thrombi on a non-enhanced CT), suffers from low sensitivity. When translated into the clinical setting, direct thrombus imaging is likely to enable individualized acute stroke therapy by allowing clinicians to detect the thrombus with high sensitivity, assess the size and nature of the thrombus more precisely, serially monitor the therapeutic effects of thrombolysis, and detect post-treatment recurrence. This review is intended to provide recent updates on stroke-related direct thrombus imaging using MR imaging, positron emission tomography, or CT.

Force-induced on-rate switching and modulation by mutations in gain-of-function von Willebrand diseases

Significance Binding of von Willebrand factor (VWF) to platelets is regulated by hydrodynamic forces in the vasculature. VWF can sense force and can bind when the hydrodynamics change due to bleeding. We show that force application switches the A1 domain in VWF to a second state with faster on-rate for its binding partner on platelets, GPIbα. This provides a physiological mechanism for activating VWF binding to platelets at sites of bleeding. Moreover, force increases the effects of gain-of-function mutations found in von Willebrand disease (VWD) and platelet-type VWD by mechanically stabilizing bond formation and strength. Mutations in the ultralong vascular protein von Willebrand factor (VWF) cause the common human bleeding disorder, von Willebrand disease (VWD). The A1 domain in VWF binds to glycoprotein Ibα (GPIbα) on platelets, in a reaction triggered, in part, by alterations in flow during bleeding. Gain-of-function mutations in A1 and GPIbα in VWD suggest conformational regulation. We report that force application switches A1 and/or GPIbα to a second state with faster on-rate, providing a mechanism for activating VWF binding to platelets. Switching occurs near 10 pN, a force that also induces a state of the receptor−ligand complex with slower off-rate. Force greatly increases the effects of VWD mutations, explaining pathophysiology. Conversion of single molecule kon (s−1) to bulk phase kon (s−1M−1) and the kon and koff values extrapolated to zero force for the low-force pathways show remarkably good agreement with bulk-phase measurements.

Lysine Acetylation Facilitates Spontaneous DNA Dynamics in the Nucleosome.

The nucleosome, comprising a histone protein core wrapped around by DNA, is the fundamental packing unit of DNA in cells. Lysine acetylation at the histone core elevates DNA accessibility in the nucleosome, the mechanism of which remains largely unknown. By employing our recently developed hybrid single molecule approach, here we report how the structural dynamics of DNA in the nucleosome is altered upon acetylation at histone H3 lysine 56 (H3K56) that is critical for elevated DNA accessibility. Our results indicate that H3K56 acetylation facilitates the structural dynamics of the DNA at the nucleosome termini that spontaneously and repeatedly open and close on a ms time scale. The results support a molecular mechanism of histone acetylation in catalyzing DNA unpacking whose efficiency is ultimately limited by the spontaneous DNA dynamics at the nucleosome temini. This study provides the first and unique experimental evidence revealing a role of protein chemical modification in directly regulating the kinetic stability of the DNA packing unit.

Single-Molecule Observation Reveals Spontaneous Protein Dynamics in the Nucleosome.

Structural dynamics of a protein molecule is often critical to its function. Single-molecule methods provide efficient ways to investigate protein dynamics, although it is very challenging to achieve a millisecond or higher temporal resolution. Here we report spontaneous structural dynamics of the histone protein core in the nucleosome based on a single-molecule method that can reveal submillisecond dynamics by combining maximum likelihood estimation and fluorescence correlation spectroscopy. The nucleosome, comprising ∼147 bp DNA and an octameric histone protein core consisting of H2A, H2B, H3, and H4, is the fundamental packing unit of the eukaryotic genome. The nucleosome imposes a physical barrier that should be overcome during various DNA-templated processes. Structural fluctuation of the nucleosome in the histone core has been hypothesized to be required for nucleosome disassembly but has yet to be directly probed. Our results indicate that at 100 mM NaCl the histone H2A-H2B dimer dissociates from the histone core transiently once every 3.6 ± 0.6 ms and returns to its position within 2.0 ± 0.3 ms. We also found that the motion is facilitated upon H3K56 acetylation and inhibited upon replacing H2A with H2A.Z. These results provide the first direct examples of how a localized post-translational modification or an epigenetic variation affects the kinetic and thermodynamic stabilities of a macromolecular protein complex, which may directly contribute to its functions.

Direct observation of ligand-induced receptor dimerization with a bioresponsive hydrogel

Multimerization of biomolecules is essential for biological function and thus there is a need for sensitive biochemical assays that determine whether a molecule associates with one or more other molecules in the context of biological function. In this contribution we demonstrate a simple yet versatile method for the identification of physiologically important receptor dimerization events induced by a ligand. Bioresponsive hydrogel microparticles (microgels) conjugated with a receptor, Glycoprotein Ibα (GPIbα), display large changes in optical (microscopic) appearance under conditions known for to promote thrombin-induced GPIbα dimerization. In support of X-ray crystal structures, we identify that one thrombin molecule associates with two GPIbα moieties, which may play a role in efficient hemostatic function by increasing local concentration of GPIbα on platelet surfaces. This microgel assay could provide a new way of studying important physiological and pathological mechanisms related to receptor dimerization and/or clustering.

Influence of surfactant structure on surfactant sorption and diesel removal from kaolin soil

Abstract The effects of hydrophilic and hydrophobic structures of selected nonionic surfactants on surfactant sorption and diesel removal in the system of kaolin soil were studied to have an insight into the criteria of surfactant selection for surfactant‐enhanced remediation of diesel‐contaminated soil. The capability of surfactants for diesel removal could be correlated with the extent of surfactant sorption on kaolin soil. Surfactants with higher degree of ethoxylation in their hydrophilic moiety showed less sorption and improved diesel removal. Surfactants with shorter hydrocarbon chain and unsaturated carbons in their hydrophobic moiety showed less sorption and superior diesel removal. In all cases tested in this study, less sorbed surfactants showed higher diesel removal. Among 11 nonionic surfactants we examined, Brij 35, Brij 58, and Brij 98 were the best for diesel removal and showed low extent of sorption.

Characterization of a Functional Hydrogel Layer on a Silicon-Based Grating Waveguide for a Biochemical Sensor

We numerically demonstrated the characteristics of a functional hydrogel layer on a silicon-based grating waveguide for a simple, cost-effective refractive index (RI) biochemical sensor. The RI of the functional hydrogel layer changes when a specific biochemical interaction occurs between the hydrogel-linked receptors and injected ligand molecules. The transmission spectral profile of the grating waveguide shifts depends on the amount of RI change caused by the functional layer. Our characterization includes the effective RI change caused by the thickness, functional volume ratio, and functional strength of the hydrogel layer. The results confirm the feasibility of, and set design rules for, hydrogel-assisted silicon-based grating waveguides.