Characterization of Reversible Associations by Sedimentation Velocity with UltraScan

Demeler, Borries; Brookes, Emre; Wang, Renjing; Schirf, Virgil; Kim, Chongwoo A.

doi:10.1002/mabi.200900481

Cited by 50 publications

(57 citation statements)

References 20 publications

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“…The van Holde – Weischet analysis shows a strong mass action effect with a marked shift to a higher sedimentation coefficient distribution for the higher concentration (Figure 1E). Subsequently, to calculate dissociation constant for the monomer-dimer equilibrium, both concentrations were fitted with finite element solutions of the Lamm equation that models the reversible interaction for a monomer-dimer system (Cao and Demeler, 2008; Demeler et al, 2010). The fit resulted in virtually identical KD values for both fits, averaging 43.3 μM (39.7 – 46.9 μM with 95% confidence intervals).…”

Section: Resultsmentioning

confidence: 99%

Structural and Mechanistic Insights into the Latrophilin3-FLRT3 Complex that Mediates Glutamatergic Synapse Development

Ranaivoson

Martini

et al. 2015

Structure

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SUMMARY Latrophilins (LPHNs) are adhesion-like G protein coupled receptors implicated in attention-deficit/hyperactivity disorder (ADHD). Recently, LPHN3 was found to regulate excitatory synapse number through trans interactions with fibronectin leucine-rich repeat transmembrane 3 (FLRT3). By isothermal titration calorimetry, we determined that only the olfactomedin (OLF) domain of LPHN3 is necessary for FLRT3 association. By multi-crystal native-SAD phasing, we determined the crystal structure of the OLF domain. This structure is a five-bladed β-propeller with a Ca2+ ion bound in the central pore, which is capped by a mobile loop that allows the ion to exchange with the solvent. The crystal structure of the OLF/FLRT3 complex shows that LPHN3-OLF in the closed state binds with high affinity to the concave face of FLRT3-LRR with a combination of hydrophobic and charged residues. Our study provides structural and functional insights into the molecular mechanism underlying the LPHN3/FLRT3 contribution to the development of glutamatergic synapses.

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Section: Resultsmentioning

confidence: 99%

Structural and Mechanistic Insights into the Latrophilin3-FLRT3 Complex that Mediates Glutamatergic Synapse Development

Ranaivoson

Martini

et al. 2015

Structure

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“…One of these uses the genetic algorithm optimization to optimize the model describing the appropriate reversible association to obtain solution properties of all components present in the system and was recently applied to synthetic and experimental data. 100 Demeler et al 100 also suggest limits for the accessible kinetic range. They conclude that equilibrium constants obtained from SV and SE analysis are equivalent.…”

Section: Time-derivative Methodsmentioning

confidence: 99%

Analytical ultracentrifugation of colloids

2010

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In this contribution the use of Analytical Ultracentrifugation (AUC) for the modern analysis of colloids is reviewed. Since AUC is a fractionation technique, distributions of the sedimentation coefficient, particle size and shape, molar mass and density can be obtained for particle sizes spanning the entire colloidal range. The Ångström resolution and the reliable statistics with which particle size distributions can be obtained from analytical ultracentrifugation makes this a high resolution analysis technique for the characterization of nanoparticles in solution or suspension. Several examples showing successful applications of AUC to complex problems in colloid science are given to illustrate the broad range and versatility of questions that can be answered by AUC experiments.

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“…The sedimentation velocity data were initially fitted with the twodimensional spectrum analysis, as described previously (66), to remove time-and radially-invariant noise from the raw data and to fit the meniscus position. Subsequently, the data were fitted to a discrete monomer-dimer model using the adaptive space-time finite element method (67) and genetic algorithms for the parameter optimization (68). The monomer-dimer model accounts for mass action and the reversible association behavior, fitting the thermodynamic and hydrodynamic parameters, as well as the partial specific volume while assuming the predicted molar mass for either wild type or mutant.…”

Section: Engineered Tgf-␤ Monomer That Blocks Tgf-␤ Signalingmentioning

confidence: 99%

An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

Kim¹,

Barron²,

Hinck

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellThe transforming growth factor ␤ isoforms, TGF-␤1, -␤2, and -␤3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-␤ pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-␤s in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-␤ monomer, lacking the heel helix, a structural motif essential for binding the TGF-␤ type I receptor (T␤RI) but dispensable for binding the other receptor required for TGF-␤ signaling, the TGF-␤ type II receptor (T␤RII), as an alternative therapeutic modality for blocking TGF-␤ signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-␤ monomers and bound T␤RII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-␤ signaling with a K i of 20 -70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for T␤RII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit T␤RI, enabled it to bind endogenous T␤RII but prevented it from binding and recruiting T␤RI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-␤ signaling and may inform similar modifications of other TGF-␤ family members.The transforming growth factor ␤ isoforms, TGF-␤1, -␤2, and -␤3, are small secreted signaling proteins. Their overall structures are similar and consist of two cystine-knotted monomers tethered together by a single inter-chain disulfide bond (1). They coordinate wound healing, modulate immune cell function, maintain the extracellular matrix, and regulate epithelial and endothelial cell growth and differentiation (2). The TGF-␤s are synthesized as pre-pro-proteins, and after maturation, secretion, and release from their pro-domains (3), the mature homodimeric growth factors (GFs) 3 bind and bring together two single-pass transmembrane receptors, known as T␤RI and T␤RII, to form the signaling-competent T␤RI 2 -T␤RII 2 heterotetramer (4, 5). TGF-␤ GFs assemble T␤RI 2 -T␤RII 2 heterotetramer in a sequential manner, first by binding T␤RII followed by recruitment of T␤RI (6, 7). The stepwise assembly of T␤RII and T␤RI into a heterotetramer is driven by binding of T␤RI to a composite TGF-␤/T␤RII interface (Fig. 1A) (8, 9).The disruption or dysregulation of the TGF-␤ pathway is responsible for several human diseases. These include connec-

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Characterization of Reversible Associations by Sedimentation Velocity with UltraScan

Cited by 50 publications

References 20 publications

Structural and Mechanistic Insights into the Latrophilin3-FLRT3 Complex that Mediates Glutamatergic Synapse Development

Structural and Mechanistic Insights into the Latrophilin3-FLRT3 Complex that Mediates Glutamatergic Synapse Development

Analytical ultracentrifugation of colloids

An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

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