Calculations and Publication-Quality Illustrations for Analytical Ultracentrifugation Data

Brautigam, Chad A.

doi:10.1016/bs.mie.2015.05.001

Cited by 492 publications

(401 citation statements)

References 39 publications

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“…Exothermic heats of dilution for HIF-E were treated as a single baseline parameter in SEDPHAT. For both data types, the plots were rendered with GUSSI v1.1.0 [37]. …”

Section: Methodsmentioning

confidence: 99%

On the acquisition and analysis of microscale thermophoresis data

Scheuermann

Padrick

Gardner

et al. 2016

Analytical Biochemistry

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177

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A comprehensive understanding of the molecular mechanisms underpinning cellular functions is dependent on a detailed characterization of the energetics of macromolecular binding, often quantified by the equilibrium dissociation constant, KD. While many biophysical methods may be used to obtain KD, the focus of this report is a relatively new method called “microscale thermophoresis” (MST). In an MST experiment, a capillary tube filled with a solution containing a dye-labeled solute is illuminated with an infrared laser, rapidly creating a temperature gradient. Molecules will migrate along this gradient, causing changes in the observed fluorescence. Because the net migration of the labeled molecules will depend on their liganded state, a binding curve can be constructed as a function of ligand concentration from MST data and analyzed to determine KD. Herein, simulations demonstrate the limits of KD that can be measured in current instrumentation. They also show that binding kinetics are a major concern when planning and executing MST experiments. Additionally, studies of two protein-protein interactions illustrate challenges encountered in acquiring and analyzing MST data. Combined, these approaches indicate a set of best practices for performing and analyzing MST experiments. Software for rigorous data analysis is also introduced.

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“…Exothermic heats of dilution for HIF-E were treated as a single baseline parameter in SEDPHAT. For both data types, the plots were rendered with GUSSI v1.1.0 [37]. …”

Section: Methodsmentioning

confidence: 99%

On the acquisition and analysis of microscale thermophoresis data

Scheuermann

Padrick

Gardner

et al. 2016

Analytical Biochemistry

Self Cite

150

177

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“…using absorbance detection. The collected data were analysed using continuous c(s) distribution of SEDFIT 27,28 fitting for the frictional ratio, meniscus, and time-invariant noise and using regularization level of 0.68. The buffer density and viscosity and the partial specific volumes of IZUMO1 and JUNO were calculated using the program SEDNTERP 1.09 and were 1.00852 g ml −1 and 1.0256 cP, 0.7217 cm 3 g −1 , and 0.7349 cm 3 g −1 , respectively.…”

Section: Letter Researchmentioning

confidence: 99%

Structure of IZUMO1–JUNO reveals sperm–oocyte recognition during mammalian fertilization

Ohto

Ishida

Krayukhina

et al. 2016

Nature

131

133

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Fertilization is a fundamental process in sexual reproduction, creating a new individual through the combination of male and female gametes. The IZUMO1 sperm membrane protein and its counterpart oocyte receptor JUNO have been identified as essential factors for sperm-oocyte interaction and fusion. However, the mechanism underlying their specific recognition remains poorly defined. Here, we show the crystal structures of human IZUMO1, JUNO and the IZUMO1-JUNO complex, establishing the structural basis for the IZUMO1-JUNO-mediated sperm-oocyte interaction. IZUMO1 exhibits an elongated rod-shaped structure comprised of a helical bundle IZUMO domain and an immunoglobulin-like domain that are each firmly anchored to an intervening β-hairpin region through conserved disulfide bonds. The central β-hairpin region of IZUMO1 provides the main platform for JUNO binding, while the surface located behind the putative JUNO ligand binding pocket is involved in IZUMO1 binding. Structure-based mutagenesis analysis confirms the biological importance of the IZUMO1-JUNO interaction. This structure provides a major step towards elucidating an essential phase of fertilization and it will contribute to the development of new therapeutic interventions for fertility, such as contraceptive agents.

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“…The buffer was 25 mM HEPES/NaOH, pH 7.5, 100 mM NaCl, 5 mM DTT. Sedimentation velocity data (radial scans with interference optics) were analyzed with Sedfit (24), and sedimentation coefficient distributions with s 20,w values corrected for buffer viscosity and density were plotted with GUSSI (25).…”

Section: Methodsmentioning

confidence: 99%