Sequence‐specific determination of protein and peptide concentrations by absorbance at 205 nm

Anthis, Nicholas J.; Clore, G. Marius

doi:10.1002/pro.2253

Cited by 365 publications

(269 citation statements)

References 22 publications

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“…The peptide was dissolved in double distilled H 2 O, loaded on a pre-equilibrated PD-10 desalting column, and eluted in FSI buffer (20 mM Tris/HCl, pH 7.8, 1 mM DTT, 150 mM NaCl). The FSI extinction coefficient was calculated (25) ), and concentrations were measured at 205 nm.…”

Section: Methodsmentioning

confidence: 99%

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments

Montaville¹,

Kühn²,

Compper

et al. 2016

Journal of Biological Chemistry

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Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin⅐actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end.Polarized assembly of actin filaments is pivotal in motile processes. It is precisely regulated by proteins that bind the barbed ends of actin filaments and either block or assist filament assembly using various mechanisms (1). Among these regulators, formins are acknowledged to nucleate and track filament barbed ends, mediating rapid processive elongation of filaments (2, 3). In contrast, WASP homology 2 (WH2) 3 repeatcontaining proteins display versatile barbed end capping or tracking and filament severing activities (4). In early oogenesis of Drosophila as well as in mammalian meiosis, a formin (Fmn2/Cappuccino) and a WH2-repeat protein (Spire) synergize in an intriguing fashion to stimulate massive assembly of a dynamic cytoplasmic actin meshwork, required for completion of crucial steps in axis patterning or asymmetric division (5-12). This complexity adds to the known need of profilin for rapid processive assembly of all formins, including Cappuccino (9). Like most formins, Fmn2 harbors conserved C-terminal formin homology 1 (FH1) and formin homology 2 (FH2) domains. The C-terminal FH1-FH2 domain of Fmn2 represents a constitutive active module in which the FH1 domain binds profilin and the FH2 domain to the barbed end of F-actin. The head-to-tail FH2 dimer of formins is thought, from structural data, to encircle the terminal and subterminal actin subunits that constitute the barbed end of the fil...

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Section: Methodsmentioning

confidence: 99%

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments

Montaville¹,

Kühn²,

Compper

et al. 2016

Journal of Biological Chemistry

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“…Peptide purity was analyzed via 205 nm absorbance measured in ddH 2 O with an Evolution UV/Vis detector (Thermo Scientific) and compared with predicted values using a previously published methodology. 62 …”

Section: Hydrogel Macromer Synthesismentioning

confidence: 99%

Development of Poly(Ethylene Glycol) Hydrogels for Salivary Gland Tissue Engineering Applications

Shubin

Felong

Graunke

et al. 2015

Tissue Engineering Part A

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More than 40,000 patients are diagnosed with head and neck cancers annually in the United States with the vast majority receiving radiation therapy. Salivary glands are irreparably damaged by radiation therapy resulting in xerostomia, which severely affects patient quality of life. Cell-based therapies have shown some promise in mouse models of radiation-induced xerostomia, but they suffer from insufficient and inconsistent gland regeneration and accompanying secretory function. To aid in the development of regenerative therapies, poly(ethylene glycol) hydrogels were investigated for the encapsulation of primary submandibular gland (SMG) cells for tissue engineering applications. Different methods of hydrogel formation and cell preparation were examined to identify cytocompatible encapsulation conditions for SMG cells. Cell viability was much higher after thiol-ene polymerizations compared with conventional methacrylate polymerizations due to reduced membrane peroxidation and intracellular reactive oxygen species formation. In addition, the formation of multicellular microspheres before encapsulation maximized cell-cell contacts and increased viability of SMG cells over 14-day culture periods. Thiol-ene hydrogel-encapsulated microspheres also promoted SMG proliferation. Lineage tracing was employed to determine the cellular composition of hydrogel-encapsulated microspheres using markers for acinar (Mist1) and duct (Keratin5) cells. Our findings indicate that both acinar and duct cell phenotypes are present throughout the 14 day culture period. However, the acinar:duct cell ratios are reduced over time, likely due to duct cell proliferation. Altogether, permissive encapsulation methods for primary SMG cells have been identified that promote cell viability, proliferation, and maintenance of differentiated salivary gland cell phenotypes, which allows for translation of this approach for salivary gland tissue engineering applications.

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“…For peptides containing tyrosine but no tryptophan, molar extinction coefficients at 274.5 nm were calculated assuming the value of 1340 M Ϫ1 cm Ϫ1 for each tyrosine residue. Finally, for peptides containing neither tyrosine nor tryptophan, molar extinction coefficients at 205 nm were calculated as described (35).…”

Section: Poly(a)mentioning

confidence: 99%

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

Kuzmenkov

Vassilevski

Kudryashova

et al. 2015

Journal of Biological Chemistry

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Background: Scorpion venoms are an ample source of toxins targeting potassium channels. Results: A comprehensive search for new toxins was performed by combining transcriptomics and peptidomics with a fluorescent test system. Conclusion: We identified five new high affinity potassium channel blockers in the venom of Mesobuthus eupeus. Significance: The proposed integrated approach is of general utility for potassium channel pharmacology.

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Sequence‐specific determination of protein and peptide concentrations by absorbance at 205 nm

Cited by 365 publications

References 22 publications

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments

Development of Poly(Ethylene Glycol) Hydrogels for Salivary Gland Tissue Engineering Applications

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

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