A general path for large-scale solubilization of cellular proteins: From membrane receptors to multiprotein complexes

Pullara, Filippo; Guerrero-Santoro, Jennifer; Calero, Monica; Zhang, Qiangmin; Peng, Ye; Spåhr, Henrik; Kornberg, Guy; Cusimano, Antonella; Stevenson, Hilary P.; Santamaría-Suárez, Hugo A.; Reynolds, Shelley; Brown, Ian; Monga, Satdarshan P.; Houten, Bennett Van; Rapić-Otrin, Vesna; Calero, Guillermo; Levine, Arthur S.

doi:10.1016/j.pep.2012.10.007

Cited by 19 publications

(14 citation statements)

References 37 publications

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“…1a). This extended decay is intrinsically related to the presence of larger protein aggregates in solution (Pullara et al, 2013). However, the presence of an electric field (E F of 10 V •cm − 1 ) during heating seemed to quench emergence of these large aggregates.…”

Section: Resultsmentioning

confidence: 98%

Development of iron-rich whey protein hydrogels following application of ohmic heating – Effects of moderate electric fields

Pereira

Rodrigues

Altınok

et al. 2017

Food Research International

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The influence that ohmic heating technology and its associated moderate electric fields (MEF) have upon production of whey protein isolate cold-set gels mediated by iron addition was investigated. Results have shown that combining heating treatments (90°C, 5min) with different MEF intensities let hydrogels with distinctive micro and macro properties - i.e. particle size distribution, physical stability, rheological behavior and microstructure. Resulting hydrogels were characterized (at nano-scale) by an intensity-weighted mean particle diameter of 145nm, a volume mean of 240nm. Optimal conditions for production of stable whey protein gels were attained when ohmic heating treatment at a MEF of 3V∙cm was combined with a cold gelation step using 33mmol∙L of Fe. The consistency index of hydrogels correlated negatively to MEF intensity, but a shear thickening behavior was observed when MEF intensity was increased up to 10V∙cm. According to transmission electron microscopy, ohmic heating gave rise to a more homogenous and compact fine-stranded whey protein-iron microstructure. Ohmic heating appears to be a promising technique, suitable to tailor properties of whey protein gels and with potential for development of innovative functional foods.

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Section: Resultsmentioning

confidence: 98%

Development of iron-rich whey protein hydrogels following application of ohmic heating – Effects of moderate electric fields

Pereira

Rodrigues

Altınok

et al. 2017

Food Research International

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“…The resulting nucleic acid scaffolds were mixed with Pol II (3:1 molar ratio) and excess scaffold was removed using size exclusion chromatography (Superdex200, GE LifeSciences) against Buffer A (25 mM Hepes pH 7.5, 100 mM KCl, 5 mM DTT, 0.5 mM EDTA, 10 µM ZnCl 2 ). Purification and assembly of Pol II-TC and Δ4/7-TC was achieved as previously described (Pullara et al, 2013). An SDS-PAGE of the final complex is illustrated in Fig.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble

et al. 2015

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Summary Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop-1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the Trigger Loop (TL), allowing visualization of its open state. Overall, our observations suggest that “open/closed” conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.

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“…The Intein tag was cleaved off while bound to the chitin column during purification. His-rAlx1 was processed according to previous published methods to achieve maximum solubility (Pullara et al, 2013). The protein was eluted, concentrated and desalted in 300 mM NaCl, 50 mM Tris pH 6.8, 0.1% Triton X-100 and Roche cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, 11836153001).…”

Section: Electrophoretic Mobility Shift Assaymentioning

confidence: 99%

Genome-wide identification of binding sites and gene targets of Alx1, a pivotal regulator of echinoderm skeletogenesis

2019

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Alx1 is a conserved regulator of skeletogenesis in echinoderms and evolutionary changes in Alx1 sequence and expression have played a pivotal role in modifying programs of skeletogenesis within the phylum. Alx1 regulates a large suite of effector genes that control the morphogenetic behaviors and biomineral-forming activities of skeletogenic cells. To better understand the gene regulatory control of skeletogenesis by Alx1, we used genome-wide ChIP-seq to identify Alx1-binding sites and direct gene targets. Our analysis revealed that many terminal differentiation genes receive direct transcriptional inputs from Alx1. In addition, we found that intermediate transcription factors previously shown to be downstream of Alx1 all receive direct inputs from Alx1. Thus, Alx1 appears to regulate effector genes by indirect, as well as direct, mechanisms. We tested 23 high-confidence ChIP-seq peaks using GFP reporters and identified 18 active cisregulatory modules (CRMs); this represents a high success rate for CRM discovery. Detailed analysis of a representative CRM confirmed that a conserved, palindromic Alx1-binding site was essential for expression. Our work significantly advances our understanding of the gene regulatory circuitry that controls skeletogenesis in sea urchins and provides a framework for evolutionary studies.

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A general path for large-scale solubilization of cellular proteins: From membrane receptors to multiprotein complexes

Cited by 19 publications

References 37 publications

Development of iron-rich whey protein hydrogels following application of ohmic heating – Effects of moderate electric fields

Development of iron-rich whey protein hydrogels following application of ohmic heating – Effects of moderate electric fields

Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble

Genome-wide identification of binding sites and gene targets of Alx1, a pivotal regulator of echinoderm skeletogenesis

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