βB1-Crystallin: Thermodynamic Profiles of Molecular Interactions

β-Crystallins are structural proteins maintaining eye lens transparency and opacification. Previous work demonstrated that dimerization of both βA3 and βB2 crystallins (βA3 and βB2) involves endothermic enthalpy of association (∼8 kcal/mol) mediated by hydrophobic interactions.Thermodynamic profiles of the associations of dimeric βA3 and βB1 and tetrameric βB1/βA3 were measured using sedimentation equilibrium. The homo- and heteromolecular associations of βB1 crystallin are dominated by exothermic enthalpy (-13.3 and -24.5 kcal/mol, respectively).Global thermodynamics of βB1 interactions suggest a role in the formation of stable protein complexes in the lens via specific van der Waals contacts, hydrogen bonds and salt bridges whereas those β-crystallins which associate by predominately hydrophobic forces participate in a weaker protein associations

Pichia pastoris is a valuable host for the expression of genes encoding membrane proteins from the hyperthermophilic Archeon Pyrococcus abyssi

We present here the experimental strategies, first results and identified bottlenecks of a structural genomics initiative on membrane proteins of the hyperthermophilic archaea Pyrococcus abyssi. Five ORFs coding for putative membrane proteins have been cloned and expressed in the methylotrophic Pichia pastoris expression system, using two different constructs, with or without the signal sequence alpha-mating factor of Saccharomyces cerevisiae. A c-myc epitope and 6 His codons were added at the 3'-end of the targeted genes to allow immunodetection of the recombinant proteins and to facilitate their further purification. We have selected at least one producer clone for each protein of interest and for almost every construction. All the membrane proteins were produced in Erlenmeyer flasks culture and in fed-batch cultivation for large-scale preparation. The proteins were detected in the membrane fractions of P. pastoris. Production efficiencies were relatively low in both production conditions but the quantities of biomass obtained during fed-batch cultivation have allowed us to collect sufficient amount of material for further purification. The proteins were extracted, solubilized and partially purified. Large-scale purification will be necessary for further structural work

Sub1/PC4, a multifaceted factor: from transcription to genome stability

Yeast Sub1 and human PC4, two DNA-binding proteins, were originally identified as transcriptional coactivators with a role during transcription preinitiation/initiation. Indeed, Sub1 is a PIC component, and both PC4 and Sub1 also influence the initiation-elongation transition. Moreover, in the specific case of Sub1, it has been clearly reported that it influences processes downstream during mRNA biogenesis, such as transcription elongation, splicing and termination, and even RNAPII phosphorylation/dephosphorylation. Although Sub1 mechanism of action has been mostly unknown up to date, thanks to the recent finding that Sub1 directly interacts with the RNAPII stalk domain, we can envision how it can modulate so many processes. In addition, Sub1 and PC4 participate in RNAPIII transcription as well, and much additional evidence indicates an evolutionarily conserved role for Sub1 and PC4 in the maintenance of genome stability. In this regard, the most novel function of Sub1 and PC4 has been related to the ability of these proteins to bind G-quadruplex DNA structures that may arise as a consequence of the transcription process.OC. acknowledges the Spanish Ministry of Economy and Competitiveness [MINECO; (BFU2013-48374-P)] for funding.Peer Reviewe