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Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are multidomain transmembrane proteins, which facilitate the transport of various substances across cell membranes using energy derived from ATP hydrolysis. They are important drug targets since they mediate decreased drug susceptibility during pharmacological treatments. For the methylotrophic yeast Pichia pastoris, a model organism that is a widely used host for protein expression, the role and function of its ABC transporters is unexplored. In this work, we investigated the Pichia ABC-B transporter STE6-2p. Functional investigations revealed that STE6-2p is capable of transporting rhodamines in vivo and is active in the presence of verapamil and triazoles in vitro. A phylogenetic analysis displays homology among multidrug resistance (MDR) transporters from pathogenic fungi to human ABC-B transporters. Further, we present high-resolution single-particle electron cryomicroscopy structures of an ABC transporter from P. pastoris in the apo conformation (3.1 Å) and in complex with verapamil and adenylyl imidodiphosphate (AMP-PNP) (3.2 Å). An unknown density between transmembrane helices 4, 5, and 6 in both structures suggests the presence of a sterol-binding site of unknown function. [ABSTRACT FROM AUTHOR]

To reduce global greenhouse gas emissions associated with building energy use, owners of large building portfolios such as university campuses frequently rely on building energy models (BEM) to better understand potential costs and benefits of retrofits. Model development workflows that are designed for individual buildings require a level of effort that would be time and cost prohibitive to apply to such campuses which often include hundreds of diverse-use buildings. While smaller campuses can effectively utilize the traditional BEM approach to study retrofit scenarios, this option is therefore not feasible for larger campuses. Large universities have instead utilized a combination of statistical and spreadsheet models which may not fully capture the unique architectural features, programmatic requirements, and systems configurations of individual campus buildings. With the goal of overcoming these limitations, two separate urban energy models, that employed considerably different methodologies, were developed for the campus of the Massachusetts Institute of Technology to evaluate future energy scenarios with an appreciably smaller effort vis-à-vis developing building-by-building energy models. This study reviews these two models with regards to their setup and calibration effort, ability to model individual building energy use, and the accuracy in predicted savings from implementing a variety of retrofitting measures. The paper identifies both models’ strengths and limitations and suggests best practice procedures for administrators of other campuses interested in undergoing a similar exercise. [ABSTRACT FROM AUTHOR]

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies. [ABSTRACT FROM AUTHOR]

Abstract: Human Neuromedin U receptor 1 (hNmU-R1) is a member of G protein-coupled receptor family. For structural determination of hNmU-R1, the production of hNmU-R1 in milligram amounts is a prerequisite. Here we reported two different eukaryotic expression systems, namely, Semliki Forest virus (SFV)/BHK-21 and baculovirus/Spodoptera frugiperda (Sf9) cell systems for overproduction of this receptor. In the SFV-based expression system, hNmU-R1 was produced at a level of 5 pmol receptor/mg membrane protein and the yield could be further increased to 22 pmol receptor/mg membrane protein by supplementation with 2% dimethyl sulfoxide (DMSO). Around 8 pmol receptor/mg membrane protein could be achieved in baculovirus-infected Sf9 cells. The recombinant hNmU-R1 from SFV- and baculovirus-based systems was functional, with a K d value of [125I] NmU-23 (rat) similar to that from transiently transfected COS-7 cells, where hNmU-R1 was first identified. With the aid of 1% n-dodecyl-β-d-maltoside (LM)/0.25% cholesteryl hemisuccinate (CHS), the yield of functional hNmU-R1 could reach 80%. The recombinant receptor from Sf9 cells was purified to homogeneity. The specific binding of the purified receptor to [125I] NmU-23 (rat) indicated that the receptor is bioactive. This is the first report of successful solubilization and purification of hNmU-R1, and will enable functional and structural studies of the hNmU-R1. [Copyright &y& Elsevier]

• Analyzing prevailing workflow and requirements for urban building energy modeling (UBEM). • Categorizing UBEM applications into four main use cases via examples and summary case studies. • Outlining data sources, modeling approaches, and calibration methods for UBEM applications. • Introducing the minimum viable UBEM (MVU) concept. • Outlining potential and future development for UBEM applications. Urban building energy modeling (UBEM) is a bottom-up, physics-based approach to simulate the thermal performance of new or existing neighborhoods and cities. The field has flourished in recent years, creating increasingly robust urban data streams that lead from Geographical Information Systems, CityGML, Light Detection and Ranging, and tax assessor databases to synthetic hourly building energy demand profiles for current and potential future conditions. Depending on the availability of historic building energy use data, a variety of modeling, simulation, and calibration approaches as well as use cases have been proposed. This paper provides a consolidated UBEM workflow with data and process requirements, and organizes UBEM proposals into four main application categories: urban planning and new neighborhood design, stock-level carbon reduction strategies, individual building-level recommendations, and buildings-to-grid integration. For each application, the paper further introduces a minimum viable UBEM, a novel concept for UBEMs conceptualized in the spirit of a minimum viable product. Specific case studies and/or examples are listed for each application area where available. [ABSTRACT FROM AUTHOR]

Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.