It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and ...structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.
The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and ...tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions.
A cargo-sorting DNA robot Thubagere, Anupama J.; Li, Wei; Johnson, Robert F. ...
Science (American Association for the Advancement of Science),
09/2017, Letnik:
357, Številka:
6356
Journal Article
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Two critical challenges in the design and synthesis of molecular robots are modularity and algorithm simplicity. We demonstrate three modular building blocks for a DNA robot that performs cargo ...sorting at the molecular level. A simple algorithm encoding recognition between cargos and their destinations allows for a simple robot design: a single-stranded DNA with one leg and two foot domains for walking, and one arm and one hand domain for picking up and dropping off cargos. The robot explores a two-dimensional testing ground on the surface of DNA origami, picks up multiple cargos of two types that are initially at unordered locations, and delivers them to specified destinations until all molecules are sorted into two distinct piles. The robot is designed to perform a random walk without any energy supply. Exploiting this feature, a single robot can repeatedly sort multiple cargos. Localization on DNA origami allows for distinct cargo-sorting tasks to take place simultaneously in one test tube or for multiple robots to collectively perform the same task.
Abstract
Mechanically stable specific heterodimerization between small protein domains have a wide scope of applications, from using as a molecular anchorage in single-molecule force spectroscopy ...studies of protein mechanics, to serving as force-bearing protein linker for modulation of mechanotransduction of cells, and potentially acting as a molecular crosslinker for functional materials. Here, we explore the possibility to develop heterodimerization system with a range of mechanical stability from a set of recently engineered helix-heterotetramers whose mechanical properties have yet to be characterized. We demonstrate this possibility using two randomly chosen helix-heterotetramers, showing that their mechanical properties can be modulated by changing the stretching geometry and the number of interacting helices. These helix-heterotetramers and their derivatives are sufficiently stable over physiological temperature range. Using it as mechanically stable anchorage, we demonstrate the applications in single-molecule manipulation studies of the temperature dependent unfolding and refolding of a titin immunoglobulin domain and α-actinin spectrin repeats.
The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. Although there have been advances in the de novo design of ...stable proteins with a single, deep free-energy minimum, the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.
PurposeThe purpose of this study is to discuss the principles and factors that influence the site selection of emergency medical facilities for public health emergencies. This paper discusses the ...selection of the best facilities from the available facilities, proposes the capacity of new facilities, presents a logistic regression model and establishes a site selection model for emergency medical facilities for public health emergencies in megacities.Design/methodology/approachUsing Guangzhou City as the research object, seven alternative facility points and the points' capacities were preset. Nine demand points were determined, and two facility locations were selected using genetic algorithms (GAs) in MATLAB for programing simulation and operational analysis.FindingsComparing the results of the improved GA, the results show that the improved model has fewer evolutionary generations and a faster operation speed, and that the model outperforms the traditional P-center model. The GA provides a theoretical foundation for determining the construction location of emergency medical facilities in megacities in the event of a public health emergency.Research limitations/implicationsFirst, in this case study, there is no scientific assessment of the establishment of the capacity of the facility point, but that is a subjective method based on the assumption of the capacity of the surrounding existing hospitals. Second, because this is a theoretical analysis, the model developed in this study does not consider the actual driving speed and driving distance, but the speed of the unified average driving distance and the driving distance to take the average of multiple distances.Practical implicationsThe results show that the method increases the selection space of decision-makers, provides them with stable technical support, helps them quickly determine the location of emergency medical facilities to respond to disaster relief work and provides better action plans for decision makers.Social implicationsThe results show that the algorithm performs well, which verifies the applicability of this model. When the solution results of the improved GA are compared, the results show that the improved model has fewer evolutionary generations, faster operation speed and better model than the intermediate model GA. This model can more successfully find the optimal location decision scheme, making that more suitable for the location problem of megacities in the case of public health emergencies.Originality/valueThe research findings provide a theoretical and decision-making basis for the location of government emergency medical facilities, as well as guidance for enterprises constructing emergency medical facilities.
De novo design of protein logic gates Chen, Zibo; Kibler, Ryan D; Hunt, Andrew ...
Science (American Association for the Advancement of Science),
04/2020, Letnik:
368, Številka:
6486
Journal Article
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The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, ...NOR, XNOR, and NOT gates built from de novo-designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the
gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions.
Protein-based biological circuits enable customized control of cellular functions, and de novo protein design enables circuit functionalities that are not possible by repurposing natural proteins. ...Here, I highlight recent progress in protein circuit design, including CHOMP, developed by Gao et al., and SPOC, developed by Fink et al.
Carbon nanotubes (CNTs), which exhibit stable surfaces under acidic, neutral and alkaline solutions, provide an ideal platform for studying the nature of oxygen evolution reaction (OER) mechanisms at ...different pH. Here, OER on four types of pristine carbon nanotubes (CNTs) with walls from single-walled, double-walled, three-walled to multi-walled were studied in acid, neutral and alkaline conditions. Mechanism and kinetics study reveals that the OER on all CNTs is constrained by the water deprotonation in acid and neutral conditions,resulting in high Tafel slopes close to 240 mV dec−1, high overpotentials (0.7–1 V), and approaching zeroth-reaction-order to H+/OH−. The kinetics and mechanism shift at pH ∼9–10 due to competition discharge between H2O and OH−. The Tafel slopes decrease, and the reaction orders increase with the increase of OH− concentration. The higher kinetics and enhanced activities for CNTs with 2–4 walls support the proposed tunneling effect, highlighting the favourable electron transfer pathway between the outer wall and inner tubes. The finding will provide a new direction for designing highly efficient OER catalysts.
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Sorafenib is the first-line treatment for advanced HCC, but the anti-cancer effects remain to be ...improved as indicated by its low response rates and failure to prolong the progression-free survival (PFS). Thus, it is urgent to explore approaches to improve the clinical outcome.
The effect of Sorafenib in HCC was analyzed by SRB (sulforhodamine B) assay in normoxia and hypoxia, respectively. The different dose combination effect of CT707 and sorafenib was analyzed by SRB assay in hypoxia. Flow cytometry assay was used to detect the cell apoptosis rate with CT707 and sorafenib treatment in hypoxia. Western blotting was used to detect the expression levels of apoptosis -related proteins and the mechanism of CT707 overcome the resistance of sorafenib in hypoxia.
Our study showed that the characteristic intratumor hypoxia of advanced HCC is one of the major factors which mediated the drug resistance towards sorafenib in HCC. And CT-707, a novel multi-kinase inhibitor, could sensitize the hypoxic HCC cells towards sorafenib. Further studies showed that CT-707 abolished the nuclear translocation of Yes Associate-Protein (YAP), which has been demonstrated as one of mechanism of hypoxia-mediated sorafenib-resistance in HCC.
Overall, this study not only favors the development of this novel multi-kinase inhibitor CT-707 as a therapeutic agent against HCC, but also provides a potential strategy to overcome the hypoxia-mediated resistance to sorafenib in HCC patients.
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