bims-resufa 2018-04-08 papers

Issue of 2018–04–08
five papers selected by
Josh Garlich, Apellis Pharmaceuticals

Free Radic Biol Med. 2018 Mar 28. pii: S0891-5849(18)30148-5. [Epub ahead of print]

A discrete role for alternative oxidase under hypoxia to increase nitric oxide and drive energy production.

Abhaypratap Vishwakarma, Aprajita Kumari, Luis A J Mur, Kapuganti Jagadis Gupta.

Alternative oxidase (AOX) is an integral part of the mitochondrial electron transport and can prevent reactive oxygen species (ROS) and nitric oxide (NO) production under non-stressed, normoxic conditions. Here we assessed the roles of AOX by imposing stress under normoxia in comparison to hypoxic conditions using AOX over expressing (AOX OE) and anti-sense (AOX AS) transgenic Arabidopsis seedlings and roots. Under normoxic conditions stress was induced with the defence elicitor flagellin (flg22). AOX OE reduced NO production whilst this was increased in AOX AS. Moreover AOX AS also exhibited an increase in superoxide and therefore peroxynitrite, tyrosine nitration suggesting that scavenging of NO by AOX can prevent toxic peroxynitrite formation under normoxia. In contrast, during hypoxia interestingly we found that AOX is a generator of NO. Thus, the NO produced during hypoxia, was enhanced in AOX OE and suppressed in AOX AS. Additionally, treatment of WT or AOX OE with the AOX inhibitor SHAM inhibited hypoxic NO production. The enhanced levels of NO correlated with expression of non-symbiotic haemoglobin, increased NR activity and ATP production. The ATP generation was suppressed in nia1,2 mutant and non symbiotic haemoglobin antisense line treated with SHAM. Taken together these results suggest that hypoxic NO generation mediated by AOX has a discrete role by feeding into the haemoglobin-NO cycle to drive energy efficiency under conditions of low oxygen tension.

Keywords: Alternative oxidase; Energy efficiency; Nitric oxide; Peroxynitrite; Superoxide

DOI: https://doi.org/10.1016/j.freeradbiomed.2018.03.045

Methods Mol Biol. 2018 ;1764 29-44

High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly.

Louie D Henderson, Morgan Beeby.

Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible. To illustrate the electron cryo-tomography workflow for visualizing protein complexes in situ, we describe our workflow of preparing samples, imaging, and image processing using Leginon for data collection, IMOD for image reconstruction, and PEET for subtomogram averaging.

Keywords: Electron cryo-tomography; Molecular machines; Protein self-assembly; Structural biology; Subtomogram averaging

DOI: https://doi.org/10.1007/978-1-4939-7759-8_2

Methods Mol Biol. 2018 ;1764 267-277

Observing the Assembly of Protein Complexes in Living Eukaryotic Cells in Super-Resolution Using refSOFI.

Fabian Hertel, Gary C H Mo, Peter Dedecker, Jin Zhang.

Few approaches are currently available that allow the detection of protein-protein interactions (PPIs) in super-resolution, and the observation of the assembly of protein complexes in living cells has been particularly challenging. We developed reconstituted fluorescence-based stochastic optical fluctuation imaging (refSOFI), which is based on bimolecular fluorescence complementation (BiFC) and SOFI, allowing us to detect protein complex assembly 30 min after the induction of complex formation. Here we describe how to use refSOFI to map the assembly of two proteins of interest into a complex within living cells at super-resolution.

Keywords: Bimolecular fluorescence complementation (BiFC); Protein complexes; Protein-protein interactions; Stochastic optical fluctuation imaging (SOFI); Super-resolution imaging

DOI: https://doi.org/10.1007/978-1-4939-7759-8_16

Methods Mol Biol. 2018 ;1764 3-27

Experimental Characterization of Protein Complex Structure, Dynamics, and Assembly.

Jonathan N Wells, Joseph A Marsh.

Experimental methods for the characterization of protein complexes have been instrumental in achieving our current understanding of the protein universe and continue to progress with each year that passes. In this chapter, we review some of the most important tools and techniques in the field, covering the important points in X-ray crystallography, cryo-electron microscopy, NMR spectroscopy, and mass spectrometry. Novel developments are making it possible to study large protein complexes at near-atomic resolutions, and we also now have the ability to study the dynamics and assembly pathways of protein complexes across a range of sizes.

Keywords: Cryo-electron microscopy; Mass spectrometry; NMR; Quaternary structure; Super-resolution microscopy; X-ray crystallography

DOI: https://doi.org/10.1007/978-1-4939-7759-8_1

Methods Mol Biol. 2018 ;1764 173-183

Structural Analysis of Protein Complexes by Cross-Linking and Mass Spectrometry.

Moriya Slavin, Nir Kalisman.

Cross-linking and mass spectrometry is used more and more for the structural analysis of large proteins and protein complexes. Although essentially a low-resolution method, it avoids the main drawbacks of established structural techniques. Particularly, it is largely insensitive to the inherent flexibility of the studied complexes and is applied under native conditions. It is also applicable to nearly every structural system. Therefore, cross-linking and mass spectrometry is the method of choice for elucidating the general architecture of protein complexes. Advances in instrumentation, techniques, and software now allow every lab that is working with proteins to apply the approach without much difficulty. The most specialized step in the workflow, the mass spectrometry measurement, can be done in most facilities that are performing standard proteomics. We detail here a step-by-step protocol of how to successfully apply the approach in collaboration with the mass spectrometry facility in your institution.

Keywords: Molecular machines; Protein architecture; Structural biology

DOI: https://doi.org/10.1007/978-1-4939-7759-8_11