Structural biology

About: Structural biology is a research topic. Over the lifetime, 2206 publications have been published within this topic receiving 126070 citations.

Lipidation-independent vacuolar functions of Atg8 rely on its noncanonical interaction with a vacuole membrane protein

Abstract: The ubiquitin-like protein Atg8, in its lipidated form, plays central roles in autophagy. Yet, remarkably, Atg8 also carries out lipidation-independent functions in non-autophagic processes. How Atg8 performs its moonlighting roles is unclear. Here we report that in the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, the lipidation-independent roles of Atg8 in maintaining normal morphology and functions of the vacuole require its interaction with a vacuole membrane protein Hfl1 (homolog of human TMEM184 proteins). Crystal structures revealed that the Atg8-Hfl1 interaction is not mediated by the typical Atg8-family-interacting motif (AIM) that forms an intermolecular β-sheet with Atg8. Instead, the Atg8-binding regions in Hfl1 proteins adopt a helical conformation, thus representing a new type of AIMs (termed helical AIMs here). These results deepen our understanding of both the functional versatility of Atg8 and the mechanistic diversity of Atg8 binding.

Escherichia coli as host for membrane protein structure determination: a global analysis.

Abstract: The structural biology of membrane proteins (MP) is hampered by the difficulty in producing and purifying them. A comprehensive analysis of protein databases revealed that 213 unique membrane protein structures have been obtained after production of the target protein in E. coli. The primary expression system used was the one based on the T7 RNA polymerase, followed by the arabinose and T5 promoter based expression systems. The C41λ(DE3) and C43λ(DE3) bacterial mutant hosts have contributed to 28% of non E. coli membrane protein structures. A large scale analysis of expression protocols demonstrated a preference for a combination of bacterial host-vector together with a bimodal distribution of induction temperature and of inducer concentration. Altogether our analysis provides a set of rules for the optimal use of bacterial expression systems in membrane protein production.

An Approach To Prepare Membrane Proteins for Single‐Molecule Imaging

Abstract: Membrane proteins are under represented in the database of high-resolution structures obtained from X-ray crystallographic and NMR spectroscopic methods. [1] Furthermore, they often form large and sometimes transient supramolecular complexes. Alternative approaches such as cryo-electron microscopy (EM) and Atomic force microscopy (AFM) are essential tools that are able to provide complementary information on the structure–function relationship of membrane proteins embedded in the lipid membrane. Two approaches increase the resolution of these methods, 2D crystallization and single-particle reconstruction. In EM, crystals provide electron diffraction data which can increase resolution, whereas single-particle averaging can be used for proteins > 200 kDa. High-resolution AFM topographs can reveal structural details of single native membrane proteins but, as a prerequisite, the proteins must be adsorbed to atomically flat mica and densely packed in a membrane to restrict their lateral mobility. [2] Although averaging of single particles shows their common structures, selected examples can be used to characterize structural flexibility, variability, and conformational changes. [3] Many examples of AFM analysis of self-assembled monolayers on gold have been published, [4] and thiolipids have been developed to create membrane mimetic surfaces on gold. [5–7] However, these have not yet been combined as a means to image membrane

Isotope Signatures Allow Identification of Chemically Cross-Linked Peptides by Mass Spectrometry: A Novel Method to Determine Interresidue Distances in Protein Structures through Cross-Linking

Abstract: Knowledge of protein structures and protein−protein interactions is essential for understanding of biological processes. Recent advances in protein cross-linking and mass spectrometry (MS) have shown significant potential to contribute to this area. Here we report a novel method to rapidly and accurately identify cross-linked peptides based on their unique isotope signature when digested in the presence of H218O. This method overcomes the need for specially synthesized cross-linkers and/or multiple MS runs required by other techniques. We validated our method by performing a “blind” analysis of 5 proteins/complexes of known structure. Side chain repacking calculations using Rosetta show that 17 of our 20 positively identified cross-links fit the published atomic structures. The remaining 3 cross-links are likely due to protein aggregation. The accuracy and rapid throughput of our workflow will advance the use of protein cross-linking in structural biology.