Detection of Known and Novel Small Proteins in Using a Combination of Bottom-Up and Digest-Free Proteomics and Proteogenomics.

Small proteins of around 50 aa in length have been largely overlooked in genetic and biochemical assays due to the inherent challenges with detecting and characterizing them. Recent discoveries of their critical roles in many biological processes have led to an increased recognition of the importance of small proteins for basic research and as potential new drug targets. One example is CcoM, a 36 aa subunit of the -type oxidase that plays an essential role in adaptation to oxygen-limited conditions in , a model for the clinically relevant, opportunistic pathogen .

Insights into the sulfur metabolism of Chlorobaculum tepidum by label-free quantitative proteomics.

Chlorobaculum tepidum is an anaerobic green sulfur bacterium which oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. It can also oxidize sulfide to produce extracellular S globules, which can be further oxidized to sulfate and used as an electron donor. Here, we performed label-free quantitative proteomics on total cell lysates prepared from different metabolic states, including a sulfur production state (10 h post-incubation [PI]), the beginning of sulfur consumption (20 h PI), and the end of sulfur consumption (40 h PI), respectively.

Essential protein P116 extracts cholesterol and other indispensable lipids for Mycoplasmas.

Mycoplasma pneumoniae, responsible for approximately 30% of community-acquired human pneumonia, needs to extract lipids from the host environment for survival and proliferation. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213). Single-particle cryo-electron microscopy of P116 reveals a homodimer presenting a previously unseen fold, forming a huge hydrophobic cavity, which is fully accessible to solvent.

Isolation of a novel heterodimeric PSII complex via strep-tagged PsbO.

The multi-subunit membrane protein complex photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag.

Conformational changes in mitochondrial complex I of the thermophilic eukaryote .

Mitochondrial complex I is a redox-driven proton pump that generates proton-motive force across the inner mitochondrial membrane, powering oxidative phosphorylation and ATP synthesis in eukaryotes. We report the structure of complex I from the thermophilic fungus , determined by cryoEM up to 2.4-Å resolution.

Top-Down Identification and Sequence Analysis of Small Membrane Proteins Using MALDI-MS/MS.

Identification and sequence determination by mass spectrometry have become routine analyses for soluble proteins. Membrane proteins, however, remain challenging targets due to their hydrophobicity and poor annotation. In particular small membrane proteins often remain unnoticed as they are largely inaccessible to Bottom-Up proteomics.

Cysteine oxidation and disulfide formation in the ribosomal exit tunnel.

Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel.

Site-Specific Detection of Arginine Methylation in Highly Repetitive Protein Motifs of Low Sequence Complexity by NMR.

Post-translational modifications of proteins are widespread in eukaryotes. To elucidate the functional role of these modifications, detection methods need to be developed that provide information at atomic resolution. Here, we report on the development of a novel Arg-specific NMR experiment that detects the methylation status and symmetry of each arginine side chain even in highly repetitive RGG amino acid sequence motifs found in numerous proteins within intrinsically disordered regions.

Elucidating the control and development of skin patterning in cuttlefish.

Few animals provide a readout that is as objective of their perceptual state as camouflaging cephalopods. Their skin display system includes an extensive array of pigment cells (chromatophores), each expandable by radial muscles controlled by motor neurons. If one could track the individual expansion states of the chromatophores, one would obtain a quantitative description-and potentially even a neural description by proxy-of the perceptual state of the animal in real time.