The biogenesis of mitochondria relies on the import of hundreds of different precursor proteins from the cytosol. Most of these proteins are synthesized with N-terminal presequences which serve as mitochondrial targeting signals. Presequences consistently form amphipathic helices, but they considerably differ with respect to their primary structure and length. Here we show that presequences can be classified into seven different groups based on their specific features. Using a test set of different presequences, we observed that group A presequences endow precursor proteins with improved in vitro import characteristics. We developed IQ-Compete (for Import and de-Quenching Competition assay), a novel assay based on fluorescence de-quenching, to monitor the import efficiencies of mitochondrial precursors in vivo. With this assay, we confirmed the increased import competence of group A presequences. Using mass spectrometry, we found that the presequence of the group A protein Oxa1 specifically recruits the tetratricopeptide repeat (TPR)-containing protein TOMM34 to the cytosolic precursor protein. TOMM34, and the structurally related yeast co-chaperone Cns1, apparently serve as presequence-specific targeting factors which increases the import efficiency of a specific subset of mitochondrial precursor proteins. Our results suggest that presequences contain a protein-specific priority code that encrypts the targeting mechanism of individual mitochondrial precursor proteins.

• MeSH
• Nuclear Proteins MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondrial Proteins * metabolism   genetics   MeSH
• Mitochondria * metabolism   MeSH
• Protein Precursors metabolism   MeSH
• Electron Transport Complex IV MeSH
• Saccharomyces cerevisiae Proteins * metabolism   genetics   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Protein Transport MeSH
• Mitochondrial Membrane Transport Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nuclear Proteins MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondrial Proteins * MeSH
• OXA1 protein MeSH Browser
• Protein Precursors MeSH
• Electron Transport Complex IV MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• Mitochondrial Membrane Transport Proteins MeSH

In proteomics, postproline cleaving enzymes (PPCEs), such as Aspergillus niger prolyl endopeptidase (AnPEP) and neprosin, complement proteolytic tools because proline is a stop site for many proteases. But while aiming at using AnPEP in online proteolysis, we found that this enzyme also displayed specificity to reduced cysteine. By LC-MS/MS, we systematically analyzed AnPEP sources and conditions that could affect this cleavage preference. Postcysteine cleavage was blocked by cysteine modifications, including disulfide bond formation, oxidation, and alkylation. The last modification explains why this activity has remained undetected so far. In the same experimental paradigm, neprosin mimicked this cleavage specificity. Based on these findings, PPCEs cleavage preferences should be redefined from post-Pro/Ala to post-Pro/Ala/Cys. Moreover, this evidence demands reconsidering PPCEs applications, whether cleaving Cys-rich proteins or assessing Cys status in proteins, and calls for revisiting the proposed enzymatic mechanism of these proteases.

• Publication type
• Journal Article MeSH

Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

• Keywords
• Antibody, Biosimilars, Hydrogen/deuterium exchange, Mass spectrometry, Protein structure and dynamics, Proteolysis,
• MeSH
• Deuterium MeSH
• Mass Spectrometry MeSH
• Antibodies * MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry * MeSH
• Hydrogen MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Antibodies * MeSH
• Hydrogen MeSH

Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no limitations in terms of protein size, flexibility, or reaction conditions and can thus be performed in solution at different pH values and temperatures under controlled redox conditions. Thanks to its coupling with mass spectrometry (MS), it is also straightforward to perform and has relatively high throughput, making it an excellent complement to the high-resolution methods of structural biology. Given the recent expansion of artificial intelligence-aided protein structure modeling, there is considerable demand for techniques allowing fast and unambiguous validation of in silico predictions; HDX-MS is well-placed to meet this demand. Here we present a protocol for HDX-MS and illustrate its use in characterizing the dynamics and structural changes of a dimeric heme-containing oxygen sensor protein as it responds to changes in its coordination and redox state. This allowed us to propose a mechanism by which the signal (oxygen binding to the heme iron in the sensing domain) is transduced to the protein's functional domain.

• Keywords
• Globin-coupled histidine kinase, Heme-containing oxygen sensors, Hydrogen/deuterium exchange, Ligand binding, Mass spectrometry, Protein conformational dynamics, Signal transduction,
• MeSH
• Deuterium MeSH
• Heme chemistry   MeSH
• Hemeproteins * MeSH
• Mass Spectrometry methods   MeSH
• Oxygen metabolism   MeSH
• Artificial Intelligence MeSH
• Deuterium Exchange Measurement methods   MeSH
• Hydrogen chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Heme MeSH
• Hemeproteins * MeSH
• Oxygen MeSH
• Hydrogen MeSH