Alkaliphiles thrive in environments with a pH of 8.5 or above, while maintaining an internal pH closer to neutral. Thus, alkaliphilic microorganisms have a proton gradient inverted with respect to the normal orientation. Intuitively, this would nullify the potential to generate energy via respiration with regularly oriented respiratory chains that rely on proton-coupled ATP synthases. Yet, alkaliphilic respiratory chains are oriented traditionally and are actively used. The question therefore is how they are able to create conditions conducive to such behaviour. In addition, attempts to answer that question will hopefully also clarify how alkaliphiles acidify their cytoplasm with respect to the exterior milieu in the first place. This thesis details methods required to study these questions and provides some answers regarding alkaliphilic life. This thesis focuses on a single category of alkaliphiles: the low-salt gram positive alkaliphiles. These microbes have just a single membrane, the proteins therein, and a cell wall to generate conditions suitable for energy generation and other transport mechanisms. In short, it can be regarded as the most basic system to study an alkaline, or basic, problem....

The rise of multi-drug resistant bacterial infections is a worldwide growing concern. Despite being one of the greatest medical advances of the 20th century, classical antibiotics are no longer effective against such organisms. Unfortunately, patients infected with drug-resistant bacteria are more likely to experience worse clinical outcomes, as well as have higher mortality rates. The bacterial respirasome is one of the most promising target spaces in the search for novel antibiotics, since many pathogenic organisms display unique respiratory adaptations: e.g., a change in the pathogens’ metabolism upon infection of the host. More importantly, these adaptations are often essential for the cell viability of the pathogen, what makes them excellent candidates for targeted inhibition.
These drug targets frequently are membrane-bound respiratory proteins. For this reason, they have a significantly different biochemical and biophysical behavior than that of cytoplasmic or other water-soluble proteins, and so they are notoriously difficult to study. Most remarkably, membrane proteins are amphipathic, interacting with the aqueous environment, but also surrounded by a highly hydrophobic environment. Lipid composition is extremely complex: lipids of different polarities, hydrophobic electron carriers, etc. Altogether, a multicomponent system that ought to be considered if we aim to design efficient drugs that will interact with these enzymes (i.e., inhibit them) in their native environments.

Structural and functional characterization of membrane proteins (MPs) requires extraction from the native lipid bilayer, which generally necessitates the formation of water-soluble MPs structures or complexes suitable for biochemical assays. These structures are often detergent micelles that form protein-detergent complexes (PDCs), due to detergents being straightforward
in implementation and providing homogenous yields. However, obtaining physiologically relevant PDCs remains a challenge, because most detergents do not keep the native structure/function of the protein intact, jeopardizing its stability, and disrupting the proton pressure and electric gradient even after reincorporation in artificial lipid membranes. To achieve native-like
physiology (i.e. to maintain native protein-associated lipids and structurally relevant ligands or cofactors), it is essential to choose the right extraction parameters. Nevertheless, the field of membrane biochemistry is still missing a holistic, interdependent understanding of these systems, too often neglecting the evident, key role that detergents play. In this review, we aim to obtain a more fundamental understanding of detergent-solubilized membrane proteins by pinpointing the most relevant assay parameters (i.e. the most influential on the biochemical properties) involving MPs.