High resolution RAMAN in membrane protein research

Membrane proteins facilitate the passage of various molecules across cell membranes, physical barriers made of lipids that encapsulate either the whole cell or individual cell compartments such as organelles or vacuoles. The composition of these bio-membranes is spatially and temporally heterogeneous and they not only form a physical barrier in which highly selective and specialized proteins are embedded, but their composition has also been shown to regulate the function, stability and structure of these proteins. Despite their importance, most methods used to study the structure and stability of membrane proteins neglect the effects of the lipid bilayer. Raman spectroscopy allows the investigation of the complete chemical structure of biological molecules under physiological conditions and thus provides a "fingerprint" of all chemical bonds of the lipid bilayer, their components and their interaction under physiological conditions. With this method, membrane proteins in lipid membranes will be examined either in solution or in the vicinity of a membrane support in order to elucidate structures of membrane proteins and structural changes within an intact lipid bilayer in a time- and cost-efficient manner. More than half of the population worldwide is infected with Helicobacter pylori, a pathogen that colonizes the gastric mucus or the gastric mucosa. In many patients, the infection leads to gastritis or a duodenal ulcer. The urea channel HpUreI of H. pylori is located inside two membranes that surround the bacterium. In the acidic environment of the stomach, the channel opens and transports gastric urea from the compartment between the two membranes into the interior of the cell. There, urea is split into ammonia and carbon dioxide by an enzyme, the urease. These two substances, in turn, neutralize the acidic environment, creating a viable microenvironment for the pathogen. In the project, UreI, a small pH-controlled urea channel consisting of six identical subunits, representing the "life insurance" of H. pylori, is overexpressed and purified. Raman spectra of UreI will be recorded pH-dependently in and without the lipid network and will lead to unique insights into how the opening and closing behavior is influenced or controlled by the lipid bilayer or specific lipid interactions. This results in important insights for the research efforts to specifically switch off HpUreI with drugs. A potential strategy to eliminate H. pylori as a replacement for standard therapies that must cope with increasing antibiotic resistance. The project is carried out in cooperation between the project leader Assoc. Prof. Dr. Andreas Horner (Johannes Kepler University Linz, Institute for Biophysics) and co-project leader Univ.-Prof. Dr. Sabine Hild (Johannes Kepler University Linz, Institute for Polymer Science).

Helicobacter pylori (Hp) is a bacterium that chronically infects the stomachs of more than half the world's population. It is a leading cause of gastric diseases, including ulcers and stomach cancer. Current treatments, which combine acid-reducing medications and antibiotics, are losing effectiveness as Hp increasingly develops antibiotic resistance. There is an urgent need for new therapeutic strategies. A key to Hp's survival in the stomach's harsh, acidic environment (as low as pH 1) is the protein HpUreI, an acid-gated channel in its inner membrane. HpUreI allows urea to enter the bacterial cell, where it is converted into ammonia and carbon dioxide to neutralize the surrounding acid. Without this protein, Hp cannot survive in the stomach. Understanding how HpUreI opens and closes in response to acidity and its environment is critical for designing new treatments, but until now, this mechanism has remained unclear. In this study, we employed Raman spectroscopy, a laser-based technique that enables the characterization of protein chemical structure under near-native conditions. This method is particularly valuable because fast, reliable tools for studying membrane proteins in physiologically relevant environments are rare. Our results identified distinct changes in the protein's Raman signal at specific spectral positions. These changes cannot be attributed solely to individual amino acids gaining or losing protons due to shifts in acidity. Instead, they indicate larger conformational shifts in the protein's overall structure as the channel opens and closes in response to pH. This work demonstrates that Raman spectroscopy can provide real-time structural insights into membrane proteins, even under challenging conditions. It paves the way for using vibrational spectroscopy to explore how factors like the lipid membrane environment or buffer conditions affect protein structure and function. Ultimately, these insights could inform the development of novel therapies that disrupt Hp's ability to thrive in the stomach, offering a potential solution to the growing problem of antibiotic resistance.