¹²⁹Xe NMR-Protein Sensor Reveals Cellular Ribose Concentration

Current methods for measuring cellular ribose levels lack specificity and sensitivity. While ribose is a key metabolite in cellular processes, no reliable analytical tool exists for its quantification in complex biological matrices. Prior research has shown that ribose plays a role in energy metabolism and nucleotide synthesis. However, detecting it remains a challenge due to structural similarities with other sugars. Existing techniques often lack the resolution to distinguish ribose from related molecules like glucose. This gap motivated the development of a more precise method. The need for a tool that can quantify ribose without interference is critical. The development of a sensor based on ribose-binding proteins could address this limitation.

The study aimed to develop a novel ribose sensor using ribose-binding protein (RBP) and hyperpolarized 129Xe NMR. The goal was to create a tool capable of measuring ribose in complex biological samples with high specificity. The researchers focused on leveraging the conformational changes in RBP upon ribose binding. They sought to use these changes to detect ribose via NMR signal shifts. The study also aimed to test the sensor’s performance in real biological fluids. The team wanted to confirm that the sensor could distinguish ribose from other sugars. They aimed to demonstrate the sensor’s applicability in mammalian cell lysates and serum. The ultimate purpose was to provide a new analytical platform for metabolic studies.

The team engineered ribose-binding protein (RBP) to bind xenon gas. They used hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST) for detection. The RBP was modified to allow xenon binding in its closed conformation. The sensor was tested in various concentration ranges of ribose. The conformational change of RBP upon ribose binding was monitored via NMR. The researchers compared the sensor’s response to other sugars like glucose. They analyzed the sensor’s performance in mammalian cell lysates and serum. The study used a combination of protein engineering and NMR spectroscopy techniques.

The RBP sensor detected ribose concentrations from nM to mM with high specificity. The sensor’s signal was minimally affected by other sugars like glucose or ribose-6-phosphate. The hyper-CEST technique captured conformational changes in RBP upon ribose binding. The sensor’s response was linear across a wide concentration range. The study found low-mM ribose levels in HeLa cell lysates. The method showed minimal background interference from endogenous sugars. The sensor’s specificity was confirmed through comparative tests. The results suggest the sensor’s potential for use in biological fluids and tissues.

The study demonstrates the feasibility of using RBP as a ribose sensor via hyper-CEST NMR. The sensor’s specificity for ribose was confirmed through comparative analysis. The method’s ability to detect ribose in complex samples was validated. The researchers propose that this approach could be applied to other metabolites. The findings suggest that genetically encoded proteins can serve as analytical tools. The study highlights the potential for using periplasmic binding proteins in metabolic research. The authors suggest that this method could improve the accuracy of ribose quantification. They emphasize the importance of further testing in diverse biological contexts.