ONEST: a web-based platform for the rapid and robust analysis of protein excited states through CEST spectroscopy

<h4>Summary</h4>The biological function of proteins is often driven by "invisible" excited states-transient, low-population conformations that remain undetectable by conventional structural methods. Chemical Exchange Saturation Transfer (CEST) NMR spectroscopy is a powerful technique for characterizing these states; however, the complexity of data analysis and the computational cost of numerical fitting have hindered its widespread adoption. To address these challenges, we present ONEST (Optimized Novel Exchange Saturation Transfer), a user-friendly web server designed to automate and accelerate CEST analysis. ONEST utilizes a simultaneous multi-field fitting algorithm that leverages exact analytical solutions for two-state exchange (Baldwin model) rather than computationally intensive numerical integration. This approach incorporates a rigorous correction for radio-frequency (RF) field inhomogeneity and resolves parameter degeneracy by jointly fitting datasets acquired at distinct RF field strengths. Validation against synthetic datasets yielded reduced c2 values near unity (∼1.05), confirming that the analytical approach recovers kinetic parameters with accuracy comparable to full Bloch-McConnell simulations but at a fraction of the computational cost. Furthermore, application to the anti-HIV lectin OAA successfully characterized slow conformational exchange (kex = 279 s-1) involving a minor population of 3%. By streamlining the extraction of kinetic and thermodynamic parameters, ONEST significantly lowers the technical barrier to entry, enabling a broader range of researchers to investigate protein dynamics at atomic resolution.<h4>Contact</h4>[email protected].<h4>Availability and implementation</h4>ONEST is available through the web server at http://onest.ai.kr.