We present RASP, a method that derives protein backbone resonance assignments from chemical shift predictions based on known protein structure. The ambiguities arising from imprecise predictions are largely resolved by exploiting the sequential information available in the triple resonance experiments conventionally used for backbone assignment. Using this method, robust assignments can be obtained from a minimal set of only the most sensitive triple-resonance experiments, even for spectroscopically challenging proteins. Over a test set of 154 proteins RASP assigns 88 % of residues with an accuracy of 99.7 %, using only information available from HNCO and HNCA spectra. Tested on experimental data from a challenging 34 kDa protein, RASP assigns 90 % of manually assigned residues using only 40 % of the experimental data used for the manual assignment.
We also describe the application of RASP to Apical Membrane Antigen 1 (AMA1), a protein found on the surface of Plasmodium and related parasites. AMA1 is an essential component of the tight junction formed when these parasites invade host cells, and is a target for the development of novel anti-malarial therapies.1 Despite extensive effort, functional constructs derived from the AMA1 ectodomain have to date resisted assignment by conventional means.2,3,4 Using RASP we have assigned a 38 kDa AMA1 construct that binds its physiological ligand, as well as several characterised inhibitors. These assignments will enable the structural characterisation of small-molecule inhibitors currently under development.5
RASP has the potential to significantly accelerate the backbone assignment process for a wide range of proteins for which structural information is available, including those for which conventional assignment strategies are not feasible.