Abstract
We report an integrated workflow that allows mass spectrometry-based high-resolution hydroxyl radical protein footprinting (HR-HRPF) measurements to accurately measure the absolute average solvent accessible surface area (<SASA>) of amino acid side chains. This approach is based on application of multi-point HR-HRPF, electron-transfer dissociation (ETD) tandem MS (MS/MS) acquisition, measurement of effective radical doses by radical dosimetry, and proper normalization of the inherent reactivity of the amino acids. The accuracy of the resulting <SASA> measurements was tested by using well-characterized protein models. Moreover, we demonstrated the ability to use <SASA> measurements from HR-HRPF to differentiate molecular models of high accuracy (<3 Å backbone RMSD) from models of lower accuracy (>4 Å backbone RMSD). The ability of <SASA> data from HR-HRPF to differentiate molecular model quality was found to be comparable to that of <SASA> data obtained from X-ray crystal structures, indicating the accuracy and utility of HR-HRPF for evaluating the accuracy of computational models.
Highlights
A variety of new methods in HRPF allow for high resolution maps of changes in protein topography to be made with many amino acids probed in a single experiment
We introduce a method for generating accurate side chain absolute values for a wide variety of amino acids using Fast Photochemical Oxidation of Proteins (FPOP) high-resolution hydroxyl radical protein footprinting (HR-HRPF) data by integrating a variety of technological improvements for FPOP previously described by our group, as well as by developing improved methods to normalize for inherent amino acid reactivity
In order to meet this goal, several obstacles needed to be addressed: (1) HR-HRPF apparent oxidation rates must be accurately measured at the amino acid level; (2) the measured oxidation must be normalized by the concentration of radical generated and the scavenging properties of the solution; (3) the inherent reactivity of the amino acid in its sequence context must be accurately measured or estimated; (4) the quantitative relationship between normalized amino acid reactivity and must be established
Summary
A variety of new methods in HRPF allow for high resolution maps of changes in protein topography to be made with many amino acids probed in a single experiment. Using the technologies described here, we present data using the model protein system lysozyme demonstrating that we can clearly differentiate between high-quality molecular models (4 Å backbone RMSD) based on the agreement of the model’s values with those measured experimentally using HR-HRPF With these advances, the generation of accurate molecular models of protein structure validated by MS HR-HRPF data becomes truly possible
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
