Pulsed electron beam water radiolysis for submicrosecond hydroxyl radical protein footprinting.
Hydroxyl radical footprinting is a valuable technique for studying protein structure, but care must be taken to ensure that the protein does not unfold during the labeling process due to oxidative damage. Footprinting methods based on submicrosecond laser photolysis of peroxide that complete the labeling process faster than the protein can unfold have been recently described; however, the mere presence of large amounts of hydrogen peroxide can also cause uncontrolled oxidation and minor conformational changes. We have developed a novel method for submicrosecond hydroxyl radical protein footprinting using a pulsed electron beam from a 2 MeV Van de Graaff electron accelerator to generate a high concentration of hydroxyl radicals by radiolysis of water. The amount of oxidation can be controlled by buffer composition, pulsewidth, dose, and dissolved nitrous oxide gas in the sample. Our results with ubiquitin and beta-lactoglobulin A demonstrate that one submicrosecond electron beam pulse produces extensive protein surface modifications. Highly reactive residues that are buried within the protein structure are not oxidized, indicating that the protein retains its folded structure during the labeling process. Time-resolved spectroscopy indicates that the major part of protein oxidation is complete in a time scale shorter than that of large scale protein motions.
Watson
Caroline
C
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA.
Janik
Ireneusz
I
Zhuang
Tiandi
T
Charvátová
Olga
O
Woods
Robert J
RJ
Sharp
Joshua S
JS
D000042:Absorption; D002853:Chromatography, Liquid; D004583:Electrons; D005583:Fourier Analysis; D037502:Galectin 3 / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D017665:Hydroxyl Radical / Q000737:chemistry* / Q000494:pharmacology; D007782:Lactoglobulins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D023302:Protein Footprinting / Q000379:methods*; D011675:Pulse Radiolysis; D013056:Spectrophotometry, Ultraviolet; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D025801:Ubiquitin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D014867:Water / Q000737:chemistry*
D037502:Galectin 3; D007782:Lactoglobulins; D025801:Ubiquitin; D014867:Water; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D014357:Trypsin