19F-NMR and crystallographic studies of 5-fluorotryptophan labeled anthrax protective antigen and effects of receptor on stability.
Related Articles 19F-NMR and crystallographic studies of 5-fluorotryptophan labeled anthrax protective antigen and effects of receptor on stability.
Biochemistry. 2014 Jan 3;
Authors: Chadegani F, Lovell SW, Mullangi V, Miyagi M, Battaile KP, Bann JG
Abstract
The anthrax protective antigen (PA) is an 83 kDa protein that is one of three protein components of the anthrax toxin, an AB toxin secreted by Bacillus anthracis. PA is capable of undergoing several structural changes, including oligomerization to either a heptameric or octameric structure called the pre-pore, and at acidic pH undergoes a major conformational change to form a membrane spanning pore. In order to follow these structural changes at a residue-specific level, we have carried out initial studies where we have biosynthetically incorporated 5-fluorotryptophan (5-FTrp) into PA, and we have studied the influence of 5-FTrp labeling on the structural stability of PA and on binding to the host receptor capillary morphogenesis protein 2 (CMG2) using 19F-NMR. There are seven tryptophans in PA, but of the four domains in PA, only two contain tryptophans: domain 1 (Trp65, 90, 136, 206 and 226) and domain 2 (Trp346 and 477). Trp346 is of particular interest because of its close proximity to the CMG2 binding interface, and because it forms part of the membrane spanning pore. We show that the 19F resonance of Trp346 is sensitive to changes in pH, consistent with crystallographic studies, and that receptor binding significantly stabilizes Trp346 to both pH and temperature. In addition, we provide evidence which suggests that resonances from tryptophans distant from the binding interface, are also stabilized by the receptor. Our studies highlight the positive impact of receptor binding on protein stability, and the use of 19F-NMR in gaining insight into structural changes in a large molecular weight protein.
PMID: 24387629 [PubMed - as supplied by publisher]
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