Jack F. Eichler, J. C. Cramer, K. Kirk
Dec 2, 2005
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Influential Citations
34
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ChemBioChem
Abstract
Histidine is important for carrying out a number of protein functions. For instance, it can act as a general acid/base in enzymatic catalysis, it plays a role in ligating metal cations in metalloproteins, and stabilizes protein structure by metal binding, hydrogen bonding, or electrostatic interactions. One noteworthy example of histidine’s potential role in protein stability is observed in the pathogenesis of the anthrax toxin, where protonation of His side chains in one of the proteins leads to a large structural perturbation and subsequent toxicity. Given the unique role of this amino acid in such processes, the development of methods that probe the structural and mechanistic features of His in proteins would be extremely valuable. The biosynthetic incorporation of unnatural amino acids into proteins provides the experimentalist with a variety of methods that can probe protein structure and function. In particular, fluorinated amino acids can be used to achieve a relatively isosteric change (by replacing a single hydrogen with fluorine) that results in quite different electronic properties. Additionally, F NMR can be used to monitor changes in protein conformation in response to changes in the environment that are sometimes not detectable by other techniques. Although incorporating fluorine-labeled amino acids is not a new idea, access to an expanding tool box of fluorinated protein building blocks has promoted a renaissance in this area of research. Over 30 years ago, a photochemical Schiemann reaction was developed for synthesizing 2-fluorohistidine (2-FHis) and 4-fluorohistidine (4-FHis). To our knowledge this still represents the only fluorination procedure available for accessing these imidazole derivatives. The pKa of the side chain of both 2-FHis and 4-FHis has been measured previously, and is decreased from approximately 6.0–6.5 to 1 and 3, respectively. Because of this, these analogues provide a means for verifying the role of native His in pH-dependent processes. To this end, 4-FHis has been incorporated into the S peptide of ribonuclease, and into full-length ribonuclease A with chemical synthetic methods. Early experiments demonstrated that tritium-labeled 2-FHis could be incorporated into bacterial protein, and these analogues (2-FHis more so than 4-FHis) had an inhibitory effect on E. coli growth. However, in order to achieve high levels of incorporation for structural studies, it is typical to employ the use of bacterial auxotrophs. While the use of auxotrophs for the biosynthetic incorporation of novel His analogues into E. coli has been reported, a similar protocol for the incorporation of fluorohistidine derivatives has yet to be described. Herein, we provide unequivocal evidence for the incorporation of both 4-FHis and 2-FHis into a mutant form of the chaperone PapD by using an E. coli strain that is auxotrophic for His. PapD is the prototype for a wide variety of highly homologous chaperones that utilize the chaperone-usher pathway for the assembly of P-pili, and has been previously labeled with fluorophenylalanine and fluorotryptophan in protein-folding studies. The wild-type (WT) protein does not contain any His residues. Thus, site-specific labeling can be accomplished by the introduction of a single His residue by site-directed mutagenesis, and biosynthetic labeling can be performed according to previously described protocols. In this work, we used site-directed mutagenesis to introduce a single His residue at Arg200 in PapD. Among the chaperones that are homologous to PapD, the most similar is PmFD from Proteus mirabilis (47% identity), which possesses a His residue at position 200. Therefore, an R200H substitution was not expected to alter the structure and stability of PapD. To confirm this, urea denaturation studies were performed on PapD (R200H) and it was found to have a similar stability as that previously reported for WT PapD; PapD(WT): DG8=8.95 kcal [a] Dr. J. F. Eichler, Prof. Dr. J. G. Bann Department of Chemistry, Wichita State University Wichita, KS 67226 (USA) Fax: (+1)316-978-7373 E-mail : jim.bann@wichita.edu [b] Dr. J. C. Cramer, Dr. K. L. Kirk Laboratory of Bioorganic Chemistry National Institute of Diabetes & Digestive & Kidney Diseases National Institutes of Health Bethesda, MD 20892 (USA) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.