21 Scopus citations


Murine adenosine deaminase (mADA) is a 40 kDa (β/α) 8-barrel protein consisting of eight central β-strands and eight peripheral α-helices containing four tryptophan residues. In this study, we investigated the urea-dependent behavior of the protein labeled with 6-fluorotryptophan (6-19F-Trp). The 19F NMR spectrum of 6-19F-Trp-labeled mADA reveals four distinct resonances in the native state and three partly overlapped resonances in the unfolded state. The resonances were assigned unambiguously by site-directed mutagenesis. Equilibrium unfolding of 619F-Trp-labeled mADA was monitored using 19F NMR based on these assignments. The changes in intensity of folded and unfolded resonances as a function of urea concentration show transition midpoints consistent with data observed by far-UV CD and fluorescence spectroscopy, indicating that conformational changes in mADA during urea unfolding can be followed by 19F NMR. Chemical shifts of the 19F resonances exhibited different changes between 1.0 and 6.0 M urea, indicating that local structures around 6-19F-Trp residues change differently. The urea-induced changes in local structure around four 6-19F-Trp residues of mADA were analyzed on the basis of the tertiary structure and chemical shifts of folded resonances. The results reveal that different local regions in mADA have different urea-dependent behavior, and that local regions of mADA change sequentially from native to intermediate topologies on the unfolding pathway.

Original languageEnglish
Pages (from-to)1432-1439
Number of pages8
Issue number6
StatePublished - Feb 17 2004


Dive into the research topics of 'Urea-Dependent Unfolding of Murine Adenosine Deaminase: Sequential Destabilization As Measured by 19F NMR'. Together they form a unique fingerprint.

Cite this