Urea-dependent unfolding of murine adenosine deaminase: sequential destabilization as measured by 19F NMR.


Murine adenosine deaminase (mADA) is a 40 kDa (beta/alpha)(8)-barrel protein consisting of eight central beta-strands and eight peripheral alpha-helices containing four tryptophan residues. In this study, we investigated the urea-dependent behavior of the protein labeled with 6-fluorotryptophan (6-(19)F-Trp). The (19)F NMR spectrum of 6-(19)F-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 6-(19)F-Trp-labeled mADA was monitored using (19)F 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 (19)F NMR. Chemical shifts of the (19)F resonances exhibited different changes between 1.0 and 6.0 M urea, indicating that local structures around 6-(19)F-Trp residues change differently. The urea-induced changes in local structure around four 6-(19)F-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. Study holds ProTherm entries: 16852, 16853, 16854, 16855, 16856, 16857, 16858, 16859, 16860, 16861, 16862 Extra Details: 2mM DTT was added in the experiment. (beta/alpha)8-barrel protein; tryptophan; 6-fluorotryptophan; conformational changes

Submission Details


Submitter: Connie Wang

Submission Date: April 24, 2018, 8:48 p.m.

Version: 1

Publication Details
Shu Q;Frieden C,Biochemistry (2004) Urea-dependent unfolding of murine adenosine deaminase: sequential destabilization as measured by 19F NMR. PMID:14769019
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Adenosine deaminase P03958 ADA_MOUSE
94.0 Adenosine deaminase Q920P6 ADA_RAT