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


Abstract

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

ID: MLMNB2sR

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 PDB Entries

Structure ID Release Date Resolution Structure Title
3MVI 2010-11-03 1.6 Crystal structure of holo mADA at 1.6 A resolution
1A4M 1998-10-14 1.95 ADA STRUCTURE COMPLEXED WITH PURINE RIBOSIDE AT PH 7.0
3KM8 2010-10-20 2.0 Crystal structuore of adenosine deaminase from mus musculus complexed with 9-deazainosine
3MVT 2010-10-13 2.2 Crystal structure of apo mADA at 2.2A resolution
3T1G 2012-02-08 2.35 Engineering of organophosphate hydrolase by computational design and directed evolution
1FKX 1996-08-01 2.4 MURINE ADENOSINE DEAMINASE (D296A)
1UIP 1997-06-24 2.4 ADENOSINE DEAMINASE (HIS 238 GLU MUTANT)
2ADA 1995-03-31 2.4 ATOMIC STRUCTURE OF ADENOSINE DEAMINASE COMPLEXED WITH A TRANSITION-STATE ANALOG: UNDERSTANDING CATALYSIS AND IMMUNODEFICIENCY MUTATIONS
1UIO 1997-06-24 2.4 ADENOSINE DEAMINASE (HIS 238 ALA MUTANT)
1FKW 1996-08-01 2.4 MURINE ADENOSINE DEAMINASE (D295E)
1ADD 1994-01-31 2.4 A PRE-TRANSITION STATE MIMIC OF AN ENZYME: X-RAY STRUCTURE OF ADENOSINE DEAMINASE WITH BOUND 1-DEAZA-ADENOSINE AND ZINC-ACTIVATED WATER
1A4L 1998-10-14 2.6 ADA STRUCTURE COMPLEXED WITH DEOXYCOFORMYCIN AT PH 7.0

Relevant UniProtKB Entries

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