Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k.


Abstract

The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF-hand calcium-binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ -binding sites. The Ca2+-loaded forms of the wild-type and mutant calbindins are too stable to be significantly denatured by heating at 90 degrees C or by adding 8 M urea. For the Ca2+-free (apo) forms thermal unfolding appears to be only half complete at 90 degrees C, while denaturation is complete in 7-8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu-17----Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state. According to X-ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr-13) is hydrogen-bonded to the carboxyl group of Glu-35, thus linking the two alpha helices flanking the N-terminal Ca2+ site. The pK of ionization of the Tyr-13 hydroxyl group was over 13 for calcium forms of the wild-type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms. Study holds ProTherm entries: 2744, 2745, 2746, 2747 Extra Details: EGTA(1 mM) was added in the experiment calbindin D9k; thermal stability; amino acid substitutions;,deletions; alpha helices; hydrogen-bonded; electrostatic

Submission Details

ID: Lbi67SBN

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:19 p.m.

Version: 1

Publication Details
Wendt B;Hofmann T;Martin SR;Bayley P;Brodin P;Grundström T;Thulin E;Linse S;Forsén S,Eur. J. Biochem. (1988) Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k. PMID:3409879
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
1RT0 2003-12-10T00:00:00+0000 0 12-mer from site II calbindin D9K (DKNGDGEVSFEE) coordinating Zn(II)
4ICB 1991-08-27T00:00:00+0000 1.6 PROLINE CIS-TRANS ISOMERS IN CALBINDIN D9K OBSERVED BY X-RAY CRYSTALLOGRAPHY
1CLB 1995-02-08T00:00:00+0000 0 Determination of the solution structure of apo calbindin D9K by nmr spectroscopy
1N65 2002-11-08T00:00:00+0000 0 FAMILY OF NMR SOLUTION STRUCTURES OF CA CE CALBINDIN D9K IN DENATURATING CONDITIONS
1HT9 2000-12-29T00:00:00+0000 1.76 DOMAIN SWAPPING EF-HANDS
1IG5 2001-04-17T00:00:00+0000 1.5 BOVINE CALBINDIN D9K BINDING MG2+
3ICB 1986-09-09T00:00:00+0000 2.3 THE REFINED STRUCTURE OF VITAMIN D-DEPENDENT CALCIUM-BINDING PROTEIN FROM BOVINE INTESTINE. MOLECULAR DETAILS, ION BINDING, AND IMPLICATIONS FOR THE STRUCTURE OF OTHER CALCIUM-BINDING PROTEINS
2BCB 1993-08-18T00:00:00+0000 0 HIGH-RESOLUTION SOLUTION STRUCTURE OF CALCIUM-LOADED CALBINDIN D9K
2BCA 1993-08-18T00:00:00+0000 0 HIGH-RESOLUTION SOLUTION STRUCTURE OF CALCIUM-LOADED CALBINDIN D9K
1QX2 2003-09-04T00:00:00+0000 1.44 X-ray Structure of Calcium-loaded Calbindomodulin (A Calbindin D9k Re-engineered to Undergo a Conformational Opening) at 1.44 A Resolution

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Protein S100-G P02633 S100G_BOVIN