Hydrophobic core substitutions in calbindin D9k: effects on stability and structure.


Abstract

The effects of hydrophobic core mutations on the stability and structure of the four-helix calcium-binding protein, calbindin D9k, have been investigated. Eleven mutations involving eight residues distributed within the hydrophobic core of calbindin D9k were examined. Stabilities were measured by denaturant and thermal induced unfolding monitored by circular dichroism spectroscopy. The mutations were found to exert large effects on the stability with midpoints in the urea induced unfolding varying from 1.8 M for Leu23 --> Gly up to 6.6 M for Val70 --> Leu and free energies of unfolding in the absence of denaturant ranging from 6.6 to 27.4 kJ/mol for the Phe66 --> Ala mutant and the wild-type, respectively. A significant correlation was found between the difference in free energy of unfolding (Delta Delta GNU) and the change in the surface area of the side chain caused by the mutation, in agreement with other studies. Notably, both increases and decreases in side-chain surface area caused quantitatively equivalent effects on the stability. In other words, a correlation between the absolute value of the change in the surface of the side chain and Delta DeltaGNU was observed with a value of approximately 0.14 kJ M-1 A-2. The generality of this observation is discussed. Significant effects on the cooperativity of the unfolding reaction were also observed. However, a correlation between the cooperativity and Delta Delta GNU, which has been reported in other systems as an indication of effects of mutations on the unfolded state, was not observed for calbindin D9k. Despite the large effects on Delta Delta GNU and cooperativity, the structures of the mutants in the native form remained intact as indicated by circular dichroism, NMR, and fluorescence measurements. The structural response to calcium-binding was also conserved. The following paper in this issue [Kragelund, B. B., et al. (1998) Biochemistry 37, 8926-8937] examines the effects of these mutations on the calcium binding properties of calbindin D9k. Study holds ProTherm entries: 9501, 9502, 9503, 9504, 9505, 9506, 9507, 9508, 9509, 9510, 9511, 9512 Extra Details: EGTA(0.5 mM) was added in the experiment hydrophobic core; fourhelix calcium-binding protein;,free energies of unfolding; cooperativity

Submission Details

ID: ZjXQoiYp3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
Julenius K;Thulin E;Linse S;Finn BE,Biochemistry (1998) Hydrophobic core substitutions in calbindin D9k: effects on stability and structure. PMID:9636033
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
1CLB 1995-04-20 Determination of the solution structure of apo calbindin D9K by nmr spectroscopy
1B1G 1998-11-25 SOLVATED REFINEMENT OF CA-LOADED CALBINDIN D9K
1BOC 1993-10-31 THE SOLUTION STRUCTURES OF MUTANT CALBINDIN D9K'S, AS DETERMINED BY NMR, SHOW THAT THE CALCIUM BINDING SITE CAN ADOPT DIFFERENT FOLDS
1KQV 2002-01-16 Family of NMR Solution Structures of Ca Ln Calbindin D9K
2BCA 1993-10-31 HIGH-RESOLUTION SOLUTION STRUCTURE OF CALCIUM-LOADED CALBINDIN D9K
1D1O 2000-03-08 COOPERATIVITY IN EF-HAND CA2+-BINDING PROTEINS: EVIDENCE OF SITE-SITE COMMUNICATION FROM BINDING-INDUCED CHANGES IN STRUCTURE AND DYNAMICS OF N56A CALBINDIN D9K
2MAZ 2013-08-07 Backbone 1H, 13C, and 15N Chemical Shift Assignments for Bovine Apo Calbindin
1RT0 2005-07-26 12-mer from site II calbindin D9K (DKNGDGEVSFEE) coordinating Zn(II)
1BOD 1993-10-31 THE SOLUTION STRUCTURES OF MUTANT CALBINDIN D9K'S, AS DETERMINED BY NMR, SHOW THAT THE CALCIUM BINDING SITE CAN ADOPT DIFFERENT FOLDS
1KCY 2001-11-21 NMR solution structure of apo calbindin D9k (F36G + P43M mutant)
1KSM 2002-01-23 AVERAGE NMR SOLUTION STRUCTURE OF CA LN CALBINDIN D9K
2BCB 1993-10-31 HIGH-RESOLUTION SOLUTION STRUCTURE OF CALCIUM-LOADED CALBINDIN D9K
1N65 2003-11-18 FAMILY OF NMR SOLUTION STRUCTURES OF CA CE CALBINDIN D9K IN DENATURATING CONDITIONS
1CDN 1995-11-14 Solution structure of (CD2+)1-calbindin D9K reveals details of the stepwise structural changes along the apo--> (CA2+)II1--> (CA2+)I,II2 binding pathway
1QX2 2004-05-25 1.44 X-ray Structure of Calcium-loaded Calbindomodulin (A Calbindin D9k Re-engineered to Undergo a Conformational Opening) at 1.44 A Resolution
1IG5 2001-04-25 1.5 BOVINE CALBINDIN D9K BINDING MG2+
4ICB 1993-10-31 1.6 PROLINE CIS-TRANS ISOMERS IN CALBINDIN D9K OBSERVED BY X-RAY CRYSTALLOGRAPHY
1HT9 2001-05-09 1.76 DOMAIN SWAPPING EF-HANDS
1IGV 2001-04-25 1.85 BOVINE CALBINDIN D9K BINDING MN2+
3ICB 1986-10-24 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

Relevant UniProtKB Entries

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