Site-directed mutagenesis of either the highly conserved Trp-22 or the moderately conserved Trp-95 to a large, hydrophobic residue reduces the thermodynamic stability of a spectrin repeating unit.


Abstract

As reported previously (MacDonald, R. I., Musacchio, A., Holmgren, R. A., and Saraste, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1299-1303), an unfolded peptide was obtained by site-directed mutagenesis of Trp-22 to Ala in the cloned, wild type 17th repeating unit (alpha17) of chicken brain alpha-spectrin. Trp occurs in position 22 of nearly all repeating units of spectrin. In the present study, Trp-22 was mutated to Phe or to Tyr to compare thermodynamic stabilities of urea-induced unfolding of alpha16 and mutants thereof. alpha16 was chosen for this study instead of alpha17, because alpha16 has two tryptophans, allowing urea-induced unfolding to be tracked by the fluorescence of the Trp remaining in each mutant peptide and by circular dichroism in the far UV. The free energies of unfolding of W22Y and W22F were 50% that of alpha16, showing that Trp-22 is crucial in stabilizing the triple helical bundle motif of the spectrin repeating unit. Mutation of the moderately conserved Trp-95 of alpha16 to Val, which occupies position 95 in alpha17, also yielded a peptide with 50% of the free energy of unfolding of alpha16. Thus, the thermodynamic stability of a given spectrin repeating unit may depend on both moderately and highly conserved tryptophans. Different structural roles of Trp-22 and Trp-95 in alpha16 are suggested by the slightly higher wavelength of maximum emission of Trp-22, the greater acrylamide quenching of Trp-95 than Trp-22, and the longer lifetime of Trp-95. For comparison with alpha16, urea-induced unfolding of spectrin dimer isolated from human red cells was monitored by far UV-CD and by tryptophan fluorescence. Thermodynamic parameters could not be rigorously derived for the stability of spectrin dimer because unfolding of spectrin dimer involved more than two states, unlike unfolding of cloned repeating units. However, the similar midpoints of CD-monitored denaturation curves of alpha16 and spectrin dimer, i. e. 2.7 and 3.2 M urea, respectively, indicate that investigation of cloned repeating units of spectrin can provide physiologically relevant information on these structures. Study holds ProTherm entries: 2969, 2970, 2971, 2972, 2973 Extra Details: additive : EDTA(0.1 mM), thermodynamic stability; site-directed mutagenesis;,alpha-spectrin; hydrophobic residue; structural role

Submission Details

ID: ZHWuvGsm

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:20 p.m.

Version: 1

Publication Details
Pantazatos DP;MacDonald RI,J. Biol. Chem. (1997) Site-directed mutagenesis of either the highly conserved Trp-22 or the moderately conserved Trp-95 to a large, hydrophobic residue reduces the thermodynamic stability of a spectrin repeating unit. PMID:9261107
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
3THK 2011-08-19T00:00:00+0000 1.7 Structure of SH3 chimera with a type II ligand linked to the chain C-terminal
3FB2 2008-11-18T00:00:00+0000 2.3 Crystal structure of the human brain alpha spectrin repeats 15 and 16. Northeast Structural Genomics Consortium target HR5563a.
6ZEH 2020-06-16T00:00:00+0000 1.3 Structure of PP1-spectrin alpha II chimera [PP1(7-304) + linker (G/S)x9 + spectrin alpha II (1025-1039)] bound to Phactr1 (516-580)
3F31 2008-10-30T00:00:00+0000 2.3 Crystal Structure of the N-terminal region of AlphaII-spectrin Tetramerization Domain
2FOT 2006-01-13T00:00:00+0000 2.45 Crystal structure of the complex between calmodulin and alphaII-spectrin
5FWC 2016-02-12T00:00:00+0000 1.4 Human Spectrin SH3 domain D48G, E7A, K60A
5FWB 2016-02-12T00:00:00+0000 1.5 Human Spectrin SH3 domain D48G, E7F, K60F
5FW9 2016-02-12T00:00:00+0000 1.55 Human Spectrin SH3 domain D48G, E7Y, K60Y
5M6S 2016-10-25T00:00:00+0000 4.8 folding intermediate of spectrin R16
1SHG 1993-05-19T00:00:00+0000 1.8 CRYSTAL STRUCTURE OF A SRC-HOMOLOGY 3 (SH3) DOMAIN

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.3 Spectrin alpha chain, non-erythrocytic 1 P16086 SPTN1_RAT
97.3 Spectrin alpha chain, non-erythrocytic 1 P16546 SPTN1_MOUSE
98.2 Spectrin alpha chain, non-erythrocytic 1 Q13813 SPTN1_HUMAN
100.0 Spectrin alpha chain, non-erythrocytic 1 P07751 SPTN1_CHICK