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
2RMO 2008-09-30 Solution structure of alpha-spectrin_SH3-bergerac from Chicken
2KR3 2010-09-15 Solution structure of SHA-D
2JMC 2007-04-24 Chimer between Spc-SH3 and P41
1M8M 2002-11-20 SOLID-STATE MAS NMR STRUCTURE OF THE A-SPECTRIN SH3 DOMAIN
2KXD 2011-04-20 The structure of SH3-F2
2LJ3 2011-12-07 PFBD: High-throughput Strategy of Backbone fold Determination for small well-folded proteins in less than a day
2ROT 2009-04-28 Structure of chimeric variant of SH3 domain- SHH
2JMA 2007-04-24 R21A Spc-SH3:P41 complex
1AJ3 1997-07-07 SOLUTION STRUCTURE OF THE SPECTRIN REPEAT, NMR, 20 STRUCTURES
2JM9 2007-04-24 R21A Spc-SH3 bound
1AEY 1997-05-15 ALPHA-SPECTRIN SRC HOMOLOGY 3 DOMAIN, SOLUTION NMR, 15 STRUCTURES
2JM8 2007-04-24 R21A Spc-SH3 free
3NGP 2010-09-08 1.08 High resolution structure of alpha-spectrin SH3 domain mutant with a redesigned core
3M0R 2011-01-19 1.1 Crystal Structure of the R21D mutant of alpha-spectrin SH3 domain. Crystal obtained in ammonium sulphate at pH 6.
3M0U 2011-01-19 1.1 Crystal Structure of the R21D mutant of alpha-spectrin SH3 domain. Hexagonal crystal obtained in sodium formate at pH 6.5.
1G2B 2000-11-01 1.12 ALPHA-SPECTRIN SRC HOMOLOGY 3 DOMAIN, CIRCULAR PERMUTANT, CUT AT N47-D48
5IHK 2017-03-29 1.35 Crystal Structure of the alpha spectrin SH3 domain mutant N47A
5FWC 2016-12-28 1.4 Human Spectrin SH3 domain D48G, E7A, K60A
3I9Q 2009-12-15 1.45 Crystal Structure of the triple mutant S19G-P20D-R21S of alpha spectrin SH3 domain
5IHI 2017-03-29 1.45 Crystal Structure of the alpha spectrin SH3 domain double mutant V46G-D48G
1U06 2005-01-13 1.49 crystal structure of chicken alpha-spectrin SH3 domain
5IHN 2017-03-29 1.5 Crystal Structure of the alpha spectrin SH3 domain mutant N47G
5FWB 2016-12-28 1.5 Human Spectrin SH3 domain D48G, E7F, K60F
5FW9 2016-12-28 1.55 Human Spectrin SH3 domain D48G, E7Y, K60Y
4F17 2013-05-08 1.55 Crystal Structure of the alpha spectrin SH3 domain at pH 9
3M0S 2011-01-19 1.6 Crystal Structure of the R21D mutant of alpha-spectrin SH3 domain. Crystal obtained in ammonium sulphate at pH 7
2F2X 2006-10-31 1.6 alpha-spectrin SH3 domain R21G mutant
2F2W 2006-10-31 1.7 alpha-spectrin SH3 domain R21A mutant
3THK 2011-11-23 1.7 Structure of SH3 chimera with a type II ligand linked to the chain C-terminal
3M0T 2011-01-19 1.7 Crystal Structure of the R21D mutant of alpha-spectrin SH3 domain. Crystal obtained in ammonium sulphate at pH 9.
3M0Q 2011-01-19 1.75 Crystal Structure of the R21D mutant of alpha-spectrin SH3 domain. Crystal obtained in ammonium sulphate at pH 5.
1TUD 1996-08-01 1.77 ALPHA-SPECTRIN SRC HOMOLOGY 3 DOMAIN, CIRCULAR PERMUTANT, CUT AT N47-D48
1PWT 1999-05-11 1.77 THERMODYNAMIC ANALYSIS OF ALPHA-SPECTRIN SH3 AND TWO OF ITS CIRCULAR PERMUTANTS WITH DIFFERENT LOOP LENGTHS: DISCERNING THE REASONS FOR RAPID FOLDING IN PROTEINS
1QKX 2000-12-15 1.8 Alpha-spectrin Src Homology 3 domain, N47A mutant in the distal loop.
1SHG 1993-10-31 1.8 CRYSTAL STRUCTURE OF A SRC-HOMOLOGY 3 (SH3) DOMAIN
2F2V 2006-10-31 1.85 alpha-spectrin SH3 domain A56G mutant
2NUZ 2007-05-15 1.85 crystal structure of alpha spectrin SH3 domain measured at room temperature
2OAW 2008-04-08 1.9 Structure of SHH variant of 'Bergerac' chimera of spectrin SH3
4F16 2013-05-08 1.93 Crystal Structure of the alpha spectrin SH3 domain at pH 5
1HD3 2001-11-01 1.98 A-SPECTRIN SH3 DOMAIN F52Y MUTANT
1QKW 2000-08-18 2.0 Alpha-spectrin Src Homology 3 domain, N47G mutant in the distal loop.
1CUN 1999-10-06 2.0 CRYSTAL STRUCTURE OF REPEATS 16 AND 17 OF CHICKEN BRAIN ALPHA SPECTRIN
1U5P 2004-10-19 2.0 Crystal Structure of Repeats 15 and 16 of Chicken Brain Alpha Spectrin
1BK2 1999-02-16 2.01 A-SPECTRIN SH3 DOMAIN D48G MUTANT
1E6H 2002-05-23 2.01 A-SPECTRIN SH3 DOMAIN A11V, M25I, V44I, V58L MUTANTS
1TUC 1996-08-01 2.02 ALPHA-SPECTRIN SRC HOMOLOGY 3 DOMAIN, CIRCULAR PERMUTANT, CUT AT S19-P20
3FB2 2008-11-25 2.3 Crystal structure of the human brain alpha spectrin repeats 15 and 16. Northeast Structural Genomics Consortium target HR5563a.
3F31 2009-10-13 2.3 Crystal Structure of the N-terminal region of AlphaII-spectrin Tetramerization Domain
1NEG 2003-01-14 2.3 Crystal Structure Analysis of N-and C-terminal labeled SH3-domain of alpha-Chicken Spectrin
1E6G 2002-05-23 2.3 A-SPECTRIN SH3 DOMAIN A11V, V23L, M25I, V53I, V58L MUTANT
2FOT 2006-09-05 2.45 Crystal structure of the complex between calmodulin and alphaII-spectrin
1U4Q 2004-10-19 2.5 Crystal Structure of Repeats 15, 16 and 17 of Chicken Brain Alpha Spectrin
2CDT 2007-02-20 2.54 alpha-SPECTRIN SH3 DOMAIN A56S MUTANT
1UUE 2004-02-19 2.6 a-SPECTRIN SH3 DOMAIN (V44T, D48G MUTANT)
3M0P 2011-01-19 2.6 Crystal Structure of the R21D mutant of alpha-spectrin SH3 domain. Crystal obtained in ammonium sulphate at pH 4.
1H8K 2002-05-23 2.7 A-SPECTRIN SH3 DOMAIN A11V, V23L, M25V, V53I, V58L MUTANT
1E7O 2003-05-21 3.2 A-SPECTRIN SH3 DOMAIN A11V, V23L, M25V, V44I, V58L MUTATIONS
5M6S 2017-01-11 4.8 folding intermediate of spectrin R16

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