A thermodynamic and kinetic analysis of the folding pathway of an SH3 domain entropically stabilised by a redesigned hydrophobic core.


Abstract

The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammond's postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein. Study holds ProTherm entries: 16447, 16448, 16449, 16450, 16451, 16452, 16453, 16454, 16455, 16456, 16457, 16458, 16459, 16460, 16461, 16462, 16463, 16464, 16465, 16466, 16467, 16468, 16469, 16470 Extra Details: SH3 domain SH3 domain; folding; transition state; hydrophobic core packing; protein design

Submission Details

ID: tpvffgCA

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Cobos ES;Filimonov VV;Vega MC;Mateo PL;Serrano L;Martínez JC,J. Mol. Biol. (2003) A thermodynamic and kinetic analysis of the folding pathway of an SH3 domain entropically stabilised by a redesigned hydrophobic core. PMID:12684010
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
2JM8 2007-04-24 R21A Spc-SH3 free
2LJ3 2011-12-07 PFBD: High-throughput Strategy of Backbone fold Determination for small well-folded proteins in less than a day
1AJ3 1997-07-07 SOLUTION STRUCTURE OF THE SPECTRIN REPEAT, NMR, 20 STRUCTURES
2ROT 2009-04-28 Structure of chimeric variant of SH3 domain- SHH
1M8M 2002-11-20 SOLID-STATE MAS NMR STRUCTURE OF THE A-SPECTRIN SH3 DOMAIN
2JM9 2007-04-24 R21A Spc-SH3 bound
2JMA 2007-04-24 R21A Spc-SH3:P41 complex
2KR3 2010-09-15 Solution structure of SHA-D
2KXD 2011-04-20 The structure of SH3-F2
2JMC 2007-04-24 Chimer between Spc-SH3 and P41
1AEY 1997-05-15 ALPHA-SPECTRIN SRC HOMOLOGY 3 DOMAIN, SOLUTION NMR, 15 STRUCTURES
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
1SHG 1993-10-31 1.8 CRYSTAL STRUCTURE OF A SRC-HOMOLOGY 3 (SH3) DOMAIN
1QKX 2000-12-15 1.8 Alpha-spectrin Src Homology 3 domain, N47A mutant in the distal loop.
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
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
1QKW 2000-08-18 2.0 Alpha-spectrin Src Homology 3 domain, N47G mutant in the distal loop.
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
1NEG 2003-01-14 2.3 Crystal Structure Analysis of N-and C-terminal labeled SH3-domain of alpha-Chicken Spectrin
3F31 2009-10-13 2.3 Crystal Structure of the N-terminal region of AlphaII-spectrin Tetramerization Domain
3FB2 2008-11-25 2.3 Crystal structure of the human brain alpha spectrin repeats 15 and 16. Northeast Structural Genomics Consortium target HR5563a.
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
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.
1UUE 2004-02-19 2.6 a-SPECTRIN SH3 DOMAIN (V44T, D48G MUTANT)
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
100.0 Spectrin alpha chain, non-erythrocytic 1 P16086 SPTN1_RAT
100.0 Spectrin alpha chain, non-erythrocytic 1 P16546 SPTN1_MOUSE
100.0 Spectrin alpha chain, non-erythrocytic 1 Q13813 SPTN1_HUMAN
100.0 Spectrin alpha chain, non-erythrocytic 1 P07751 SPTN1_CHICK