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
3THK 2011-08-19T00:00:00+0000 1.7 Structure of SH3 chimera with a type II ligand linked to the chain C-terminal
2FOT 2006-01-13T00:00:00+0000 2.45 Crystal structure of the complex between calmodulin and alphaII-spectrin
3F31 2008-10-30T00:00:00+0000 2.3 Crystal Structure of the N-terminal region of AlphaII-spectrin Tetramerization Domain
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.
5FW9 2016-02-12T00:00:00+0000 1.55 Human Spectrin SH3 domain D48G, E7Y, K60Y
5FWB 2016-02-12T00:00:00+0000 1.5 Human Spectrin SH3 domain D48G, E7F, K60F
5FWC 2016-02-12T00:00:00+0000 1.4 Human Spectrin SH3 domain D48G, E7A, K60A
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)
1AEY 1997-03-02T00:00:00+0000 0 ALPHA-SPECTRIN SRC HOMOLOGY 3 DOMAIN, SOLUTION NMR, 15 STRUCTURES
1AJ3 1997-05-14T00:00:00+0000 0 SOLUTION STRUCTURE OF THE SPECTRIN REPEAT, NMR, 20 STRUCTURES

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