Dramatic stabilization of an SH3 domain by a single substitution: roles of the folded and unfolded states.


Abstract

The N-terminal SH3 domain of the Drosophila drk protein (drkN SH3) exists in equilibrium between folded and unfolded states under non-denaturing buffer conditions. In order to examine the origins of this instability, we have made mutations in the domain and characterized the thermodynamics and kinetics of folding. Results of substitutions of negatively charged residues to neutral amino acid residues suggest that the large electrostatic potential of the domain does not play a dominant role in the instability of the domain. Sequence alignment of a large number of SH3 domains reveals that the drkN SH3 domain has a threonine (T22) at a position corresponding to an otherwise highly conserved glycine residue in the diverging beta-turn connecting the beta3 and beta4 strands. Mutation of T22 to glycine results in significant stabilization of the drkN SH3 domain by 2.5 kcal/mole. To further characterize the basis for the stabilization of the T22 mutant relative to wild-type, we made additional mutant proteins with substitutions of residue T22. A strong correlation is seen between protein stability or folding rate and propensity for native beta-turn structure at this position. Correlation of folding rates with AGADIR predictions of non-native helical structure in the diverging turn region, along with our previous NMR evidence for non-native structure in this region of the unfolded state of the drkN SH3 domain, suggests that the free energy of the unfolded state also plays a role in stability. This result highlights the importance of both folded and unfolded states for understanding protein stability. Study holds ProTherm entries: 10843, 10844, 10845, 10846, 10847, 10848, 10849, 10850, 10851, 10852, 10853, 10854, 10855, 10856, 10857, 10858, 10859, 10860, 10861 Extra Details: N-terminal SH3 domain protein stability; folding kinetics; SH3 domain; unfolded state structure;,non-native residual structure

Submission Details

ID: zYgFREGt

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:41 p.m.

Version: 1

Publication Details
Mok YK;Elisseeva EL;Davidson AR;Forman-Kay JD,J. Mol. Biol. (2001) Dramatic stabilization of an SH3 domain by a single substitution: roles of the folded and unfolded states. PMID:11273710
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
2AZS 2005-12-13 NMR structure of the N-terminal SH3 domain of Drk (calculated without NOE restraints)
2A36 2005-12-13 Solution structure of the N-terminal SH3 domain of DRK
2A37 2005-12-13 Solution structure of the T22G mutant of N-terminal SH3 domain of DRK (DRKN SH3 DOMAIN)
2AZV 2005-12-13 Solution structure of the T22G mutant of N-terminal SH3 domain of DRK (calculated without NOEs)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Protein enhancer of sevenless 2B Q6YKA8 DRK_DROSI
100.0 Protein enhancer of sevenless 2B Q08012 DRK_DROME