The folding/unfolding equilibrium of the alpha-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the beta-strands, the short 3(10) helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the beta-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53-57) and a highly unstable region, the loop between residues 34-41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential scanning calorimetry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reactions. A structure-based thermodynamic analysis has allowed us to predict correctly the thermodynamics of the global unfolding of the domain and to define the ensemble of conformational states that quantitatively accounts for the observed pattern of hydrogen exchange protection. These results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independent local folding/unfolding reactions indicates that under native conditions the scale of cooperative interactions is regional rather than global. Study holds ProTherm entries: 23822, 23823, 23824, 23825, 23826, 23827, 23828, 23829, 23830, 23831, 23832, 23833, 23834 Extra Details: folding/unfolding equilibrium, alpha-spectrin SH3 domain, differential scanning calorimetry.
ID: fEawmRs73
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:54 p.m.
Version: 1
Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
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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 |
Percent Identity | Matching Chains | Protein | Accession | Entry Name |
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100.0 | Spectrin alpha chain, non-erythrocytic 1 | P07751 | SPTN1_CHICK | |
100.0 | Spectrin alpha chain, non-erythrocytic 1 | Q13813 | SPTN1_HUMAN | |
100.0 | Spectrin alpha chain, non-erythrocytic 1 | P16546 | SPTN1_MOUSE | |
100.0 | Spectrin alpha chain, non-erythrocytic 1 | P16086 | SPTN1_RAT |