PEDS Advance Access originally published online on July 17, 2009
Protein Engineering Design and Selection 2009 22(10):597-606; doi:10.1093/protein/gzp041
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Evaluation of folding co-operativity of a chimeric protein based on the molecular recognition between polyproline ligands and SH3 domains
1Departamento de Quimica Fisica e Instituto de Biotecnologia, Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain 2Present address: Division of Molecular Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
3 To whom correspondence should be addressed. E-mail: jcmh{at}ugr.es
In previous work, we designed a chimeric protein, named SPCp41, to evaluate the thermodynamics of the interaction between SH3 domains and proline-rich ligands by combining thermal unfolding measurements and mutagenesis. Here, we have investigated the energetic integrity of the chain extension corresponding to the ligand sequence into the native structure, since the opposite will produce changes in the folding mechanism of the SH3 domain that may give rise to undesirable contributions to the thermodynamic parameters. We have analysed the folding–unfolding kinetics under standard conditions (50 mM phosphate pH 7). Kinetic evolutions are well described by a bi-exponential where, on top of the main kinetic phase, a low-populated slower phase appears as a consequence of cis–trans isomerisation of Pro39, as demonstrated by the influence of prolyl isomerases and by mutational analysis. There is also a burst phase possibly due to a productive formation of some helical ensembles. The main evolution, accounting for the true folding kinetics of SPCp41, can be considered as a two-state process, where the folding transition state produces essentially the same picture shown by the circular permutant S19-P20s (the ‘nucleus’ of the design) and the ligand will dock at the latter stages of the two-state process. Thus, all conclusions argue in favour of the effectiveness of SPCp41 to study energetic, dynamic and structural aspects of SH3–ligand interactions.
Keywords: folding kinetics/folding transition state/proline isomerisation/SH3 domains/SH3–ligand interactions
Received April 24, 2009; revised June 9, 2009; accepted June 13, 2009.
Abbreviations: SH3, Src-homology 3 domain; Spc-SH3, SH3 domain from chicken
-spectrin; NMR, nuclear magnetic resonance; WT, wild type; S19-P20s, circular permutant of Spc-SH3 opened between residues Ser19 and Pro20; hCyp18, human cyclophilin 18.