title: Evaluation of folding co-operativity of a chimeric protein based on the molecular recognition between polyproline ligands and SH3 domains.
abstract: 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.
authors.affiliation: Departamento de Quimica Fisica e Instituto de Biotecnologia, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
authors.forename: Adela M
authors.initials: AM
authors.lastname: Candel
authors.forename: Eva S
authors.initials: ES
authors.lastname: Cobos
authors.forename: Francisco
authors.initials: F
authors.lastname: Conejero-Lara
authors.forename: Jose C
authors.initials: JC
authors.lastname: Martinez
mesh_terms: D000595:Amino Acid Sequence; D021983:Cyclophilins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry; D018909:src Homology Domains
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; C437224:cyclophilin hCyp-18; C011083:polyproline; D014508:Urea; D021983:Cyclophilins