title: Modulating the folding stability and ligand binding affinity of Pin1 WW domain by proline ring puckering.
abstract: The pyrrolidine side chain makes proline play a unique role in protein structure and function. The C(γ) ring pucker preference and the cis-trans peptidyl bond ratio can be mediated via stereoelectronic effects. Here we used a compact triple-stranded antiparallel β-sheet protein, the human Pin1 WW domain, to study the consequences of implanting a preorganized C(γ) ring pucker on protein structure and function. The conserved Pro37 is a key residue involved in one hydrophobic core, plays an important role in the WW domain, and adopts a C(γ) -endo ring pucker in the native structure. Pro37 was replaced with C(γ) -exo biased pucker derivatives: (2S,4R)-4-hydroxyproline (4R-Hyp), (2S,4R)-4-fluoroproline (4R-Flp), (2S,4R)-4-methoxyproline (4R-Mop), and C(γ) -endo biased pucker derivatives: (2S,4S)-4-hydroxyproline (4S-hyp), (2S,4S)-4-fluoroproline (4S-flp), (2S,4S)-4-methoxyproline (4S-mop) to examine how a preorganized pucker affects the folding stability and ligand-binding affinity. Circular dichroism measurements indicate that among the variants, only the one with 4S-flp substitution (P37flp) is more stable than the wild type, suggesting that the stabilization effects originated from preorganization of the backbone conformation and the hydrophobicity of C - F group. Analysis of ligand-binding affinity using isothermal titration calorimetry revealed that only P37flp has a stronger ligand affinity than the wild type, showing that 4S-flp can stabilize the WW domain and increase its ligand affinity. Together we have used 4-substituted proline derivatives and the WW domain to demonstrate that proline ring puckering can be a key factor in determining the folding stability of a protein but the choice of the derivative groups is also critical.
authors.affiliation: Department of Chemistry, National Tsing Hua University, Hsinchu, 30013, Taiwan.
authors.forename: Hsu-Cheng
authors.initials: HC
authors.lastname: Tang
authors.forename: Yu-Ju
authors.initials: YJ
authors.lastname: Lin
authors.forename: Jia-Cherng
authors.initials: JC
authors.lastname: Horng
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D002151:Calorimetry; D002942:Circular Dichroism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D013050:Spectrometry, Fluorescence
chemical_list: D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D011392:Proline; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase
keywords: circular dichroism; fluoroproline; hydrophobic effect; hydroxyproline; isothermal titration calorimetry; methoxyproline; steric effect