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Modulating the folding stability and ligand binding affinity of Pin1 WW domain by proline ring puckering
Author(s) -
Tang HsuCheng,
Lin YuJu,
Horng JiaCherng
Publication year - 2014
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.24359
Subject(s) - chemistry , ww domain , antiparallel (mathematics) , isothermal titration calorimetry , stereochemistry , circular dichroism , ligand (biochemistry) , steric effects , crystallography , proline , ring size , ring (chemistry) , folding (dsp implementation) , biochemistry , amino acid , receptor , physics , organic chemistry , quantum mechanics , magnetic field , gene , electrical engineering , engineering
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: (2 S ,4 R )‐4‐hydroxyproline (4 R ‐Hyp), (2 S ,4 R )‐4‐fluoroproline (4 R ‐Flp), (2 S ,4 R )‐4‐methoxyproline (4 R ‐Mop), and C γ ‐ endo biased pucker derivatives: (2 S ,4 S )‐4‐hydroxyproline (4 S ‐hyp), (2 S ,4 S )‐4‐fluoroproline (4 S ‐flp), (2 S ,4 S )‐4‐methoxyproline (4 S ‐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 4 S ‐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 CF 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 4 S ‐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. Proteins 2014; 82:67–76. © 2013 Wiley Periodicals, Inc.