title: Influence of hPin1 WW N-terminal domain boundaries on function, protein stability, and folding.
abstract: An N-terminally truncated and cooperatively folded version (residues 6-39) of the human Pin1 WW domain (hPin1 WW hereafter) has served as an excellent model system for understanding triple-stranded beta-sheet folding energetics. Here we report that the negatively charged N-terminal sequence (Met1-Ala-Asp-Glu-Glu5) previously deleted, and which is not conserved in highly homologous WW domain family members from yeast or certain fungi, significantly increases the stability of hPin1 WW (approximately 4 kJ mol(-1) at 65 degrees C), in the context of the 1-39 sequence based on equilibrium measurements. N-terminal truncations and mutations in conjunction with a double mutant cycle analysis and a recently published high-resolution X-ray structure of the hPin1 cis/trans-isomerase suggest that the increase in stability is due to an energetically favorable ionic interaction between the negatively charged side chains in the N terminus of full-length hPin1 WW and the positively charged epsilon-ammonium group of residue Lys13 in beta-strand 1. Our data therefore suggest that the ionic interaction between Lys13 and the charged N terminus is the optimal solution for enhanced stability without compromising function, as ascertained by ligand binding studies. Kinetic laser temperature-jump relaxation studies reveal that this stabilizing interaction has not formed to a significant extent in the folding transition state at near physiological temperature, suggesting a differential contribution of the negatively charged N-terminal sequence to protein stability and folding rate. As neither the N-terminal sequence nor Lys13 are highly conserved among WW domains, our data further suggest that caution must be exercised when selecting domain boundaries for WW domains for structural, functional, or thermodynamic studies.
authors.affiliation: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Marcus
authors.initials: M
authors.lastname: Jäger
authors.forename: Houbi
authors.initials: H
authors.lastname: Nguyen
authors.forename: Maria
authors.initials: M
authors.lastname: Dendle
authors.forename: Martin
authors.initials: M
authors.lastname: Gruebele
authors.forename: Jeffery W
authors.initials: JW
authors.lastname: Kelly
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
chemical_list: D000072340:NIMA-Interacting Peptidylprolyl Isomerase; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase