Unfolding and double-stranded DNA binding of the cold shock protein homologue Cla h 8 from Cladosporium herbarum.
The cloning, purification, and biophysical characterization of the first eukaryotic cold shock protein homologue, Cla h 8, expressed as single functional polypeptide is reported here. It was discovered as a minor allergen of the mold Cladosporium herbarum by phage display using a library selectively enriched for IgE-binding proteins. Based on the sequence homology of Cla h 8 with bacterial cold shock proteins (CSPs), a homology-based computer model of the allergen was computed indicating an all-beta structure of Cla h 8. This major structural feature was confirmed by CD spectroscopy. Despite the structural similarities with bacterial CSPs, the DNA-binding and unfolding behavior of Cla h 8 exhibited unique and previously undescribed characteristics. High affinities of Cla h 8 for single-stranded DNA as well as for double-stranded DNA corresponding to the human Y-box were detected. The affinity for double-stranded DNA increased significantly with decreasing temperature, which was paralleled by an increase in the beta sheet content of the protein. Temperature-dependent fluorescence anisotropy and far-UV CD measurements revealed different unfolding transitions at 28 and at 35.7 degrees C, respectively, indicating a multistate transition, which is uncommon for CSPs. The enhanced affinity for DNA at low temperatures together with the low unfolding transition refer to the functional significance of Cla h 8 at reduced temperatures.
Falsone
S Fabio
SF
Department of Protein Chemistry and Biophysics, Institute of Pharmaceutical Chemistry and Pharmaceutical Technology, University of Graz, A-8010 Graz, Austria.
Weichel
Michael
M
Crameri
Reto
R
Breitenbach
Michael
M
Kungl
Andreas J
AJ
D000485:Allergens / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D016880:Anisotropy; D052179:Antigens, Plant; D002942:Circular Dichroism; D002963:Cladosporium / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003080:Cold Temperature; D004247:DNA / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D007073:Immunoglobulin E / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
D000485:Allergens; D052179:Antigens, Plant; C456299:Cla h 8 protein, Cladosporium herbarum; D018076:DNA, Complementary; D005656:Fungal Proteins; D008024:Ligands; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D004247:DNA