Unfolding and Double-stranded DNA Binding of the Cold Shock Protein Homologue Cla h 8 from Cladosporium herbarum

S Fabio Falsone,Michael Weichel,Reto Crameri,Michael Breitenbach,Andreas J Kungl

doi:10.1074/jbc.m200833200

S Fabio Falsone, Michael Weichel + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m200833200

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Abstract

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.

Highlights

Cold shock proteins (CSPs)1 are a class of small proteins which occur in a variety of prokaryotic organisms like mesophilic, psychrotrophic, and thermophilic bacteria [1, 2]
It was shown to unfold at temperatures uncommonly low for CSPs, which is interpreted to correlate with the improved affinity of Cla h 8 for dsDNA at low temperatures referring to the functional significance of Cla h 8 at reduced temperatures
It was possible to compute a homology-based model of Cla h 8 based on the three-dimensional structures of the CSPs of E. coli, B. subtilis, and B. caldolyticus (4 –7, 48) using for a first approach the Swiss-Model protein modeling server [45]

Summary

Introduction

Cold shock proteins (CSPs) are a class of small proteins which occur in a variety of prokaryotic organisms like mesophilic, psychrotrophic, and thermophilic bacteria [1, 2]. CspA acts as an RNA chaperone [21] in the course of translational control at decreased temperatures, binding nonspecifically to RNA in order to prevent the formation of secondary structure and thereby enabling efficient initiation of translation It regulates the synthesis of other cold stressinducible proteins through transcription antitermination [22]. Yb proteins consist of a glycine-rich N-terminal domain with unknown function, a nucleic-acid binding (cold shock) domain, and a C-terminal domain responsible for dimerization [27] They are multifunctional regulators of gene expression, and they are presumed to couple transcription to translation of mRNA [24, 25]. It was shown to unfold at temperatures uncommonly low for CSPs, which is interpreted to correlate with the improved affinity of Cla h 8 for dsDNA at low temperatures referring to the functional significance of Cla h 8 at reduced temperatures

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