Structural study on the intrinsically unstructured proteins by using NMR spectroscopy

Abstract

It has long been axiomatic that the function of a protein is directly related to its three-dimensional structure. Recently, however, the observation that numerous functional proteins lack intrinsic globular structure or contain long disordered segments under physiological conditions shifted the classical protein-structure paradigm to an alternative one such as ‘the protein trinity’ or ‘the protein quartet’ model. During last 10 years, there is a growing interest in this protein class, so called ‘the intrinsically unstructured proteins (IUPs)’. The IUPs play critical roles in the important cellular processes including cell signaling and regulation. Here we report structural study on the IUPs by using NMR spectroscopy. The Arabidopsis HY5 protein is a basic leucine zipper (bZIP) transcription factor that promotes photomorphogenesis. HY5 binds directly to the promoters of light responsible element containing the G-box and thus regulates their transcriptional activity. The level and activity of HY5 are negatively regulated, in a light-dependent manner, by interaction with the COP1 protein, which targets HY5 for proteasome-mediated degradation in the nucleus. Despite its essential roles in plant development, no structural information exists for HY5. Here, we report the first structural and biophysical characterization of HY5. Using limited proteolysis in combination with mass spectrometry, circular dichroism and nuclear magnetic resonance spectroscopy, we have deduced that the N-terminal 77 amino acids of HY5 form a premolten globular structure, while amino acids 78-110, which constitute the basic region (BR) of the protein, exist in a molten globule state. Our studies also revealed that the overall structural features of full-length HY5 are dominated largely by the disordered N-terminal domain, despite the existence of a bZIP domain at its C-terminus. We propose that HY5 is a member of the intrinsically unstructured protein (IUP) family, and that HY5 functions a...