Structural studies of human naked2: A biologically active intrinsically unstructured protein - Analytical Methods & Tools for Lyophilization

Structural studies of human naked2: A biologically active intrinsically unstructured protein
Received 15 September 2006. Available online 2 October 2006.
Tianhui Hua, Andrzej M. Krezelb, c, Cunxi Lia and Robert J. Coffeya
Biochemical and Biophysical Research Communications
ScienceDirect
Copyright ? 2006 Elsevier Inc. All rights reserved.
aDepartment of Medicine and Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
bDepartment of Biological Sciences and Center For Structural Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
cDepartment of Veterans Affairs Medical Center, Nashville, TN 37232, USA
Abstract
Naked1 and 2 are two mammalian orthologs of Naked Cuticle, a canonical Wnt signaling antagonist in Drosophila. Naked2, but not Naked1, interacts with transforming growth factor-a (TGFa) and escorts TGFa-containing vesicles to the basolateral membrane of polarized epithelial cells. Full-length Naked2 is poorly soluble. Since most functional domains, including the Dishevelled binding region, EF-hand, vesicle recognition, and membrane targeting motifs, reside in the N-terminal half of the protein, we expressed and purified the first 217 residues of human Naked2 and performed a functional analysis of this fragment. Its circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra showed no evidence of secondary and/or tertiary structure. The fragment did not bind calcium or zinc. These results indicate that the N-terminal half of Naked2 behaves as an intrinsically unstructured protein.
Keywords: Naked2; Intrinsically unstructured protein; Circular dichroism; Nuclear magnetic resonance; Transforming growth factor-a

Abbreviations: IUP, intrinsically unstructured protein; CD, circular dichroism; GST, glutathione S-transferase; NMR, nuclear magnetic resonance; IPTG, isopropyl-?-d-thiogalactopyranoside; TGFa, transforming growth factor-a.
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