A backbone-reversed form of an all-beta alpha-crystallin domain from a small heat-shock protein (retro-HSP12.6) folds and assembles into structured multimers.

Shukla, Anshuman and Raje, Manoj and Guptasarma, Purnananda (2003) A backbone-reversed form of an all-beta alpha-crystallin domain from a small heat-shock protein (retro-HSP12.6) folds and assembles into structured multimers. The Journal of biological chemistry, 278 (29). pp. 26505-10. ISSN 0021-9258

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Abstract

The structural consequences of polypeptide backbone reversal ("retro" modification) remain largely unexplored, in particular, for the retro forms of globular all-beta-sheet proteins. To examine whether the backbone-reversed form of a model all-beta-sheet protein can fold and adopt secondary and tertiary structure, we created and examined the recombinant retro form of a 110-residue-long polypeptide, an alpha-crystallin-like small heat-shock protein, HSP12.6, from C. elegans. Following intracellular overexpression in fusion with a histidine affinity tag in Escherichia coli, purification under denaturing conditions, and removal of denaturant through dialysis, retro-HSP12.6 was found to fold to a soluble state. The folded protein was examined using fluorescence and CD spectroscopy, gel filtration chromatography, non-denaturing electrophoresis, differential scanning calorimetry, and electron microscopy and confirmed to have adopted secondary structure and assembled into a multimer. Interestingly, like its parent polypeptide, retro-HSP12.6 did not aggregate upon heating; rather, heating led to a dramatic increase in structural content and the adoption of what would appear to be a very well folded state at high temperatures. However, this was essentially reversed upon cooling with some hysteresis being observed resulting in greater structural content in the heated-cooled protein than in the unheated protein. The heated-cooled samples displayed CD spectra indicative of structural content comparable to that of any naturally occurring globular protein. Attempts are being made to refine crystallization conditions for the folded protein.

Item Type:	Article
Additional Information:	Copyright of this article belongs to ASBMB.
Subjects:	Q Science > QD Chemistry
Depositing User:	Dr. K.P.S.Sengar
Date Deposited:	05 Jan 2012 15:03
Last Modified:	05 Jan 2012 15:03
URI:	http://crdd.osdd.net/open/id/eprint/247

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