Influence of drying on the secondary structure of intrinsically disordered and globular proteins Michaela Hundertmark a,1,4 , Antoaneta V. Popova a,2,4 , Saskia Rausch a,3 , Robert Seckler b , Dirk K. Hincha a,⇑ a Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Potsdam, Germany b Institut für Physikalische Biochemie, Universität Potsdam, Karl-Liebknecht-Str. 24-25, D-14476 Potsdam, Germany article info Article history: Received 8 November 2011 Available online 1 December 2011 Keywords: Desiccation CD spectroscopy FTIR spectroscopy Intrinsically disordered proteins LEA proteins Protein secondary structure abstract Circular dichroism (CD) spectroscopy of five Arabidopsis late embryogenesis abundant (LEA) proteins constituting the plant specific families LEA_5 and LEA_6 showed that they are intrinsically disordered in solution and partially fold during drying. Structural predictions were comparable to these results for hydrated LEA_6, but not for LEA_5 proteins. FTIR spectroscopy showed that verbascose, but not sucrose, strongly affected the structure of the dry proteins. The four investigated globular proteins were only mildly affected by drying in the absence, but strongly in the presence of sugars. These data highlight the larger structural flexibility of disordered compared to globular proteins and the impact of sugars on the structure of both disordered and globular proteins during drying. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction LEA proteins have been found in plants, invertebrates and bacte- ria and have been associated with cellular dehydration tolerance. Most LEA proteins have been predicted to be IDPs, i.e. to have no stable structure under physiological, fully hydrated conditions [1,2]. The genome of Arabidopsis thaliana contains 51 genes encod- ing LEA proteins [3]. Three of these proteins have been shown to be unstructured in solution, while they fold into a-helices upon drying [4,5], similar to other LEA proteins [1]. Here we report the charac- terization of the secondary structure of five additional Arabidopsis LEA proteins forming the two small plant specific families LEA_5 (LEA20 and LEA35, Pfam PF00477) and LEA_6 (LEA15, LEA16 and LEA17, Pfam PF10714). The first LEA_6 gene (PvLEA18) was characterized as drought induced in the common bean (Phaseolus vulgaris) [6]. Apart from the fact that the protein does not stabilize enzymes during desicca- tion in vitro [7] it is functionally and structurally uncharacterized. Of the three homologous genes in Arabidopsis, LEA15 is expressed specifically in seeds and strongly induced by the phytohormone ABA. LEA16 is induced under salt stress in leaves, while LEA17 is only highly expressed in flower buds, but not regulated by any stress treatments [3]. The LEA_5 group in Arabidopsis comprises only two genes. LEA20 (EM6) is expressed constitutively in all investigated tissues, but is induced in leaves under salt stress and after ABA treatment, while LEA35 (EM1) is seed specific [3]. LEA_5 proteins from other plant species are unstructured in solution [8–11] and protect the enzyme lactate dehydrogenase against inactivation during desicca- tion [12]. Due to the lack of a stable secondary structure, IDPs are gener- ally more flexible than globular proteins [13], but the structure of globular proteins can also be influenced by drying and sugars can stabilize the structure and function of globular proteins [14,15]. The influence of sugars on the structure of dry LEA proteins has only rarely been reported. To investigate the differences in the structural responses of globular proteins and IDPs to drying in the absence or presence of sugars, we used CD and FTIR spectros- copy to compare the secondary structures of the five Arabidopsis LEA proteins of the LEA_5 and LEA_6 families with those of four globular proteins known to contain different amounts of a-helices and b-sheets. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.11.067 Abbreviations: ABA, abscisic acid; BSA, bovine serum albumin; CD, circular dichroism; FTIR, Fourier-transform infrared; GRAVY, grand average of hydropathy; IDP, intrinsically disordered protein; LEA, late embryogenesis abundant; LG, b-lactoglobulin; RFO, raffinose-family oligosaccharide; RNaseA, ribonuclease A; Suc, sucrose; Thau, thaumatin; Ver, verbascose. ⇑ Corresponding author. Fax: +49 331 567 8250. E-mail address: hincha@mpimp-golm.mpg.de (D.K. Hincha). 1 Present address: UMR 1191 Physiologie Moléculaire des Semences, 16 Bvd Lavoisier, 49045 Angers Cedex 1, France. 2 Permanent address: Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. 3 Present address: Institut für Chemie, Technische Universität Berlin, Franklinstr. 28/29, D-10587 Berlin, Germany. 4 These authors contributed equally. Biochemical and Biophysical Research Communications 417 (2012) 122–128 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc