Mimicking the plant-cell interior under water stress by ... · Jean-Marie Mouillon1#, Sylvia K...

Mimicking the plant-cell interior under water stress by

macromolecular crowding: disordered dehydrin proteins are highly

resistant to structural collapse.

Corresponding author:

Pia Harryson

Department of Biochemistry and Biophysics

Arrhenius Laboratories for Natural Sciences

Stockholm University

S-106 91 Stockholm, Sweden

Tel: + 46 8 164238 Fax: + 46 8 153679

[email protected]

Research area:

Biochemistry and/or Environmental stress and adaptation

1

Plant Physiology Preview. Published on October 10, 2008, as DOI:10.1104/pp.108.124099

Copyright 2008 by the American Society of Plant Biologists

www.plantphysiol.orgon July 7, 2018 - Published by Downloaded from Copyright © 2008 American Society of Plant Biologists. All rights reserved.

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http://www.plantphysiol.org

Jean-Marie Mouillon1#, Sylvia K Eriksson* and Pia Harryson*

1 Umeå Plant Science Centre

Department of Plant Physiology

Umeå University

S-901 87 UMEÅ

SWEDEN

# Present address:

Fluxome Sciences A/S

Diplomvej 378

DK-2800 Lyngby

Denmark

* Department of Biochemistry and Biophysics

Arrhenius Laboratories for Natural Sciences

Stockholm University

S-106 91 Stockholm, Sweden

Tel: + 46 8 164238 Fax: + 46 8 153679

2 www.plantphysiol.orgon July 7, 2018 - Published by Downloaded from

Copyright © 2008 American Society of Plant Biologists. All rights reserved.


Financial source:

This project has been funded by the Swedish Research Council for the Environment,

Agricultural Sciences and Spatial Planning (P.H.) and the Carl Tryggers Foundation (P.H.)

and the Lawski Foundation (S.E.).

Corresponding author:

[email protected] Fax: + 46 8 153679



mailto:[email protected]


ABSTRACT

The dehydrins are a class of drought-induced proteins in plants that lack a fixed three-

dimensional structure. Their specific molecular action, as well as the reason for their

disordered character, is as yet poorly understood. It has been speculated, however, that the

dehydrins are tuned to acquire a biologically active structure only under the conditions at

which they normally function, i.e. upon dehydration. To test this hypothesis, we here

investigate the effect of reduced water content and macromolecular crowding on three

dehydrins from Arabidopsis thaliana. As a simplistic model for mimicking cellular

dehydration we used polyethylene glycol (PEG), glycerol, and sugars which plants naturally

employ as compatible solutes, i.e. sucrose and glucose. Macromolecular crowding was

induced by the large polysaccharides ficoll and dextran. The results show that the dehydrins

are remarkably stable in their disordered state and are only modestly affected by the solvent

alterations. A notable exception is the dehydrin Cor47 which shows a small, intrinsic increase

in helical structure at high concentrations of osmolytes. We also examined the effect of

phosphorylation but found no evidence that such post-translational modifications of the

dehydrin sequences modulate their structural response to osmolytes and crowding agents.

The results suggest that the dehydrins are highly specialised proteins that have evolved to

maintain their disordered character under conditions where unfolded states of several globular

proteins would tend to collapse.

Key words: dehydrin, intrinsically disordered protein, macromolecular crowding, circular

dichroism (CD)




INTRODUCTION

The loss of water from plant cells during drought and freezing triggers a series of adaptation

processes. One of these is the production of osmolytes such as sucrose, proline and betaine

(Hasegawa et al., 2000; Wang et al., 2003). As a consequence, the already crowded interior

of the cell gets even denser and the physiological processes need to adjust to elevated

backgrounds of non-reacting macromolecules and steric confinement. In some cases the

increase in macromolecular density can be radical, going from 300–400 g/L under ideal

growth conditions to >900 g/L upon severe desiccation (Ellis, 2001; Bryant et al., 2005).

Such cellular crowding does not only present a challenge in terms of modulating diffusion-

based chemical reactions (Ellis, 2001; Minton, 2005; 2006), but will also affect structural

integrity. Crowding promotes protein assembly by favouring compact conformations over

extended ones, and the reduction in bulk water will directly influence membrane topology and

dynamics. The molecular conditions in water-starved plant cells are therefore very different

from those normally used to characterise biochemical processes in vitro. It has been put

forward that in such situations the biological function of proteins could be regulated through

crowding-induced conformational changes (Hall, 2006; Hall and Dobson, 2006). Under these

extreme conditions the plant cells respond by expressing a characteristic class of stress

proteins, the dehydrins (Close, 1997; Garay-Arroyo et al., 2000) (Fig. 1).

The function of dehydrins is as yet unknown. A complicating factor, from a biochemical

perspective, is that they seem to lack ordered structure (Lisse et al., 1996; Soulages et al.,

2003; Mouillon et al., 2006). Even so, all dehydrins contain, by definition, at least one copy

of a highly conserved sequence segment, the K-seg, and may or may not include other

conserved sequences called the S-seg, the Y-seg (Close, 1996) and the ChP-1 (Mouillon et al.,

2006). Since the dehydrins accumulate at very high concentrations inside cells (Bartels and

Salamini, 2001) they are unlikely to be signal molecules or conventional enzymes. The role

of the dehydrins is probably of a more general nature. For example, they may stabilise

membranes, act as chaperones or by other means buffer the altered solvent properties inside

water-stressed cells (Close, 1996; Garay-Arroyo et al., 2000; Boudet et al., 2006). As a clue

to how the stress response is orchestrated molecularly, it has been suggested that the

functional structure of the dehydrins is induced by their hydration status (Boudet et al., 2006).

Accordingly, the disordered appearance of the dehydrins may be converted into active, three-

dimensional structures provided that the conditions inside a drought-stressed plant cell are

sufficiently well reproduced. The disordered conformations seen under dilute conditions




would then represent the denatured state and, consequently, tell very little about the

dehydrin’s actual biological function. This idea is supported by the observations that water

can modulate ionisation potentials, pKA values and protein-binding potentials (Kornblatt and

Kornblatt, 2002). Other classes of disordered proteins have been found to participate in

molecular recognition, regulation and cell signalling (Dunker et al., 2002; Uversky et al.,

2005). Function is, then, induced by folding of the disordered protein into a structured state

where the functional groups are placed at the right locations for interactions. Folding can be

initiated (or templated) by binding to a biological partner such as other proteins, DNA, RNA,

or metals (Dunker et al., 2002; Tompa and Csermely, 2004).

Of particular interest with respect to the dehydrins is that the conformational changes are

sometimes conditional, i.e. the protein structure responds to changes in the environment such

as temperature (McNulty et al., 2006), pH (Tornroth-Horsefield et al., 2006), availability of

water (Luo and Baldwin, 1997) or macromolecular crowding (Minton, 2005a; 2005b; Hall,

2006). Disorder is used to allow the function to be switched on and off, e.g. a 10% change in

the concentration of intracellular proteins can lead to changes up to a factor 10 in the activity

of molecular regulatory species (Al-Habori, 2001). Computational studies have recently

demonstrated that the effect of crowding, despite its underlying complexity, can be simply

mimicked by encapsulation of the proteins in spherical pores (Klimov et al., 2002; Cheung

and Thirumalai, 2007). This freedom in how the crowding pressure can be applied suggests

that simplified experiments where crowding effects are tested by addition of large branched

polymers, such as ficoll and dextran, could produce valid approximations of the in vivo

condition. In fact, it has recently been verified by nuclear magnetic resonance (NMR) that the

effect of such crowding agents in vitro reproduces the intracellular conditions fairly well

(Selenko et al., 2006).

In this study, we examine whether the addition of macromolecular crowding agents in vitro

can force the dehydrins to fold. The progressive substitution of water with various polymers

is performed as a simplistic model of how the cellular interior changes upon

drought/desiccation. As representative crowding agents we chose a series of natural and

synthetic osmolytes, the effects of which are well characterised in other systems. To

distinguish the response of the full-length dehydrins from that of their constituent K- and

ChP-1 segments, the latter have been analysed separately in the form of peptides. The results

show that both the full-length dehydrins, and the isolated K- and ChP-1 segments, retain their




disordered character also at extremely high levels of osmolytes. Moreover, the propensity of

the dehydrins to undergo unspecific collapse, c.f. amorphous glass, appears even lower than

that of denatured states of globular proteins. Taken together, these observations suggest that

the dehydrin sequence is highly evolved and adapted to remain disordered also under

conditions of severe dehydration. In this respect, the dehydrins are different from the class of

disordered proteins that rely on folding to become functional. The function of the dehydrins

is therefore likely to lie in the interactions of the conserved segments with their specific

biological targets.




RESULTS

Analysis of structural transitions, using circular dichroism. Circular dichroism is a

common spectroscopic method for detecting secondary structure in proteins, where α-helices,

β-sheets and random coils display characteristic spectra at wavelengths 190–250 nm (Fig. 2).

Accordingly, the CD spectrum of a typical folded protein is a characteristic structural

fingerprint made up by the sum of its constituent secondary-structure elements. In

physiological buffer the CD spectra of the dehydrins, however, resemble those of unfolded

global proteins (Mouillon et al., 2006) (Figs. 2 and 3). This signal differs somewhat from that

of the pure random coil since it contains contributions from dynamic, residual secondary-

structure and polyproline helices II (Mouillon et al., 2006). In this study we used this coil-

like signal as a reference to test whether a more ordered structure is induced upon addition of

solutes that mimic the solvent conditions experienced inside a living plant cell. For example,

the induction of helical structure in the dehydrins upon addition of trifluoroethylene (TFE)

(Mouillon et al., 2006) is seen as a characteristic decrease in the CD signal around 222 nm in

combination with an increase at around 200 nm (cf. Fig. 2).

The natural stress solutes have little impact on the dehydrin structure. Under conditions

of stress, plants are known to produce a variety of compatible solutes such as betaine (Chen

and Murata, 2002), proline (Yoshiba et al., 1997) and different sugars (Ingram and Bartels,

1996; Hoekstra et al., 2001). Of particular interest for this study are sucrose and glucose

which are naturally produced in response to low temperature and drought (Hoekstra et al.,

2001). To investigate whether increased levels of these sugars contribute to modulating the

conformational properties of the dehydrins, we undertook titration experiments (followed by

CD) in which the sucrose and glucose content was varied between 0% and 80%.

Interestingly, the results suggest that sucrose and glucose have only a marginal effect on the

disordered character of Cor47, Lti29 and Lti30 (Fig. 3). Even at very high concentrations of

sugar, the CD signal at 222 nm remains largely the same as in physiological buffer, apart from

a small but significant decrease in ellipticity at between 210 nm and 230 nm for Cor47. At

shorter wavelengths the characteristic minimum of the random-coil signal tends to be

progressively weakened. Under the extreme solvent conditions used here, however, these

spectral changes should be considered relatively modest. The increased CD signal at 200 nm

possibly reflects a small increase in helical structure. Such increased helical propensity is

expected upon weakening of the hydrogen bonding between water and the protein backbone




at high concentrations of solute. Even so, the strength of this signal is too small to imply that

the observed effect has any bearing on the physiological function of the dehydrin proteins.

The effect of glycerol follows the intrinsic α-helical propensities of the polypeptide

chains. In comparison with the small sugars sucrose and glucose, the structural effect of

adding glycerol is much more pronounced. Above 50% glycerol, the CD spectra of all

proteins in this study indicate a significant increase in the occupancy of α-helical structure

(Fig. 3). Moreover, this effect seems to be larger for Cor47 than for Lti29 and Lti30. The

result is in good accord with the intrinsic helical propensities of the three proteins. The

relative response of the different dehydrins to additions of glycerol follows precisely that of

the helical inducer TFE (Mouillon et al., 2006). In other words, the magnitude of the glycerol

response can be correlated to the protein’s intrinsic α-helical propensity, i.e. Cor47 > Lti30 =

Lti29. To illustrate this correspondence more clearly, in Figure 4 we compare the effect of

adding 50% glycerol with that of adding 50% of the strong helical inducer TFE. Although the

magnitude of the glycerol response is in general much smaller than that of the TFE response,

the features of the difference spectra appear very much the same. Analysis of the different

CD spectra using the structural prediction program Dichroweb (Whitmore and Wallace, 2004)

shows that Cor47 is 15% α-helical in 50% glycerol while for corresponding value of Lti29

and Lti30, is 7.5%. Upon comparison with other proteins, it is apparent that this low α-helical

propensity matches the background levels of most polypeptide sequences and supports the

notion that the extent of residual α-helical structure observed in this study is unlikely to

constitute the key to the molecular action of the dehydrins in vivo.

The reason why the α-helical response is higher with glycerol than with sucrose and glucose

could simply be that the former is a stronger helical inducer (Table 1). Even so, we observed

that Cor47, which is most sensitive to glycerol, displays a CD change that deviates from an

ideal two-state transition into α-helical structure. Upon titration with glycerol the CD

spectrum of Cor47 does not display a single iso-elliptic point, as is typical for TFE titrations,

but acquires the α-helical signal in two steps, i.e. A ⇔ B ⇔ C (Fig. 5). First, the CD

difference spectra indicate a mixed α-helical structure at between 0% and 75% glycerol.

Then, the spectrum of Cor47 undergoes a second transition to a more characteristic α-helical

signal at >80% glycerol. Apparently, Cor47 first responds to glycerol by adopting low levels

of a mixed α -helical structure. Finally, at very high glycerol concentrations, this mixed




structure is converted to more purely α-helical structures (Fig. 5). These α-helical structures

could first form locally and then extend to cover a larger part of the sequence. Analysis using

Dichroweb (Whitmore and Wallace, 2004) shows 22% helical structures at 75% glycerol and

48% helical structure at 95% glycerol. Thus, by quantitative comparison, the results suggest

that the degree of α-helical structure induced by 95% glycerol is similar to that in 25% TFE

(Mouillon et al., 2006). It should also be noted that at 25% TFE the level of α-helical

structure of Cor47, as analysed using CDPro has levelled out at a maximum value of 47%

(Mouillon et al., 2006). Therefore, Cor47 is likely to acquire a significant content of local

structure in 95% glycerol.

The corresponding signal for Lti29 and Lti30 is much smaller. This indicates that the

sensitivity to glycerol is a specific feature of the Cor47 sequence and not a common,

functional property of the dehydrin proteins as a group.

Structural collapse in the presence of polyethylene glycol: comparison with the unfolded

states of globular proteins. The tendency of the polypeptide chain to collapse into compact

states at high concentrations of osmolytes such as glycerol is not unique to Cor47 but has

generally been observed for small peptides (Venkatesu et al., 2007) and denatured states of

globular proteins (Fig. 2) (Foord and Leatherbarrow, 1998; Silow and Oliveberg, 2003). If

the pressure to minimise the surface area is sufficiently high, any unfolded polypeptide is

expected to collapse into compact forms that need not always resemble the native structures

(Bryngelson and Thirumalai, 1996). The ability to resist such unspecific coil collapse under

physiological conditions is a key feature that distinguishes natural proteins from randomly

assembled polypeptides (Bryngelson and Thirumalai, 1996). This property renders the

denatured states of natural proteins flexible and facilitates their folding into unique native

structures. In an attempt to benchmark the collapse propensity of the dehydrin proteins along

such a general scale, we have monitored the effect of adding PEG, following the conditions

used in protein folding studies. Again, the structural response was highest for Cor47,

followed by Lti30 (Fig. 6). Least affected was Lti29. As the effect of PEG 4,000 and PEG

6,000 resembles that of glycerol, the mode of action of these osmolytes is most likely similar.

Analysis of the different PEG spectra using Dichroweb reveals that the induced structure is a

mix of β-sheets and α-helices (14% helical in 50% PEG 6,000). It is interesting to note,

however, that the susceptibility of the dehydrins to change their structure in the presence of

osmolytes generally seems lower than for denatured states of small globular proteins (c.f.




Figs. 2 and 6). Although the globular proteins typically adopt their folded states in highly

concerted two-state transitions, i.e. without populating intermediates, their denatured states

have been observed to become trapped in compact, sterically restricted conformations already

at 30% PEG or ethylene glycol (EG) (Qu et al., 1998; Silow and Oliveberg, 2003). In the

case of the archetypical two-state protein CI2, the acid-denatured state shows striking changes

in the CD spectrum at 70% PEG (Fig. 2) (Silow and Oliveberg, 2003). Comparable effects

are only seen with Cor47, while the impact on the CD spectra of Lti29 and Lti30 is less

pronounced. Consistently, the dehydrins also show very limited response to sulphate, which

is another well-known misfolding and aggregation agent for globular proteins (Otzen and

Oliveberg, 1999). Therefore, when it comes to resisting unspecific collapse, it is apparent that

the dehydrins are equally, if not more, robust than the unfolded states of globular proteins.

The response of the dehydrins to crowding agents ficoll and dextran. The preferred way

to examine the effect of macromolecular crowding would be to subject the dehydrins to high

concentrations of other proteins, RNA and colloidal particles. In vivo, the total concentration

of proteins and RNA is in excess of 300 mg/mL (Zimmerman and Trach, 1991). An intrinsic

problem with such experiments, however, is that the spectroscopic handle of the dehydrin

structure, in this case the CD signal, would disappear against the much larger background of

the co-solutes. As an alternative, we used the large polysaccharides ficoll (70 kDa) and

dextran (40 kDa and 70 kDa). Interestingly, the presence of these crowding agents showed no

appreciable effect on the dehydrin proteins. At 50% ficoll or dextran the CD spectra of Lti29

and Lti30 were virtually identical to those in pure buffer while the spectrum of Cor47 showed

an increase in the 200 nm region similar to that in glucose and sucrose (Fig. 6). Although the

changes in the lower wavelengths of the Cor47 spectrum are difficult to rationalise

structurally (Mouillon et al., 2006), the results on the whole underline the dehydrins’

resistance to altering their disordered character in response to co-solute exposure. Therefore,

the disordered character of these proteins could be a key requirement for their function and

this property seems to be maintained in the crowded interior in the dehydrated plant cell.

Combinations of sugars and osmolytes. Although tests of the pure, individual osmolytes

and crowding agents are required for experimental stringency, these experiments need not

represent the complex conditions inside stressed cells where a multitude of co-solutes act in

concert. To examine whether there are any synergistic effects of mixing sugars, osmolytes

and crowding agents, we also screened the response of the dehydrins to a series of mixtures.




As a base, we used 25% glycerol, and added to this an additional 25% of the other

compounds. Notably, these systematic mixtures did not contribute to enhancing or changing

the effect on the dehydrin CD spectra: in all cases the observed effect was the combination of

the signals from the isolated compounds. To further rule out the possibility of a delayed

response to the solvent perturbations, the samples were re-run after 24 hours but without any

detectable difference in the CD spectra (data not shown). These findings are in line with the

recent conclusions by Holthauzen and Bolen (2007).

The conserved segments behave approximately like the full-length proteins. Even though

the response of the full-length proteins to solvent additives is generally small, it is possible

that this response stems from a strong structural effect in a small part of the sequence. The

prime candidates for such local structure formation would then be the sequence regions

containing the conserved segments. These are the K-segment (K-seg:

EKKGIMDKIKEKLPG) and the charged-peptide 1 (ChP-1: EEGEDGEKKKKEKKKKI), in

other words, the charged part following the serine-rich segment found in Cor47 and Lti29, i.e.

in the SKn type of dehydrins (c.f. Fig. 1). The function of these characteristic and repeatedly

occurring sequence stretches is still unknown. In partial contrast to the full-length dehydrins,

peptides of the K-seg and ChP-1 show the most pronounced structural response to PEG 6,000

and not to glycerol (Fig. 7). At 50% PEG 6,000, structural prediction analysis using

Dichroweb (Whitmore and Wallace, 2004) suggests an α-helical content of 9% for K-seg and

11% for ChP-1. By comparison, the effect of PEG 4,000 is substantially smaller than that of

PEG 6,000 and similar to the structural response induced by glycerol.

The different effects of PEG 6,000 and PEG 4,000 are interesting since the PEGs are

substantially larger than the peptide fragments and the full-length dehydrins and would,

accordingly, be expected to affect these in an equivalent way. Ficoll and dextran are also

considerably larger than either peptide but give as low a response as with the full-length

dehydrins. Upon titration with glycerol, the K-segment and ChP-1 both show an

approximately linear decrease of the CD signal at 222 nm. Therefore, K-seg and ChP-1

behave very much like the full-length proteins Lti29 and Lti30 and lack the characteristic late

transition of Cor47 at 95% glycerol (data not shown).

Phosphorylation has only a marginal effect on the dehydrin structures. Recombinantly

expressed dehydrins are not phosphorylated when purified from E. coli (data not shown).




However, dehydrins are known to undergo phosphorylation both in vivo and in vitro (Jiang

and Wang, 2004; Alsheikh et al., 2005; Brini et al., 2007; Rohrig et al., 2006). To investigate

the structural impact of phosphorylation, we treated the dehydrins with casein kinase II. Both

Cor47 and Lti29, but not Lti30, became phosphorylated. Such selective phosphorylation of

the different dehydrins is in good agreement with previous reports (Alsheikh et al., 2005).

The results show that phosphorylation of Lti29 has no detectable effect on the CD spectra

under any of the solvent conditions tested in this study (data not shown). In the presence of

50% glycerol, however, phosphorylation of Cor47 produces a small decrease in the coil

content, indicated by the CD spectrum below 210 nm (Fig. 8). Notably, this signal cannot be

seen in the presence of glucose or PEG 4,000, suggesting that the structural relevance is after

all very small (data not shown). The effect of phosphorylation on the dehydrin structure is

therefore very small and does not significantly enhance the response to osmolytes or crowding

agents in vitro.




DISCUSSION

Crowding has no observable role in modulating the dehydrin structures. How the

molecular function of proteins and membranes is maintained in organisms during

environmental stress is a poorly understood area of biology that relates to the detailed

interplay between solvent and solute molecules. The latter include supra-structures of lipids,

proteins and carbohydrates. In plants, drought can reduce the water content to only a fraction

of the normal levels, often accompanied by compensating production of osmolytes and

changes in protein composition. The benefit of such stress-induced adjustments of the intra-

cellular conditions is to safeguard biological viability. At a molecular level, however, not

only is it challenging to reproduce this complex response in vitro but the response is also

challenging to rationalise mechanistically: the interior of cells can as yet not be treated

exactly as in vivo and simplifications are needed. In the present study of the dehydrin

proteins, one such simplification is to parameterise the experimental conditions in terms of

macromolecular crowding or excluded volume effects. As the water content of the cell is

decreased, the background of other molecules is enriched, leading to a geometric restriction of

the available space for polypeptide chains. Typically, proteins function under conditions

where the solute concentration is around 400 g/L (Zimmerman and Minton, 1993) and in

severely dehydrated plant cells this number can be substantially higher (Bartels and Salamini,

2001). Such increased background of crowding molecules, i.e. the osmolytes and all other

solvent components taken together, is expected to have an important impact on the cellular

processes (Ellis, 2001a; 2001b). By simply competing for volume, macromolecular crowding

favours compact states of the polypeptide chain and increases the stability of native proteins,

provided that no competing misfolded conformations are produced. Accompanying this

general stability effect, crowding increases also the occupancy of compact denatured species,

which, eventually, leads to a retardation of the protein folding rates due to increased

reconfiguration times. As the normally flexible and dynamic polypeptide chain gets

entangled in compact conformations its internal movements become energetically more

cumbersome (Bryngelson and Thirumalai, 1996; Oliveberg and Wolynes, 2005). It has been

put forward that such collapse/expansion of certain inert biopolymers could actually be used

as a means to regulate function of proteins in biological environments via crowding (Hall,

2006; Hall and Dobson, 2006).




The effect of macromolecular crowding on the protein structure is therefore expected to

resemble that of small stabilising agents such as sodium sulphate, Na2SO4. However, the

efficiency of macromolecular crowding in promoting protein stability is relatively modest and

is substantially smaller than that of Na2SO4. The equilibrium constant for RNAse T1 was

only shifted 7.5-fold towards the native state in 400 mg/mL dextran 70 (Qu and Bolen, 2002),

while the corresponding effect of Na2SO4 on the protein L23 is several times larger (Hedberg

and Oliveberg, 2004). Consistent with the earlier observation that Na2SO4 has no appreciable

impact on the dehydrin structures (Mouillon et al., 2006), we could not discern any

compelling effect upon crowding by the large branched sugars ficoll and dextran (Fig. 6).

The only outcome is a small change in the low wavelength region of the CD spectra. This

change could possibly reflect a relaxation of PII helices towards more flexible coil

conformations. By comparison, induction of a classic secondary structure, such as α-helices

or β-sheets, would lead to a reduction in the 200 nm signal with a simultaneous increase in the

minimum around 220 nm (c.f. Fig. 2). However, it is necessary to consider also the size of

the tested crowding agent in relation to that of the dehydrins. At a first approximation, a

small crowding agent is expected to get closer to the protein and, hence, occupy volume more

efficiently compared with a large crowding agent. Even so, the dehydrin structures are

equally indifferent to the smaller sugars glucose and sucrose (Figs. 3 and 4).

We could not distinguish any correspondence between changes in CD spectra and the size of

any of the added solutes in this study. The disordered nature of the dehydrin polypeptide

seems to persist also when the available space is geometrically restricted. A plausible

explanation for this result is that the dehydrins do not easily adapt compact conformations,

either in the form of uniquely defined folded states, as has been observed for several other

disordered proteins (Dawson et al., 2003; Love et al., 2004; Fink, 2005), or in the form of

unspecifically collapsed conformations that are sometimes observed in the denatured states of

globular proteins (Lattman et al., 1994; Otzen and Oliveberg, 2002; Ittah et al., 2004). The

behaviour is in good agreement with earlier reports on the disordered c-Fos and p27Kip1

proteins, where crowding with differently sized dextrans (9 kDa, 37.5 kDa or 77 kDa) and

ficoll 70 at concentrations of up to 250 g/L was not sufficient to induce ordered structure

(Flaugh and Lumb, 2001). The dehydrins resemble also the Parkinson-related α-synuclein

that remains disordered in the crowded interior of E. coli cells and at high concentrations of

BSA (McNulty et al., 2006). In some contrast, the transcription factor regulator FlgM partly




gains structure under the corresponding conditions (Dedmon et al., 2002), and FlgM also

gains structure upon binding to its biological target (Daughdrill et al., 1997). These

differences have been taken to exemplify two classes of disordered proteins: those that are

functionally tuned to remain disordered in the crowded interior of the cell (Flaugh and Lumb,

2001), and those that are not. According to the data in Figures 3–8, the dehydrins belong to

the former category. Taken together, the effect of macromolecular crowding on disordered

protein conformations therefore seems to be variable and needs to be tested in each separate

case. A brief summary of the crowding effects on other disordered proteins and denatured

globular proteins is given in Table 1. Another effect of water loss is increased ionic strength.

We have earlier demonstrated, however, that increased ionic strength or elevated levels of

metal ions has no appreciable impact on the dehydrin structure in vitro (Mouillon et al.,

2006).

Preferential protein-solute exclusion increases the sampling of the local α-helical

structure. The conformational transitions underlying the altered CD signals of Cor47 in the

presence of glycerol and PEGs are therefore likely to have a molecular origin different from

pure crowding. From a functional perspective it is reasonable to examine whether these

molecular factors are somehow coupled to changes in the accessibility of water. For example,

they may be manifested in altered structure of the protein hydration shells. However, such

water-mediated effects are intrinsically difficult to mechanistically rationalise because of the

complexity of aqueous solutions. A simplified view is offered by weak interaction models.

These models assume that the solute molecules, typically osmolytes, are either accumulated

or excluded from the protein-solvent interface. In the event that the osmolyte is preferentially

attracted to the protein surface, this will favour extended conformations and promote

destabilisation according to mass action (Schellman, 2002). Examples of such destabilising

osmolytes are the denaturants urea and guanidium chloride (GdmCl). Conversely, if the

osmolyte is preferentially excluded from the protein surface, this will lead to minimisation of

the protein-solvent interface area and protein stabilisation (Bolen and Baskakov, 2001). Such

stabilising, or protective, osmolytes are sugars, free amino acids and glycerol. The reason for

the “affinity differences” between the osmolytes seems partly related to the degree by which

they can form favourable polar interactions with the polypeptide backbone (Street et al.,

2006). Computational (Street et al., 2006) and experimental (Qu and Bolen, 2002) analysis

suggests that the order of stabilising capacity of the osmolytes used in this study is sucrose >

sorbitol > proline > glycerol. From this perspective, it is interesting to note that the osmolyte




that most efficiently perturbed the CD spectra of the dehydrin Cor47 is glycerol (Fig. 5). The

effect of 80% sucrose, on the other hand, is still relatively modest. An explanation for this

reversed order could lie in the somewhat unusual amino-acid composition of the dehydrin

sequences.

The computational ranking of the osmolyte effect is obtained from backbone interactions

alone (Street et al., 2006), which is in good accord with the stability effect on RNAse T1 (Qu

and Bolen, 2002) with a hydrophobic side-chain content typical of globular proteins (cf. pdf

structure 1bu4). In contrast, the dehydrin sequences contain a relatively high proportion of

charged and polar side chains, presenting a higher degree of hydrogen bonding opportunities

with the surrounding solution molecules. This peculiarity could also explain why α-

synuclein, in contrast to the dehydrins, collapses upon addition of glucose (Morar et al.,

2001): α-synuclein contains approximately 40% hydrophobic residues while the dehydrins

contain just 15% (Lti30) to 30% (Cor47 and Lti29).

Even so, and regardless of the mechanistic origin of the accentuated glycerol effect of the

dehydrins, it is apparent from the CD data that the structural impact of adding osmolytes is

only small at physiologically relevant concentrations (Figs. 3 and 5). The same is true for the

fragments of their conserved segments (Fig. 7). The glycerol effect is not clearly manifested

unless the glycerol content is increased above 30%, a level that is likely to greatly exceed the

physiological concentrations. By comparison, the highest reported level of sucrose in vivo is

40% of the dry weight and this in a resurrection plant that more or less tolerates complete

desiccation (Ingram and Bartels, 1996). Moreover, the structural transition observed at

extreme concentrations of glycerol corresponds mainly to an increased sampling of the α-

helical structure, which is in good accordance with the intrinsic α-helical propensity of the

different dehydrin proteins (Fig. 1). The largest effect was observed for Cor47, followed by

Lti29 and Lti30, which precisely matched the gain of the α-helical structure in TFE titrations

(Mouillon et al., 2006). Consistently, a similar structural response was observed upon

addition of PEG 4,000 and PEG 6,000. These polymers are well-known protein stabilisers

(Kornblatt and Kornblatt, 2002; Kozer and Schreiber, 2004; Ren et al., 2006) that have also

been observed to unspecifically collapse the denatured states of globular proteins (Silow and

Oliveberg, 2003). On this basis, we conclude that the exertion of pressure to minimise the

solvent-accessible surface area of the dehydrin chains has no appreciable effects on their




tertiary structures. They generally remain disordered. The effect on the local α-helical

content merely follows the intrinsic α-helical propensity of the polypeptide chains. The latter,

however, is more likely to reflect the generic properties of the polypeptide chain than specific

function traits.

Possible interactions between dehydrins and lipid membranes. A frequently proposed

function of dehydrins and other LEA proteins is as membrane stabilisers. In essence, the

dehydrins would interact with membranes to modulate their topology and phase transitions

under stress, and in this process the dehydrins could also gain ordered structure. The idea is

supported by several findings of interactions between lipid vesicles and dehydrins and other

LEA proteins (Soulages et al., 2002; Koag et al., 2003; Soulages et al., 2003; Tolleter et al.,

2007; Kovacs et al., 2008). In some cases, as with the maize dehydrin DHN1, the membrane

interaction leads to conformational changes in the protein’s secondary structure, i.e. an

increase in the α –helical content (Koag et al., 2003). However, it is not clear to what extent

these effects can be transferred to other members of the protein family as, in contrast, the

interaction between Lti29 and negative charged lipids did not result in any conformational

changes (Kovacs et al., 2008). Also, the presence of lipids fail to induce any structural effect

on the soybean LEA protein GmD-19 and dehydrin GmDHN1 (Soulages et al., 2002;

Soulages et al., 2003). Although the involvement of the dehydrins in safeguarding membrane

structure during stress is indeed an attractive possibility, its generality remains to be

established.

Phosphorylation does not generate any significant structural changes of dehydrins.

Phosphorylation is a post-translational modification that is essential for regulation of cellular

functions. It was recently shown that most phosphorylation sites, 86%, are within disordered

regions of proteins (Collins et al., 2008). Even so, there are few studies on the effect of

phosphorylation on the structure of disordered proteins. Of particular interest for this study is

the fact that phosphorylation of the dehydrins occurs in vivo and has been reported to

modulate the coordination of calcium ions (Jiang and Wang, 2004; Alsheikh et al., 2005;

Brini et al., 2007; Rohrig et al., 2006). In this study only Cor47 and Lti29 could be

phosphorylated by casein kinase II. The result of this phosphorylation, however, had no

significant bearing on the structural behaviour of Cor47 and Lti29. Recent NMR studies of α-

synuclein have shown that phosphorylation at Ser 129 by casein kinase II increases the




disordered character of the protein. It was further shown that this structural alteration

increased the proteins’ tendency to self-associate (Sasakawa et al., 2007). As seen in Figure

8, the structure of Cor47 is left unaltered upon phosphorylation and also shows no tendency to

aggregate. Crowding by glycerol makes no difference. Even though Cor47 and Lti29 seem to

be structurally unchanged, it is important to note that phosphorylation can still play a key role

as regulatory factor for the interaction with other cellular targets.

Conclusions. From a physiological standpoint, the separate tests of crowding agents and

osmolytes constitute a fairly crude representation of the intracellular conditions where spatial

confinement and the presence of complex mixtures of background molecules act in concert on

the dehydrin structures. However, we failed to observe any synergistic structural effects in

mixtures of glycerol and the other solutes used in this study, lending further support to the

idea that the dehydrin structures are generally insensitive to changing solvent conditions.

Based on previous results, the structural insensitivity includes also changes of ionic strength

and levels of metal ions (Mouillon et al., 2006). This suggests that the dehydrin sequences

are evolved to remain flexible also under severe stress conditions, and that this property is an

integral requirement of their physiological function. Possibly, the high content of charged

and polar residues of the dehydrins effectively prevents structural collapse due to the

accompanying desolvation penalties of ionic moieties and hydrogen-bond partners. Such

transitions into compact glass-like species are otherwise characteristic of random polymers

(Bryngelson and Thirumalai, 1996) and to some extent also of denatured states of globular

proteins (Silow and Oliveberg, 2003). In both instances, the affected sequences generally

contain higher fractions of hydrophobic side chains than observed for the dehydrins.

Additional indications that the dehydrins are tuned to remain flexible under in vivo conditions

come from their high solubility and resistance to precipitation (Mouillon et al., 2006). As a

contrasting example, α-synuclein readily assembles in ordered fibrillar aggregates at elevated

protein concentrations or upon addition of PEG/osmolytes (Goers et al., 2003; Fink, 2006). In

discussions about the biological function of the dehydrins, it has been pointed out that their

unfolded nature would effectively maximise the coordination of water. According to NMR

experiments, this water is more tightly bound than in other disordered proteins (Bokor et al.,

2005). Some of the water seems tied up in bridging interactions within PII helices (Mouillon

et al., 2006). Upon removal of the coordinated water by evaporation, other proteins in the

LEA family have been observed to undergo structural rearrangements (Wolkers et al., 2001;




Goyal et al., 2003; Goyal et al., 2005). For example, the LEA protein AavLea1 increases its

content of α-helical structure (Goyal et al., 2003). Could then the dehydrins act as water

reservoirs that expel coordinated water to the surrounding medium (Boudet et al., 2006)?

Considering the finding that the in vivo concentration of dehydrins does not seem to exceed

2–4% of cytoplasmic proteins, however, the benefit of such water expulsion is likely to be

marginal (Roberts et al., 1993; Ceccardi et al., 1994; Close, 1996), at least in moderate

drought. More likely, the molecular action of the dehydrins is more specific. One possibility

is that the dehydrins provide structural support, either spatially by cross-linking with other

macromolecular constituents (Abu-Abied et al., 2006) or laterally by adhering to membrane

surfaces (Tolleter et al., 2007). Desiccation of the cell will not only alter the molecular

composition of the aqueous compartments of the cell but will also distort its physical

dimensions, including the shape and proximity of membrane structures.

It is easy to envisage that such global alterations could have a pronounced effect on the

colloidal processes, for example by promoting phase transitions, topological frustration and

altered dynamics of membrane fusion and budding. The role of the dehydrins may be to

intervene with these processes by filling out, and perhaps topologically organising, crevices in

bulging membranes and thereby to safeguard their structural integrity. Alternatively, the

dehydrins could support macromolecular structures of proteins, carbohydrates or nucleotides.

In any case, the elements of recognition with the cellular target are likely to be the conserved

segments. The remarkable capacity of the dehydrins to remain coil-like suggests that the high

flexibility of the sequence regions connecting these segments is an important part of their

function (Fig. 1): the conserved segments are organised as “hooks on a string” (Mouillon et

al., 2006). Notably, this arrangement not only restricts the spatial location of the conserved

elements but also increases their local concentration.




MATERIALS AND METHODS

Expression and heat fractionation. Expression, purification and identification of the

recombinant Arabidopsis thaliana dehydrin proteins were as described previously (Svensson

et al., 2000). Glycerol stocks of the different Escherichia coli strains were made and 200 μL

was spread on Luria agar (LA) plates with 150 μg ampicillin. The plates were kept at 37°C

and grown overnight. The cells were suspended and added to 1 litre (L) of LB medium

containing 50 μg/mL ampicillin. Expression was induced at an optical density (OD) of 0.3 by

adding isopropyl ß-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The

suspensions were kept at 37°C until grown to an OD of 0.8–1.0 (i.e. 2–4 hours). Cells were

harvested by centrifugation at 4,000 g for 45 min and the pellet stored at –20°C. The thawed

cells from 1 L cultures were resuspended in 25 mL of 20 mM Na2HPO4, pH 7.2, 150 mM

NaCl. To this was added 1 mM (final concentration) phenylmethylsulfonyl fluoride (PMSF)

and 0.1 mg/mL lysosyme and the suspension was left for 30 min on ice. Lysated cells were

sonicated 6 x 15 s and centrifuged at 9,000 g for 30 min. The supernatants were placed in a

water bath at 70°C for 20 min. To precipitate heat-denatured proteins the sample was

centrifuged for 30 min at 9,000 g and the supernatant stored at –80°C.

Purification of dehydrins. Purification of dehydrins on immobilised metal ion affinity

chromatography (IMAC) and the following step on ion exchange chromatography was

according to Svensson et al. (2000). The supernatants from heat precipitation were diluted

1:2 with 20 mM NaHPO4, pH 7.2, 1.85 M NaCl and 1 mM PMSF. The samples were loaded

on a 5 mL HiTrap IDA-Sepharose column (Pharmacia) connected to a FPLC system

(Pharmacia) and charged with 2 mL of 3 mg/mL CuSO4. Absorbance was read at 280 nm.

Before applying the sample, five volumes of 20 mM Na2HPO4, pH 7.2, and 1.0 M NaCl were

used to equilibrate the column. The same buffer (40 volumes) was used to wash off unbound

sample from the column. Fractions of 5 mL were collected for analysis during the whole run.

Elution was performed with 2 M NH4Cl in 20 mM Na2HPO4, pH 7.2, 1.0 M NaCl, in one

step. The column was then equilibrated with ten volumes of 20 mM Na2HPO4, pH 7.2,

followed by elution of the Cu with 10mM EDTA in 20 mM Na2HPO4, pH 7.2. Precipitation

of protein was done with 80% (NH4)2SO4 and proteins were collected by centrifugation at

18,000 g for 30 min. The different dehydrins were resuspended in 2 mL of the following




buffers: Lti29 and Cor47 in 20 mM Bistris, pH 6.0, Rab18 in 20 mM Tris-HCl, pH 8.0, and

Lti30 in 50 mM glycine, pH 9.0. The dehydrins were desalted on a PD-10 column

(Pharmacia) by the resuspension buffers. The achieved 3 mL fractions were put on an anionic

exchange column (1-mL Mono Q HR 5/5) (Pharmacia) in the case of Cor47 and Lti29, and on

a cationic exchange column (1-ml Mono S HR 5/5, Pharmacia) connected to an FPLC in the

case of Lti30, and absorbance was read at 280 nm. The columns were equilibrated with the

resuspension buffers and elution by a NaCl gradient from 0 to 0.5 M in respective buffer over

30 volumes. Fractions of 0.5 mL were collected during the runs for analysis. The purity was

tested by SDS-PAGE gel electrophoresis, and without exception the purified material was run

as untreated dehydrins from crude cell extracts (data not shown). An extensive identification

of the recombinant dehydrins has previously been published by Svensson et al. (2000).

Analysis by circular dichroism. Circular dichroism (CD) measurements were carried out

using a JASCO J-810 spectropolarimeter (JASCO, Japan) and presented as mean ellipticity

per residue. The scan rate was 20 nm/min at a band pass of 0.2 nm, and 20 mdeg sensitivity.

All samples were mixed 1 hour prior to the CD analysis and centrifuged at 12,000 g for 2 min

before filling the cuvettes. Protein concentration was 1 mg/mL in 20 mM MES (0.2 mm

cuvette). Unless stated otherwise, all runs were performed at 25°C.

Preparation of samples. Unless otherwise stated, concentrations are given as % referring to

g/L. Dextrans (average molecular weight of 40 kDa or 70 kDa) (Sigma), ficolls (average

molecular weight of 70 kDa) (Sigma) and polyethylene glycol (PEG) (average molecular

weights of 4,000 or 6,000 Da) (Sigma) were used in concentrations of up to 500–800 g/L. All

osmolytes, sugars and crowding agents were titrated in steps of 10–25%. Each titration series

was repeated at least twice. The exception was Lti30 where the titration steps were 15–25%

and titration ended at 50% due to low amount of high-purity Lti30 protein.

Phosphorylation of dehydrins. The dehydrins were phosphorylated by 5.4 units/mL of

casein kinase II (CKII) (Sigma) in 1 mM ATP, 20 mM Tris-HCl, 50 mM KCl and 10 mM

MgCl2 at pH 7.5 for 10 hours. The phosphorylation status of the dehydrins was tested by Pro-

Q Diamond Phosphoprotein Gel stain (Invitrogen) on 15% Tris-Hcl gels (Biorad) and the

background levels of protein were measured by SYPRO Ruby gel stain (Invitrogen). Stained

gels were visualised by an FLA-3000G (Fuji Photo Film Co. Ltd) and an ultraviolet (UV)

transilluminator. The phosphorylation status was measured on three different batches of




dehydrins, with identical results. For the structural analysis, the CKII was separated from the

dehydrins by precipitation at 70°C, followed by centrifugation. The effect of solvent

additives on the phosphorylated dehydrins was measured as described above.




LEGENDS TO FIGURES

Figure 1. Distribution of the conserved segments in the sequences of the dehydrins Cor47,

Lti29 and Lti30 from Arabidopsis. The dehydrins constitute group 2 of the large family of the

hydrophilic LEA proteins (Close, 1996; Bartels and Salamini, 2001).

The amino acid sequences are as follows:

COR47: MAEEYKNNVPEHETPTVATEESPATTTEVTDRGLFDFLGKKEEEVKPQETTTLESEFDH

KAGISEPELAAEHEEVKENKITLLEELGEKTEEDEENKPSVIEKLHRSNSSSSSSSDEEGE

EKKEKKKKIVEGEEDKKGLVEKIKEKLPGHHDKTAEDDVPVSTTIPVPVSESVVEHDHP

E IKEKLPGYHAKTTEEEVKKEKESDD

LTI29: MAEEYKNTYPEGETPKVATEESSAPEIKERGMFDFLKKKEEVKPQETTTLASEFEH

KTGISEPESFVAKHEEEEHKPTLLEGLHQKHEEEEENKPSLLDKLHRSNSSSSSSSDEE

GEDGEKPEEEEKKGFMDKIKEKLPGHSKKPEDSGVVNTTPLVETATPIADIPEEKKGF

MDKIKEKLPGYHAKTTGEEEKKEKVSD

LTI30: MNSHQNQTGVQKKGITEKIMEKLPGHHGPTNTGVVHHEKKGMTEKVMEQLPGHHGA

TGTGGVHHEKKGMTEKVMEQLPGHHGSHQTGTNTTYGTTNTGGVHHEKKSVTEKVM

EKLPGHHGSHQTGTNTAYGTNTNVVHHEKKGIAEKIKEGLPGHHGTHKTGTTTSYGNT

GVVHHENKST

Figure 2. Reference circular dichroism (CD) spectra of the common types of secondary

structure and random coil conformations. A. Pure α-helical structure (dotted line), pure β-

sheet structure (striped line) and random coil (solid line). B. The acid-denatured state of the

globular protein CI2 in pure buffer and in 70% of EG and PEG. Notably the structural

induction of CI2 by EG and PEG is not along the normal folding pathway but seems to

involve non-native interactions (Silow and Oliveberg, 2003).

Figure 3. Circular dichroism (CD) signal of Cor47, Lti29 in the presence of 80% sucrose,

glucose and glycerol, and Lti30 in the presence of 50% sucrose and glycerol, illustrating a

difference in response to the different sugars.

Figure 4. (Top panel): Difference spectra of Cor47, Lti29 and Lti30 in 50% glycerol.

(Bottom panel): Difference spectra of Cor47, Lti29 and Lti30 in 50% of the strong helical

inducer TFE. The results indicate that the effects of both glycerol and TFE reflect the

intrinsic helical propensity of the dehydrin sequences.




Figure 5. (Top panel): Circular dichroism (CD) spectra of the glycerol titration of Cor47.

The spectra show from top at 220 nm, 0%, 10%, 25%, 50%, 60%, 75% and 95% glycerol.

(Middle panel): Circular dichroism difference spectra showing that the structural induction of

Cor47 with glycerol occurs in two steps. Between 0% and 75%, glycerol Cor47 displays a

mix of β-sheet and α-helices while above 75% it displays mainly α-helices. (Bottom panel):

Changes in the CD signal at 200 nm and 222 nm upon titration of Cor47 with glycerol,

illustrating the transitions to mixed and pure α-helical structures, respectively.

Figure 6. Crowding of Cor47, Lti29 and Lti30 with polymers. The concentration of

polymers is 50%.

Figure 7. Circular dichroism (CD) difference spectra of the conserved segments of the

dehydrins, showing the effect of adding 50% polymers. (Top panel): The K-segment

(EKKGIMDKIKEKLPG) is present in all dehydrins. (Bottom panel): The ChP-1 segment

(EEGEDGEKKKKEKKKKI) is present in Cor47 and Lti29.

Figure 8. Circular dichroism (CD) spectra of phosphorylated Cor47 in pure buffer and in

50% glycerol.




Acknowledgements

We are most grateful to Professor Mikael Oliveberg, Stockholm University for the data

regarding CI2 and for helpful discussions.




Table 1. Effects of various crowding agents on intrinsically disordered proteins and denatured

globular proteins.




Co-solute Effect on disordered proteins

Effect on denatured globular proteins

Effect on other systems

Dextrans 1. No effects on p27KIP1 or c-FOS 3. 2. Folding of TCAM RNAse 4.

1. Increased refolding (kf) of lysosyme 1. 2. Change to molten globular state of cyt C 2.

1. Decreased unfolding (ku) of GAPDH 5.

Ficolls 1. No effects on p27KIP1 or c-FOS 3. 2. No effects on TCAM RNAse stability (KD-N) 4.

1. Minor effect on FKB12 stability 6. 2. Minor effect on refolding (kf) of lysosymes 7.

EG/PEGs 1. Accelerated fibrillation of α- synuclein 10.

1. Collapse of the coil state (decreased kf) of CI2 at high PEG 8. 2. Increased folding rate (kf) of CI2 at low PEG 8. 3. Decreased folding rate (kf) of CspB (EG) 9.

1. Decreased unfolding (ku) of GAPDH 5.

Glycerol 1. Accelerated fibrillation of α- synuclein 10.

1. Stabilised native state of RNAseA 11.

2. Increased refolding(kf) of GS with GroEL12. 3. Increased refolding (kf) of MHD 13.

Glucose 2. Induced collapse of α-synuclein 14.

1. Folding of Fe cyt C 14.

1. Stabilised dimeric chymotrypsin 15.

Sucrose 1. Contracted RCAM RNAse 17.

1. Increased refolding (kf) of GS 12. 2. Increased refolding (kf) of CI2 16. 3. Increased refolding (kf) of MHD 13.

2. Stabilised dimeric chymotrypsin 15.




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CHP-1K-seg S-seg

Cor47 SK3 (265 aa)Lti29 SK3 (260 aa)Lti30 K6 (193 aa)

Figure 1.



230 250210

wavelength (nm)

acid-denatured CI2

+70% EG

Figure 2.

A. B.

+70% PEG

random coil

α-helix

β-sheet



190 210 230 250 190 210 230 250 190 210 230 250

Buffer Suc 80%Gly 80%

Cor47

BufferSuc 80%

Gly 80% Lti29 Lti30

BufferSuc 50%

Gly 50%

-10

5

-5

-15

0

Figure 3.

wavelength (nm)



Cor47

Lti29

Lti30

Cor47Lti29

Lti30

Difference spectra Glycerol (50%)

Difference spectra TFE (50%)

0

8

6

4

2

-2

10

0

-10

-20

Figure 4.

190 210 230 250

wavelength (nm)



222nm

200nm

Cor47

Glycerol (%)

-4

-8

-12

-16

0 20 40 60 80 100

Cor47 + Glycerol 0%-95%

Difference spectraCor47-Glycerol

75% - 0%95% - 75%

5

0

-5

-10

-15

10

5

0

-5

190 210 230 250

Figure 5.

wavelength (nm)



BufferDex7000Fic7000PEG4000PEG6000

Buffer

Dex7000Fic7000PEG4000PEG6000

Dex4000 BufferDex7000Fic7000PEG4000

Cor47 Lti29 Lti305

0

-5

-10

-15

Figure 6.

190 210 230 250 190 210 230 250 190 230210 250wavelength (nm)



Dex7000Fic7000GlyPeg4000Peg6000

Dex4000

Difference spectra K-seg,Polymers 50%

Difference spectra,ChP-1Polymers 50%

Dex7000Fic7000GlyPeg4000Peg6000

3

2

1

0

-1

-2

3

2

1

0

-1

-2

Figure 7.

190 210 230 250wavelength (nm)



Cor47 phosphorylated + glycerol

0

-5

-10

-15

190 210 230 250

Cor47+Gly 50%

Cor47 phosphorylatedCor47Cor47 Phosphorylated + Gly50%

wavelength (nm)

Figure 8



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