From the Institute of Biotechnology, University of
Cambridge, Tennis Court Road, Cambridge CB2 1QT, United Kingdom, the
¶ Institute of Bioengineering and Agroecology, Department of
Biology, National University of Ireland Maynooth, Maynooth, County
Kildare, Ireland, and the
Department of Chemistry, University of
Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
Received for publication, November 25, 2002, and in revised form, February 3, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Late embryogenesis abundant (LEA) proteins are
associated with desiccation tolerance in resurrection plants and in
plant seeds, and the recent discovery of a dehydration-induced Group 3 LEA-like gene in the nematode Aphelenchus avenae suggests a
similar association in anhydrobiotic animals. Despite their
importance, little is known about the structure of Group 3 LEA
proteins, although computer modeling and secondary structure algorithms
predict a largely Although water is essential for life, a number of organisms can
survive desiccation for extended periods by entering into a state of
suspended animation. This remarkable ability, called anhydrobiosis, is
found across all biological kingdoms, including bacteria, fungi,
animals, and plants. Examples among higher eukaryotes include some
nematode worms, such as Aphelenchus avenae, a soil-dwelling fungivore amenable to laboratory culture, "resurrection" plants like Craterostigma plantagineum, and orthodox plant seeds
and pollen (1-3). The molecular mechanisms governing anhydrobiosis are
not fully characterized, but several hypotheses emphasize a major role
for non-reducing disaccharides. Trehalose, in animals, or sucrose, in
plants, accumulates to high concentrations in many anhydrobiotic
species prior to dehydration (2, 4). In vitro these sugars
have been shown to preserve enzymes, antibodies, nucleic acids, some
viruses, liposomes, and other membrane systems during and after drying
(5, 6). Largely on the basis of the in vitro data, these
sugars are proposed to act as water replacement molecules and as
thermodynamic and kinetic stabilizers of biomolecules and membranes (7,
8). However, there is remarkably little evidence from living systems in
support of these hypotheses (9). Furthermore, it is increasingly
apparent that, if disaccharides do play an important role in
vivo, they are insufficient to confer anhydrobiosis by themselves;
other adaptations are required (9-12). In desiccation-tolerant plants,
a number of genes have been identified that are induced by water stress
(13, 14), but there is little information on equivalent genes in
anhydrobiotic animals. We have therefore begun to characterize the
genes involved in the desiccation stress response in A. avenae and recently described a dehydration-responsive gene,
Aavlea1, whose cognate amino acid sequence is related to plant Group 3 late embryogenesis abundant
(LEA)1 proteins (15).
LEA proteins were first identified 20 years ago in cotton and wheat
(16-19) and are produced in abundance during seed development, comprising up to 4% of cellular protein (20). Since then, up to five
different groups of LEA proteins have been defined on the basis of
expression pattern and sequence (21, 22). Precise functions of the LEA
proteins have yet to be elucidated, but expression is linked to water
stress and the acquisition of desiccation tolerance in orthodox seeds,
pollen, and anhydrobiotic plants (13, 14, 22). They have been variously
proposed to protect cellular structures from the effects of water loss
by action as a hydration buffer, by sequestration of ions, by direct
protection of other proteins or membranes, or by renaturation of
unfolded proteins, although supporting evidence is limited (21, 22).
The Group 3 LEA proteins, comprising subgroups D-7 and D-29, are
characterized by a repeating 11-mer amino acid motif whose consensus
has been defined for plant proteins as
TAE/QAAKE/QKAXE for the D-7 family, or more broadly as
Structural studies on a number of LEA proteins have been performed to
attempt to gain insight into function, but surprisingly little
information is available for Group 3 members. No crystal structures
have been reported, but secondary structure predictions can be derived
from a number of algorithms available as online computer programs. Fig.
1 shows secondary structure predictions from seven such programs
together with a "winner takes all" summary for the A. avenae Group 3 LEA protein, suggesting that it is largely Production of Recombinant Nematode LEA Protein--
The A. avenae LEA cDNA sequence reported in our earlier study (15)
was reamplified by polymerase chain reaction (PCR) using oligonucleotide primers containing engineered NdeI
(5'-GGAATTCCATATGTCCTCTCAGCAG) and BamHI
(5'-CGGGATCCTTAGTCGCGGCCCTT) sites (underlined) and cloned
in pCR2.1-TOPO (Invitrogen). The pET15b vector (Novagen) was used to
express the protein with an N-terminal His6 tag after cloning the engineered cDNA sequence at the NdeI and
BamHI sites; the construct was verified by DNA sequence
determination. The pET15b vector encodes the protein sequence
MGSSHHHHHHSSGLVPRGSH at the N terminus, additional to the native
AavLEA1 sequence shown in Fig. 1. The
plasmid was transformed into Escherichia coli strain BL21(DE3), and a single bacterial colony was inoculated in 100 ml of
Luria Bertani broth (LB) containing 100 µg/ml carbenicillin and grown
overnight at 37 °C. 10 ml of this culture was used to inoculate 1 liter of LB plus antibiotic, and at an absorbance (A600) of
0.6, gene expression was induced with 1 mM
isopropyl- Nematode Culture and Protein Extraction--
Mass cultures of
A. avenae were grown at 20-25 °C in the dark in Duran
bottles containing wheat grains that had been autoclaved and then
sub-cultured with the fungus Rhizoctonia solani (31). To
allow aeration, holes (~3 cm) had been made in bottle caps and
covered with sterile filter paper. After sufficient growth (10-20
days), nematodes were isolated, washed with water, and filtered through
0.2-µm membranes (PALL Life Sciences). Filtered nematode samples were
resuspended in protein lysate buffer, 50 mM Tris-HCl, pH
7.5, 75 mM NaCl, 15 mM EGTA, 1 mM
dithiothreitol, 0.1% Tween 20, 60 mM SDS-PAGE and Western Blotting--
For SDS-PAGE (32), 11% SDS
slab gels were run in Bio-Rad mini-Protean 3 electrophoresis cells;
gels were stained with Coomassie Blue R-250. Apparent molecular weight
was determined relative to molecular weight standards (Sigma). For
blotting, proteins were first separated by SDS-PAGE on 11% slab gels
and transferred to a nitrocellulose membrane (Trans-Blot transfer
medium; Bio-Rad) using a Trans-Blot S.D. electrophoretic transfer cell
(Bio-Rad). Immunodetection was performed using affinity-purified
polyclonal rabbit antiserum raised against purified AavLEA1 by Harlan
Seralab. A donkey anti-rabbit IgG peroxide-linked conjugate (Amersham
Biosciences) was used for detection, and bands were visualized with
enhanced chemiluminescence (ECL) detection reagent (Amersham
Biosciences) and Biomax ML (Kodak) autoradiography film. Molecular
weight markers used were MagicMark Western standards (Invitrogen),
which comprise recombinant proteins containing an IgG binding site from
protein G and are therefore revealed by the donkey anti-rabbit immunoglobulin.
Protein Cross-linking--
Reactions based on Davies and Stark
(33) and Coggins et al. (34) were carried out at room
temperature in 0.2 M triethanolamine hydrochloride (pH 8.5)
for 3 h with a final concentration of 0.5 mg/ml AavLEA1 and
dimethyl suberimidate concentration between 0.5 and 3 mg/ml in a volume
of 100 µl. At higher suberimidate concentrations, pH was adjusted
with NaOH prior to addition of protein. Proteins were then denatured in
SDS sample buffer for 2 h at 37 °C and appropriate amounts run
on 11% SDS-slab gels, which were subsequently stained with Coomassie
Blue R-250.
Analytical Ultracentrifugation--
A Beckman XL-A analytical
ultracentrifuge was used with the temperature of the 4-hole rotor
maintained at 20 °C and an operating speed of 18,000 rpm
(equilibrium runs) or 50,000 rpm (velocity runs). Optical scanning of
the cells was performed at a wavelength of 280 nm and the output logged
to disk for subsequent analysis. For the equilibrium runs the
attainment of the equilibrium state was verified by confirming that
successive scans at 1 h intervals were identical other than for
random noise. Standard software was used (35): SVEDBERG for velocity
analysis, NONLIN for equilibrium analysis, and SEDNTERP and BIOMOLS for
estimation of partial specific volume, frictional ratios, and other
routine calculations. ProFit (Quantum Soft) was used for curve fitting
and graph plotting.
Size Exclusion Chromatography--
A Superdex 200 column
equilibrated with 50 mM Tris buffer (pH 7.0) containing 100 mM NaCl was used with an FPLC system (Amersham Biosciences)
and calibrated with proteins from the LMW and HMW gel filtration
calibration kits (Amersham Biosciences). Protein samples (50-500 µg)
in appropriate buffers were run on the column, pre-equilibrated in the
same buffer, at a flow rate of 0.25 ml/min. The apparent size and
Stokes' radius (Rs gel) of the protein was determined relative to the standards using described methods (36).
Far UV Circular Dichroism and Fluorescence Emission
Spectroscopy--
CD spectra were recorded on a Jasco J-810
spectropolarimeter using a 0.1-cm path length for wavelengths between
190 and 250 nm. Temperature was controlled using a Peltier system, and
data were acquired at different temperatures with 0.1 and 2 mg/ml of AavLEA1 in 50 mM phosphate buffer (pH 7.0). The buffer
spectrum at each temperature set point was subtracted from the sample
spectrum. Fluorescence emission spectra were recorded on a PerkinElmer
LS50B luminescence spectrometer, fitted with a temperature controlling device. Data were obtained at different temperatures using an excitation wavelength of 280 nm, slit widths of 2.5 nm, and a scan rate
of 60 nm/min. The concentrations of AavLEA1 used were 0.1 and 0.5 mg/ml. Fluorescence intensity and maximum emission wavelength
( Fourier-transform Infrared (FT-IR) Spectroscopy--
Infrared
spectra were recorded in a Bruker Equinox 55 FT-IR spectrometer
equipped with a deuterated lanthanum triglycine sulfate (DLATGS)
detector and a KBr beam-splitter and purged with a continuous flow of
N2 gas. Lyophilized protein samples were reconstituted in
2H2O at a concentration of 15 mg/ml and
incubated at room temperature for about 10 min before measurements.
Protein solutions were then passed through 0.22-µm pore syringe
filters to remove any undissolved or aggregated material, and the
supernatants were transferred to a clean tube. Samples were then placed
between a pair of CaF2 windows separated by a 50-µm Mylar
spacer. Dehydrated samples were obtained by placing 50 µl of a 15 mg/ml solution of protein (prepared and filtered as described above) on
a CaF2 window and drying under vacuum for about 30 min. In
all cases transmission spectra were collected at room temperature. For
each sample 64 interferograms were collected at a spectral resolution
of 2 cm Gel Electrophoresis and Subunit Cross-linking Demonstrate
Oligomerization of Nematode LEA Protein--
A recombinant form of
AavLEA1 with a predicted molecular mass of 18,175 Da was
produced in E. coli after cloning in the pET15b expression
vector. The protein was purified by nickel chelation and ion exchange
chromatography and had approximately the expected mass on SDS-PAGE gels
(Fig. 2A). MALDI-TOF mass
spectrometry confirmed the mass as 18,060.1 ± 0.9 Da (data not
shown), which indicated that the N-terminal methionine had been cleaved
during synthesis but which otherwise agreed with predictions. To
facilitate analysis of AavLEA1, a polyclonal antiserum against the
purified protein was produced that recognizes it in Western blotting
experiments. Intriguingly, although in some experiments AavLEA1 is
visualized by the antiserum as a single band (Fig. 2B),
oligomeric forms are also frequently observed; monomer (N)
and dimer (2N) are clearly seen in Fig. 2C.
Higher order oligomers, at least of trimer (3N), are also
faintly visible. A similar degree of oligomerization also occurs
in vivo because dimers can be visualized in Western blots of
nematode protein extracts; both monomer and dimer of the native form
run slightly ahead of the equivalent recombinant species due to the
lack of an N-terminal His tag (Fig. 2D). Small quantities of
dimeric AavLEA1 can also be seen on SDS-PAGE gels simply stained with
Coomassie Blue (data not shown). This indicates a tight association
between subunits because protein samples were boiled in SDS, strongly
denaturing conditions, prior to electrophoresis. To demonstrate the
presence of oligomers in solution, cross-linking of purified AavLEA1
using dimethyl suberimidate was performed. Conditions were used that
allow cross-links to form between the subunits of protein complexes but
not between different complexes. In Fig. 2E, three bands are
observed after incubation in dimethyl suberimidate, corresponding to
monomeric, dimeric, and trimeric forms of the protein. The intensity of
the bands representing oligomeric forms is lower than that of the
monomer, but because cross-linking does not proceed to completion, this
does not indicate the relative proportions of the oligomers observed.
Larger forms were not seen, suggesting that tetramers and higher order
oligomers were not present at significant levels in the protein sample
used.
These experiments confirm the existence of homo-oligomers of AavLEA1 in
solution and are consistent with a dimeric or trimeric coiled coil
model, similar to those proposed by Dure (23) and NDong et
al. (29). These models imply that most of the protein should adopt
a oligomeric, coiled coil conformation, but the proportion of
oligomeric forms is difficult to determine using denaturing gel
electrophoresis because protein complexes are likely to be underestimated. Conversely, Western blotting can overemphasize weak
bands in relatively long film exposures. Preliminary electrospray ionization mass spectrometry indicated that AavLEA1 was present mostly
as the monomer; the presence of dimer and possibly higher order
oligomers was detected but in small quantities, suggesting incomplete
oligomerization (data not shown). However, although these experiments
were performed under conditions designed to minimize subunit
separation, it could not be ruled out that oligomeric complexes of
AavLEA1 were largely disrupted by the procedure. If AavLEA1 is mainly
oligomeric, though, this should be demonstrable unequivocally by
analytical ultracentrifugation.
Hydrodynamic Analysis Suggests AavLEA1 Is Mostly
Monomeric--
Sedimentation velocity experiments were performed for a
range of protein concentrations, and an apparently single boundary was
observed in all cases, consistent with one predominant species being
present. The plot of the inverse of sedimentation coefficient, s, against corrected concentration, c, was fitted
using a linear regression routine (Fig.
3), and a value of
s020,w = 1.20 S was obtained after
extrapolation to infinite dilution. The relatively low value for the
sedimentation coefficient of AavLEA1 is indicative of unusually high
drag forces for a molecule of this molecular mass. The frictional ratio
(f/f0), derived from
s020,w, is given as 2.28, indicating
either a highly extended structure (typical globular proteins have a
frictional ratio close to 1.1 (36)) or an unstructured, swollen protein with a high degree of hydration, or some combination of both. The
negative regression of s with protein concentration (Fig. 3)
is most consistent with a species that is either all monomer or mostly
monomer in rapid equilibrium with a proportion of dimer. Using the
concentration dependence of the s value, deductions on the
degree of dimerization can be made from sedimentation velocity data
(37, 38). This analysis suggests, for the two structural forms
postulated, that if AavLEA1 is an extended rod then very little dimer
is needed to account for the data of Fig. 3, whereas for an
unstructured model a significant fraction of dimer (7% dimer at a
protein concentration of 2.5 mg/ml) is present. Because gel
electrophoresis experiments demonstrate that monomeric and oligomeric
forms co-exist, the latter model is more likely. Other possibilities,
including the smallest species being a dimer, can effectively be
excluded. An unequivocal value for the molecular mass of the nematode
LEA protein in solution, and hence the degree of oligomerization, can
be obtained from sedimentation equilibrium experiments. These were
performed at a range of protein concentrations and the data fit to
single-species or two-species models using the NONLIN program. Again,
the best fit was observed for a single, i.e. monomeric
species but with a small quantity of dimer present (data not shown).
Higher order oligomers were not excluded by this analysis, but they
must be present at a level of
The Stokes' radius (Rs sed) of AavLEA1 was
calculated from sedimentation analysis to be 3.91 nm. This is larger
than expected if AavLEA1 were globular in structure but is consistent
with it being an extended or highly swollen protein. Gel filtration
experiments gave a similar result, with the LEA-like protein running
very close to bovine serum albumin (66 kDa;
Rs = 3.55 nm) on a Superdex 200 column (Fig.
4); the relatively low elution volume of
AavLEA1 was maintained under a variety of running conditions, at pH
6.5-9.5, in water or 0.5 M NaCl, and in 0.5 M
sucrose or 1 M trehalose. The gel filtration column was
calibrated using globular proteins with known Rs
as standards, allowing Rs gel for AavLEA1 to be
estimated at 3.38 nm. This is somewhat lower than the value obtained
from ultracentrifugation, possibly because of matrix interaction
effects, but is still far in excess of the expected Rs value for a globular protein of similar mass
and supports the model of AavLEA1 having low compactness. In summary,
gel electrophoresis and hydrodynamic analyses suggest that the nematode
Group 3 LEA-like protein exists in solution mostly in the monomeric
form but in rapid equilibrium with a small proportion of dimer. Some
trimer is also observed.
Spectroscopic Analysis Shows That AavLEA1 Is Natively
Unfolded--
Far UV CD spectroscopy yields information on the
To confirm the largely unstructured nature of AavLEA1, fluorescence
emission spectroscopy was performed. Tryptophan residues in proteins
exhibit different fluorescence maxima (
A large proportion of the proteome of many species is predicted to
include partially or wholly unfolded proteins; in the yeast Saccharomyces cerevisiae, for example, 30% of proteins are
predicted to be partially, and 6% to be wholly, disordered (40). A
simple predictor of whether a given protein falls into this group is obtained from calculation of mean normalized hydrophobicity
(<H>) and mean net charge at neutral pH
(<R>). When these parameters were plotted by Uversky and
colleagues (41) for a set of 275 folded and 91 unfolded
proteins, it was discovered that the large majority of natively folded
and unfolded proteins fall either side of a boundary line empirically
defined by the equation <H>b = (<R> + 1.151)/2.785. Thus, for a given value of
<R>, if <H> is less than
<H>b, the protein is predicted to
be unfolded. For AavLEA1, <R> = 0.014, giving
<H>b = 0.418. Mean hydrophobicity was calculated to be 0.340, locating the nematode protein within natively unfolded space on the Uversky plot. Uversky (39) further distinguished between natively unfolded proteins containing some degree
of secondary structure, so-called pre-molten globules, and fully
unfolded random coils; these two categories of unfolded protein fall on
different lines of a plot of log(Rs)
versus log(Mr). AavLEA1 has values of
log(Rs) = 1.58 (for
Rs in Ångstroms, Rs sed = 39.1 Å) and log(Mr) = 4.26 and locates
on the line occupied by coil-like proteins rather than pre-molten
globules, agreeing with experimental data.
AavLEA1 Acquires Secondary Structure on Drying--
FT-IR spectra
collected from soluble AavLEA1 samples in 2H2O
show an amide I pattern indicative of a mainly disordered polypeptide (Fig. 7A), in agreement with
CD data. Second derivative analysis of the amide I band showed two main
peaks at 1644 cm
Interestingly, when the protein is dried under vacuum, the amide I
spectrum of AavLEA1 experiences major changes in both its overall shape
and in the distribution of spectral components, suggesting that
important conformational changes have occurred. The overall amide I
spectrum of this form of the protein exhibits a maximum at 1657 cm The gene Aavlea1 encodes a protein in the anhydrobiotic
nematode A. avenae with marked similarity to the Group 3 LEA
proteins found in many maturing plant seeds. The precise function of
LEA proteins in plants has not been defined, although their expression is associated closely with acquisition of desiccation tolerance (45).
Tomato, wheat, and barley LEA proteins have been shown to confer
increased resistance to osmotic and freeze stress when introduced into
yeast (46-49), and a barley LEA protein improved tolerance to water
deficit in transgenic rice (50) and wheat (51). In vitro, a
Group 3 LEA protein from the alga Chlorella decreased freeze
damage of the enzyme lactate dehydrogenase (52), and our unpublished
results suggest that the nematode protein behaves similarly. Therefore,
LEA proteins seem able to offer partial protection to biological
structures at the molecular and cellular level against the effects of
water loss, but how this function is linked, if at all, to structure is
unclear. This report provides the first structural information, in both
hydrated and dry states, on a fully characterized Group 3 LEA-like
protein from any species, including plants.
The literature (23, 29) and several computer programs predict that
Group 3 LEA proteins adopt a largely An intriguing feature of natively unfolded proteins and one that might
throw light on the function of LEA proteins is that although such
proteins lack structure they do not lack function. The vast majority of
proteins or protein domains in this category have recognized
activities, and almost all are known to bind specific ligands or target
molecules (40, 41). For example, the yeast nucleoporin Nup2p, which
forms part of the nuclear pore complex, interacts specifically with
importin Shifts in temperature, pH, and concentration of counter ions can also
increase folding of disordered proteins (39), and a limited temperature
effect was noted for AavLEA1 and some other LEA proteins. However,
desiccation induced a more dramatic effect on AavLEA1 structure; FT-IR
spectroscopic analysis showed that the protein became more folded,
developing a significant Dehydration apparently induces -helical monomer that forms coiled coil oligomers.
We have therefore investigated the structure of the nematode protein,
AavLEA1, in the first such analysis of a well characterized Group 3 LEA-like protein. Immunoblotting and subunit cross-linking experiments demonstrate limited oligomerization of AavLEA1, but analytical ultracentrifugation and gel filtration show that the vast majority of
the protein is monomeric. Moreover, CD, fluorescence emission, and
Fourier transform-infrared spectroscopy indicate an unstructured conformation for the nematode protein. Therefore, in solution, no
evidence was found to support structure predictions; instead, AavLEA1
seems to be natively unfolded with a high degree of hydration and low
compactness. Such proteins can, however, be induced to fold into more
rigid structures by partner molecules or by altered physiological
conditions. Because AavLEA1 is associated with desiccation stress, its
Fourier transform-infrared spectrum in the dehydrated state was
examined. A dramatic but reversible increase in
-helix and,
possibly, coiled coil formation was observed on drying, indicating that
computer predictions of secondary structure may be correct for
the solid state. This unusual finding offers the possibility that structural shifts in Group 3 LEA proteins occur on dehydration, perhaps consistent with their role in anhydrobiosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E/QX
KE/QK
XE/D/Q (where
represents
a hydrophobic residue) for the D-29 family (23, 24). Recent genome
sequencing projects have brought to light sequences related to Group 3 LEA proteins in the nematode Caenorhabditis elegans and also
in the prokaryotes Haemophilus influenzae and
Deinococcus radiodurans (24), indicating that this type of
LEA protein at least is not restricted to plant species. The
D. radiodurans genome also contains sequences related to
other LEA proteins (25). The function of non-plant LEA proteins may
also relate to water stress; mutation of two D. radiodurans genes encoding LEA-like sequences resulted in reduced desiccation tolerance (26), and an LEA-like protein was induced by dehydration in
the entomopathogenic nematode Steinernema feltiae (27), as was the Aavlea1 gene in A. avenae (15). The
A. avenae protein sequence, named AavLEA1, is very similar
to the plant Group 3 LEA proteins, as shown by data base comparisons,
and includes several 11-mer motifs (15). These motifs differ slightly
from the plant consensus, e.g. the first amino acid is often
a positively charged lysine instead of a hydrophobic residue, but they
are clearly related. It is therefore expected that AavLEA1 will adopt a
conformation similar to that of plant Group 3 LEA proteins.
-helical throughout its length. Dure (23) used computer modeling to
predict that Group 3 LEA proteins adopt amphiphilic
-helices that
dimerize in an unusual right-handed coiled coil arrangement, with a
periodicity defined by the 11-mer motif. Right-handed coiled coils
based on an 11-mer repeat were later found in a surface layer protein
from Staphylothermus marinus (28), demonstrating that this
conformation is found in nature. Larger complexes might also arise
(29), because a Group 3 LEA-like wheat protein is predicted by the
MultiCoil program (30) to form trimeric coiled coils. Indeed, MultiCoil
also predicts a 40% probability of coiled coils in AavLEA1, although
these would be, more conventionally, left-handed. Because the structure
of AavLEA1 might offer clues to its function, we decided to test the
various hypotheses resulting from computer predictions to assess
(a) whether oligomers are formed, (b) whether an
-helical polypeptide structure can be detected, and (c)
whether drying has any significant effect on structure in a recombinant
form of the protein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside for 12 h at
30 °C. Cells were harvested by centrifugation at 11,000 × g for 10 min at 4 °C, and pellets were resuspended in
50 mM MOPS buffer (pH 6.5). The cell suspension was
sonicated, and debris was removed by centrifugation at 15,000 × g for 10 min at 4 °C. Supernatant was applied to a 5-ml
nickel chelation column pre-equilibrated with 50 mM
phosphate (pH 8.0), 300 mM NaCl, 10 mM
imidazole. The bound protein was eluted batchwise using increasing
concentrations of imidazole, as described in the
QIAexpressionistTM (Qiagen, Germany). Protein fractions
were pooled and dialyzed overnight in 5 mM MOPS buffer (pH
6.5) and applied to a HiLoad Q-Sepharose column (Amersham Biosciences)
equilibrated with 20 mM MOPS buffer (pH 6.5). The adsorbed
proteins were eluted with a linear gradient from 0 to 0.5 M
NaCl using an AKTA FPLC system (Amersham Biosciences). Fractions with
AavLEA1 protein eluted as a single peak, and the purity of protein was
analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). The protein concentration was determined
spectrophotometrically using a molar extinction coefficient of 8250 M
1 cm
1, calculated using the
ProtParam program on the ExPASy server (ca.expasy.org/tools/protparam.html).
View larger version (68K):
[in a new window]
Fig. 1.
Secondary structure predictions for nematode
Group 3 LEA protein. The native LEA-like sequence from A. avenae, shown in bold at the top of each line of output
and denoted LEA, was submitted to the program PELE on the SDSC Biology
Workbench (workbench.sdsc.edu). Seven different structure
predictions are shown, with the most likely structural feature at each
residue indicated by H ( -helix), E
(
-sheet), or C (random coil). Programs used are denoted
BPS (77), D_R (78), DSC (79),
GGR (80), GOR (81), H_K (82),
K_S (83). The "winner-takes-all" joint prediction is
given by JOI and shown in bold.
-glycerophosphate,
1 mM NaF, 0.2 mM sodium orthovanadate, 2 mM sodium pyrophosphate, plus a protease inhibitor mixture
(Roche Applied Science cat. no. 1836153), and sonicated. Insoluble cellular material was removed by centrifugation for 30 min
(18,000 × g at 4 °C), leaving a cleared lysate.
max) were determined from emission spectra acquired in
the 310-400 nm range.
1, using a scanner velocity of 10 kHz. Water vapor
subtraction and baseline correction as well as additional spectra
processing as indicated below were performed using GRAMS/AI (Thermo
Galactic) software. Second derivatives of the Amide I band spectra were produced to determine the localization of the different spectral components. A peak fitting for each individual spectrum was performed using the spectral frequencies obtained by second derivative analysis and trough successive iterations using a variable mixture of Gaussian and Lorentzian bandshapes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (43K):
[in a new window]
Fig. 2.
Analysis of AavLEA1 oligomerization by gel
electrophoresis. A, purified recombinant AavLEA1 after
SDS-PAGE and Coomassie Blue staining. Molecular masses of size
standards are shown in kDa. B, Western blot of AavLEA1
separated by SDS-PAGE and transferred to nitrocellulose.
Affinity-purified rabbit polyclonal antiserum, raised against AavLEA1,
was used to reveal the protein. Sizes of molecular mass standards are
given in kDa. C, Western blot experiment, similar to
panel B but showing monomer (N), dimer
(2N), and oligomeric forms (3N). D,
Western blot comparing recombinant AavLEA1 (rec) and native
protein (nem) from an extract of A. avenae and
showing monomeric (N) and dimeric (2N) forms in
both. The larger size of the recombinant protein is due to the presence
of an N-terminal His tag and associated sequence, which augments the
monomer size by ~2 kDa. E, SDS-PAGE stained with Coomassie
Blue of AavLEA1 after cross-linking with 0.5 or 1.0 mg/ml dimethyl
suberimidate as indicated. Monomer (N), dimer
(2N), and trimer (3N) are indicated.
2% total protein.
View larger version (8K):
[in a new window]
Fig. 3.
Sedimentation velocity analysis of AavLEA1
protein. Sedimentation coefficient,
sc20,w, was determined for five
protein concentrations, c. The line fitting the inverse of
s to c was extrapolated to give the sedimentation
coefficient at infinite dilution,
s020,w; c was corrected
for radial dilution.
View larger version (7K):
[in a new window]
Fig. 4.
Gel filtration chromatography of
AavLEA1. A, purified AavLEA1 migrating on a calibrated
Superdex 200 column, where protein is detected spectrophotometrically
at 280 nm, has an elution volume of 14.25 ml in the experiment shown.
B, bovine serum albumin elutes slightly ahead of AavLEA1 in
a volume of 13.8 ml under the same conditions. C, a mixture
of both proteins, giving an absorbance profile that is the sum of those
of the separate proteins, is shown.
-helical,
-sheet, and unstructured random coil content of
proteins. If AavLEA1 is largely
-helical as predicted (Fig. 1), CD
spectra should have two characteristic minima near 208 and 222 nm. In contrast, a disordered structure would give a single minimum at about
200 nm and low ellipticity at 222 nm. Proteins with elements of both
structural forms, and indeed of
-sheet with its typical minimum at
215 nm, would give spectra combining these features. However,
significant defined secondary structure was not detected for AavLEA1;
signatures of
-helix and
-sheet were absent, with the spectrum
showing instead that the protein is most likely unstructured through a
temperature range of 4 to 75 °C (Fig.
5). A slight increase in ellipticity at
200 nm on shifting to higher temperature, together with a decrease at
220 nm, could be interpreted as an increase in
-helical content. On
cooling to 4 °C, the spectrum reverts to that seen previously at
this temperature, showing that any structural changes induced by
heating are fully reversible. This is the opposite to what is observed
in globular proteins, where heating causes unfolding, but the effect
has been noted previously with unstructured proteins and could reflect
increased strength of hydrophobic interactions at elevated temperature
(39). CD spectra did not change appreciably throughout a concentration range of 0.1 to 2 mg/ml, suggesting that any secondary structure of
AavLEA1 is not influenced by protein concentration (data not shown).
View larger version (12K):
[in a new window]
Fig. 5.
Far UV CD spectroscopy of AavLEA1. The
solid line represents the CD spectrum of AavLEA1 at
75 °C. The three dotted lines are very similar spectra at
4 and 25 °C and at 4 °C again after cooling from 75 °C.
max) dependent on
the hydrophobicity of their environment, which commonly reflects the
degree of solvent exposure of the tryptophan side chain. For fully
solvated tryptophans, a
max at 355 nm is observed;
tryptophans that are buried within the protein have a
max as low as 310 nm. The single tryptophan residue in
AavLEA1, at position 49 of 162 (allowing for the His tag, position 30 of 143 in the native nematode sequence; Fig. 1), gives rise to a
max at 4 °C of 355 nm, which is indicative of a
solvent-exposed side chain (Fig. 6). The
emission profile does not change appreciably on heating the sample to
65 °C; on cooling, the spectrum is indistinguishable from the
original (data not shown). This implies that any increased folding with temperature, as suggested by CD analysis, does not mask the tryptophan at position 49. Therefore, both far UV CD and fluorescence emission experiments are consistent with a natively unfolded structure for the
nematode Group 3 LEA protein. Preliminary proton nuclear magnetic
resonance studies (data not shown) also suggested a lack of stable
structural elements within AavLEA1.
View larger version (10K):
[in a new window]
Fig. 6.
Emission fluorescence spectroscopy of
AavLEA1. The dotted line represents the fluorescence
emission spectrum of the single tryptophan residue in AavLEA1 at
4 °C. The solid line shows fluorescence at 65 °C. Both
curves have a max at 355 nm, indicative of a fully
solvated tryptophan side chain.
1 and 1667 cm
1,
characteristic of random coil structures (42). A minor component at
1610 cm
1 could be due to side chains of amino acids being
highly represented in the sequence of the protein, such as glutamine
(43) which accounts for almost 10% of the total amino acids of
AavLEA1. Natively unfolded proteins are known to become more
structured when complexed with partner molecules or exposed to altered
physiological conditions (39-41); therefore we examined the effect of
dehydration, the natural stress vector experienced by anhydrobiotes, on
AavLEA1 structure.
View larger version (16K):
[in a new window]
Fig. 7.
FT-IR spectroscopy of AavLEA1.
Decomposition of the amide I band spectra corresponding to soluble
(A) and dried (B) protein. Second derivatives of
the respective spectra were used to obtain the different band positions
used in the curve fitting. The spectrum of rehydrated AavLEA1 overlaps
completely with that of the protein in solution, as shown in
panel A (data not shown).
1, which together with the existence of an important
component at 1658 cm
1 indicates the acquisition of
-helical structure by the protein when dehydrated (Fig.
7B; Table I). Intriguingly,
components detected at 1641, 1672, and 1689 cm
1 (and
perhaps also that at 1623 cm
1) are consistent with the
arrangement of
helical structures as coiled coils (44).
According to the existing literature, the absence of components around
1630 cm
1 together with the location of the spectral
maximum of the overall amide I band (1657 cm
1) and its
spectral weight (1659 cm
1) suggest, however, the absence
of a "superhelical pitch," i.e. any superhelical
assembly is expected to originate from the lateral association of
helices in a straight manner without any twisting, as suggested
previously for coiled coil models (44). As mentioned, the band at 1623 cm
1 could be one of those attributed to the superhelical
arrangement of dehydrated AavLEA1. Alternatively, together with the
component at 1680 cm
1, it might also account for a
residual amount of inter-molecular
-sheet structure, perhaps because
of aggregation of a small fraction of the protein during dehydration.
Finally, the bands at 1649 cm
1 and 1680 cm
1 could account for residual random conformations in
the sample. The FT-IR spectrum of rehydrated AavLEA1 is completely
superimposable with that of the original protein in solution (Fig.
7A), indicating that the structural rearrangements imposed
by desiccation are fully reversible.
FT-IR amide I components of AavLEA1 samples and their corresponding
areas
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical structure, possibly as
a coiled coil homodimer or higher order species. However, gel
electrophoresis and hydrodynamic experiments on AavLEA1 indicate that
although limited oligomerization occurs the majority species is the
monomer. The protein is apparently wholly unfolded in solution with
little evidence obtained by far UV CD, fluorescence emission, and FT-IR
spectroscopy for any defined conformation. Failure to achieve
crystallization of AavLEA12
is consistent with this. Calculations made from the hydrodynamic data
about the degree of hydration of the protein show that an ideal,
compact protein of Mr 18,060 would have a value
of Rs = 1.72 nm, but the observed value for
AavLEA1 (Rs sed) is 3.91 nm. The ratio of the
molecular volumes calculated from these radii gives
Vs/
, the ratio of
partial specific hydrated volume of the protein to partial specific
volume of the protein alone. For AavLEA1,
Vs/
= 11.8 and,
because
is estimated to be 0.705 ml/g
using the SEDNTERP application, this gives Vs = 8.32 ml/g. If we assume that volume not occupied by protein is occupied
by water with the same density as bulk phase, then the degree of hydration of AavLEA1 is Vs minus
= 7.6 ml/g, and the ratio of volume of
water per unit volume of protein is 7.6/0.705 or 10.8. This indicates a
high level of associated water, because a typical globular protein
would have a Vs/
value of around 1.5, giving a degree of hydration of 0.365 ml/g.
Therefore, in this model AavLEA1 has ~20-fold more associated water
than a typical globular protein of equivalent size, which is consistent
with the recognized hydrophilicity of LEA proteins (21, 22). On the
basis of these observations, it appears that structural predictions are
incorrect, at least for the purified, solution state of AavLEA1, and
that instead it adopts a conformation termed natively unfolded (39, 41)
or intrinsically disordered (40). One concern with the use of a
recombinant molecule is the possible interference of the N-terminal His
tag in protein folding. However, we have obtained almost identical gel
electrophoresis and hydrodynamic data with a version of AavLEA1 where
the N-terminal His tag has been removed by thrombin cleavage or by
partial proteolysis; in addition, a different recombinant form of the
protein, where a His tag is positioned at the C terminus, also exhibits
an unfolded conformation (data not shown). Although their sequence is
unrelated to that of Group 3 LEA proteins, the other main categories of LEA protein (i.e. Group 1 and Group 2, the latter frequently
referred to as dehydrins) also seem to be wholly or partially natively unfolded (53-60), suggesting that this is a general characteristic of
LEA proteins.
(Kap60p/Kap95p) and is required for nuclear import of
importin-
-dependent cargoes, is natively unfolded
(61). Substrates for disordered proteins include other proteins but
also DNA or RNA, nucleotides, and cations. Crucially, binding to
partner molecules can induce a switch between unfolded and folded
states, as with heme-induced folding of apocytochrome c (62)
and the CFP-10 major T-cell antigen of Mycobacterium tuberculosis, which is disordered in solution but becomes
structured on binding to its cognate molecule ESAT-6 (63). It is
therefore possible that LEA proteins have specific binding targets
within desiccating cells that confer more ordered secondary structure. However, the high concentrations of LEA proteins found within cells,
estimated to be >200 µM in cotton seeds prior to
desiccation (20), would necessitate an equally abundant protein binding partner, and we might expect such proteins to have been identified already. LEA proteins of different groups where they are present in the
same cell might form hetero-oligomers, although this seems unlikely
given that both partners would be unstructured. LEA proteins might also
bind small molecules, and Walters et al. (64) have reported
tight, stoichiometric binding of sugars to a mixture of wheat LEA
proteins. The effect of this binding on LEA protein structure was not
assessed by the authors, but we did not detect any difference in
mobility of AavLEA1 on gel filtration in the presence of 0.5 M sucrose or 1 M trehalose, which might be
expected if sugar binding has a dramatic effect on folding.
-helical component, when dried.
Furthermore, spectral components were present that were consistent with
the formation of superhelical, and possibly coiled coil-like,
structures. This is a highly unusual observation because protein
dehydration is most often associated with a loss of structure and
aggregation (65) rather than with an increase in structure, folding, or
subunit assembly and offers the exciting possibility that structural
shifts in Group 3 and possibly other categories of LEA proteins depend
on the availability of water. Other workers have observed that
apparently unfolded LEA (or similar) proteins become more structured
when water activity is decreased by e.g. trifluoroethanol
(56, 58, 66), high salt concentration (54), or drying in the presence
of sucrose (67). This has obvious physiological relevance in
desiccation-tolerant systems, including the anhydrobiotic nematode
A. avenae, but leads to the further question of what the
functions of the folded, presumably partially dehydrated, LEA proteins
might be. In vitro, a pollen protein increases the glass
transition temperature (Tg) of a sucrose glass
into which it is incorporated (67), prompting the consideration that
LEA proteins might stabilize the cytoplasmic sugar glasses implicated
in the protection of biomolecules during anhydrobiosis (7, 68, 69).
However, this stabilizing function is also exhibited by
poly(L)lysine (70) and bovine serum
albumin.3 Indeed, other
polymers with high intrinsic Tg, such as
hydroxyethyl starch (71), also increase the Tg
of a two-component polymer-sugar glass; the phenomenon is therefore not
specific to LEA proteins. The high concentration of protein in the
cytoplasm (80-300 mg/ml) (72,73) also means that there is no shortage
of available protein for glass formation with or without the presence
of LEA proteins.
-helix formation in AavLEA1 and
potentially coiled coil oligomerization, suggesting that computer predictions of its structure may be fulfilled in the dry, if not the
hydrated, state. Increased folding and oligomerization could be driven
by a combination of decreased protein hydration and molecular crowding
effects, which exert thermodynamic pressure on proteins to adopt a
compact structure. Crowding would be augmented by loss of water from
the cytoplasm, where a 10% reduction in cellular water could result in
an increase in thermodynamic activity of volume-excluding protein
species of up to 10-fold (74). We might speculate that AavLEA1 coiled
coils would be able to form more complex structures, reminiscent of
intermediate filaments (IFs). IFs are cytoskeletal components based on
coiled coil dimers of, for example, the various keratins or lamins that
extend throughout the cytoplasm or nucleoplasm and provide
intracellular support and physical strength to the cell (75).
Additional LEA protein-derived filaments might increase resistance to
the physical stresses imposed during desiccation that can lead to cell
deformation and collapse (76). Moreover, LEA protein filaments could
work together with sugar glasses in a manner analogous to
steel-reinforced concrete, where the filaments might increase the
tensile strength of the amorphous carbohydrate matrix. Such hypotheses,
in particular the dehydration-dependent oligomerization of
LEA proteins, are directly testable and will form the basis of future experiments.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Prof. Arthur Rowe and colleagues of the National Centre for Macromolecular Hydrodynamics Business Centre, University of Nottingham, for performing analytical ultracentrifugation experiments and for helpful advice on sedimentation analysis. Several colleagues at the University of Cambridge provided assistance during the project: Drs. Len Packman and Richard Turner, Department of Biochemistry, performed mass spectrometry; Prof. Chris Dobson, Department of Chemistry, allowed access to the spectropolarimeter and FT-IR apparatus; and Dr. Michael Wise, Department of Genetics, made valuable comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was funded by grants from the Leverhulme Trust, the Isaac Newton Trust, the Royal Irish Academy, and the Royal Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A Science Foundation Ireland Investigator. Present address: Domantis Limited, Granta Park, Cambridge CB1 6GS, UK.
** The AWG Senior Research Fellow of Pembroke College, Cambridge. To whom correspondence should be addressed: Institute of Biotechnology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QT, UK. Tel.: 44-1223-766549; Fax: 44-1223-334162; E-mail: at10004@biotech.cam.ac.uk.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M212007200
2 D. Leys, personal communication.
3 S. Ring, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: LEA, late embryogenesis abundant; CD, circular dichroism; Rs, Stokes' radius; FT-IR, Fourier transform-infrared; MOPS, 4-morpholinepropanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Crowe, J. H., Hoekstra, F. A., and Crowe, L. M. (1992) Ann. Rev. Physiol. 54, 579-599[CrossRef][Medline] [Order article via Infotrieve] |
2. | Oliver, M. J., Tuba, Z., and Mishler, B. D. (2000) Plant Ecol. 15, 85-100 |
3. | Clegg, J. S. (2001) Comp. Biochem. Physiol. 128B, 613-624 |
4. | Perry, R. N. (1999) Parasitology 119, S19-S30[Medline] [Order article via Infotrieve] |
5. | Crowe, J. H., Crowe, L. M., Carpenter, J. F., and Wistrom, C. A. (1987) Biochem. J. 242, 1-10[Medline] [Order article via Infotrieve] |
6. | Colaco, C., Sen, S., Thangavelu, M., Pinder, S., and Roser, B. (1992) Bio-Technol. 10, 1007-1011 |
7. | Crowe, J. H., Carpenter, J. F., and Crowe, L. M. (1998) Ann. Rev. Physiol. 60, 73-103[CrossRef][Medline] [Order article via Infotrieve] |
8. | Bolen, D. W., and Baskakov, I. V. (2001) J. Mol. Biol. 310, 955-963[CrossRef][Medline] [Order article via Infotrieve] |
9. | Tunnacliffe, A., and Lapinski, J. (2003) Philos. Trans. R. Soc. Lond. (B Biol. Sci.), in press |
10. | Higa, L. M., and Womersley, C. Z. (1993) J. Exp. Zool. 267, 120-129 |
11. | Womersley, C. Z., and Higa, L. M. (1998) Nematologica 44, 269-291 |
12. | Hoekstra, F. A., Golovina, E. A., and Buitink, J. (2001) Trends Plant Sci. 6, 431-438[CrossRef][Medline] [Order article via Infotrieve] |
13. | Ingram, J., and Bartels, D. (1996) Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 377-403[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Bartels, D.,
and Salamini, F.
(2001)
Plant Physiol.
127,
1346-1353 |
15. | Browne, J., Tunnacliffe, A., and Burnell, A. (2002) Nature 416, 38[CrossRef][Medline] [Order article via Infotrieve] |
16. | Dure, L., III, Greenway, S. C., and Galau, G. A. (1981) Biochemistry 20, 4162-4168[Medline] [Order article via Infotrieve] |
17. | Grzelczak, Z. F., Sattolo, M. H., Hanley-Bowdoin, L. K., Kennedy, T. D., and Lane, B. G. (1982) Can. J. Biochem. 60, 389-397[Medline] [Order article via Infotrieve] |
18. | Galau, G. A., Hughes, D. W., and Dure, L., III (1986) Plant Mol. Biol. 7, 155-170 |
19. | Baker, J. C., Steele, C., and Dure, L., III (1988) Plant Mol. Biol. 11, 277-291 |
20. |
Roberts, J. K.,
DeSimone, N. A.,
Lingle, W. L.,
and Dure, L., III
(1993)
Plant Cell
5,
769-780 |
21. |
Bray, E. A.
(1993)
Plant Physiol.
103,
1035-1040 |
22. | Cuming, A. (1999) in Seed Proteins (Shewry, P. R. , and Casey, R., eds) , pp. 753-780, Kluwer Academic Publishers, Dordrecht, Netherlands |
23. | Dure, L., III (1993) Plant J. 3, 363-369[CrossRef][Medline] [Order article via Infotrieve] |
24. | Dure, L., III (2001) Protein Peptide Lett. 8, 115-122 |
25. |
Makarova, K. S.,
Aravind, L.,
Wolf, Y. I.,
Tatusov, R. L.,
Minton, K. W.,
Koonin, E. V.,
and Daly, M. J.
(2001)
Microbiol. Mol. Biol. Rev.
65,
44-79 |
26. | Battista, J. R., Park, M.-J., and McLemore, A. E. (2001) Cryobiology 43, 133-139[CrossRef][Medline] [Order article via Infotrieve] |
27. | Solomon, A., Salomon, R., Paperna, I., and Glazer, I. (2000) Parasitology 121, 409-416[CrossRef][Medline] [Order article via Infotrieve] |
28. | Stetefeld, J., Jenny, M., Schulthess, T., Landwehr, R., Engel, J., and Kammerer, R. A. (2000) Nat. Struct. Biol. 7, 772-776[CrossRef][Medline] [Order article via Infotrieve] |
29. |
NDong, C.,
Danyluk, J.,
Wilson, K. E.,
Pocock, T.,
Huner, N. P. A.,
and Sarhan, F.
(2002)
Plant Physiol.
129,
1368-1381 |
30. |
Wolf, E.,
Kim, P. S.,
and Berger, B.
(1997)
Protein Sci.
6,
1179-1189 |
31. | Evans, A. A. F. (1970) J. Nematol. 2, 99-100 |
32. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
33. | Davies, G. E., and Stark, G. R. (1970) Proc. Natl. Acad. Sci. U. S. A. 66, 651-656[Abstract] |
34. | Coggins, J. R., Lumsden, J., and Malcolm, A. D. B. (1977) Biochemistry 16, 1111-1116[Medline] [Order article via Infotrieve] |
35. | Cole, J. L., and Hansen, J. C. (1999) J. Biomol. Tech. 10, 163-176[Abstract] |
36. | Siegal, L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346-362[Medline] [Order article via Infotrieve] |
37. | Rowe, A. J. (1977) Biopolymers 16, 2595-2611 |
38. | Rowe, A. J. (1992) in Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S. E. , Rowe, A. J. , and Horton, J. C, eds) , pp. 394-406, Royal Society of Chemistry, London |
39. |
Uversky, V. N.
(2002)
Eur. J. Biochem.
269,
2-12 |
40. | Dunker, A. K., Lawson, J. D., Brown, C. J., Williams, R. M., Romero, P., Oh, J. S., Oldfield, C. J., Campen, A. M., Ratliff, C. M., Hipps, K. W., Ausio, J., Nissen, M. S., Reeves, R., Kang, C., Kissinger, C. R., Bailey, R. W., Griswold, M. D., Chiu, W., Garner, E. C., and Obradovic, Z. (2001) J. Mol. Graph. Model. 19, 26-59[CrossRef][Medline] [Order article via Infotrieve] |
41. | Uversky, V. N., Gillespie, J. R., and Fink, A. L. (2000) Proteins Struct. Funct. Genet. 41, 415-427[CrossRef][Medline] [Order article via Infotrieve] |
42. | Byler, D. M., and Susi, H. (1986) Biopolymers 25, 469-487[Medline] [Order article via Infotrieve] |
43. | Krimm, S., and Bandekar, J. (1986) Adv. Protein Chem. 38, 181-364[Medline] [Order article via Infotrieve] |
44. | Heimburg, T., Schunemann, J., Weber, K., and Geisler, N. (1999) Biochemistry 38, 12727-12734[CrossRef][Medline] [Order article via Infotrieve] |
45. | Blackman, S. A., Obendorf, R. L., and Leopold, A. C. (1995) Physiol. Plantarum 93, 630-638[CrossRef] |
46. | Honjoh, K.-I., Oda, Y., Takata, R., Miyamoto, T., and Hatano, S. (1996) J. Plant Physiol. 155, 509-512 |
47. | Imai, R., Chang, L., Ohta, A., Bray, E. A., and Takagi, M. (1996) Gene 170, 243-248[CrossRef][Medline] [Order article via Infotrieve] |
48. | Swire-Clark, G. A., and Marcotte, W. R., Jr. (1999) Plant Mol. Biol. 39, 117-128[CrossRef][Medline] [Order article via Infotrieve] |
49. | Zhang, L., Ohta, A., Takagi, M., and Imai, R. (2000) J. Biochem. 127, 611-616[Abstract] |
50. |
Xu, D.,
Duan, X.,
Wang, B.,
Hong, B.,
Ho, T.-H. D.,
and Wu, R.
(1996)
Plant Physiol.
110,
249-257 |
51. | Sivamani, E., Bahieldin, A., Wraith, J. M., Al-Niemi, T., Dyer, W. E., Ho, T.-H. D., and Qu, R. (2000) Plant Sci. 155, 1-9[CrossRef][Medline] [Order article via Infotrieve] |
52. | Honjoh, K., Matsumoto, H., Shimizu, H., Ooyama, K., Tanaka, K., Oda, Y., Takata, R., Joh, T., Suga, K., Miyamoto, T., Iio, M., and Hatano, S. (2000) Biosci. Biotechnol. Biochem. 64, 1656-1663[Medline] [Order article via Infotrieve] |
53. | McCubbin, W. D., Kay, C. M., and Lane, B. G. (1985) Can. J. Biochem. Cell Biol. 63, 803-811 |
54. | Russouw, P. S., Farrant, J., Brandt, W., Maeder, D., and Lindsey, G. G. (1995) Seed Sci. Res. 5, 137-144 |
55. | Russouw, P. S., Farrant, J., Brandt, W., and Lindsey, G. G. (1997) Seed Sci. Res. 7, 117-123 |
56. |
Soulages, J. L.,
Kim, K.,
Walters, C.,
and Cushman, J. C.
(2002)
Plant Physiol.
128,
822-832 |
57. | Ceccardi, T. L., Meyer, N. C., and Close, T. J. (1994) Protein Expr. Purif. 5, 266-269[CrossRef][Medline] [Order article via Infotrieve] |
58. | Lisse, T., Bartels, D., Kalbitzer, H. R., and Jaenicke, R. (1996) Biol. Chem. 377, 555-561[Medline] [Order article via Infotrieve] |
59. |
Ismail, A. M.,
Hall, A. E.,
and Close, T. J.
(1999)
Plant Physiol.
120,
237-244 |
60. | Hara, M., Terashima, S., and Kuboi, T. (2001) J. Plant Physiol. 158, 1333-1339 |
61. |
Denning, D. P.,
Uversky, V.,
Patel, S. S.,
Fink, A. L.,
and Rexach, M.
(2002)
J. Biol. Chem.
277,
33447-33455 |
62. |
Stellwagen, E.,
Rysary, R.,
and Babul, G.
(1972)
J. Biol. Chem.
247,
8074-8077 |
63. |
Renshaw, P. S.,
Panagiotidou, P.,
Whelan, A.,
Gordon, S. V.,
Hewinson, R. G.,
Williamson, R. A.,
and Carr, M. D.
(2002)
J. Biol. Chem.
277,
21598-21603 |
64. | Walters, C., Ried, J. L., and Walker-Simmons, M. K. (1997) Seed Sci. Res. 7, 125-134 |
65. | Dong, A., Prestrelski, S. J., Allison, S. D., and Carpenter, J. F. (1995) J. Pharm. Sci. 84, 415-424[Medline] [Order article via Infotrieve] |
66. |
Boothe, J. G.,
Sönnichsen, F. D.,
de Beus, M. D.,
and Johnson-Flanagan, A. M.
(1997)
Plant Physiol.
113,
367-376 |
67. | Wolkers, W. F., McReady, S., Brandt, W. F., Lindsey, G. G., and Hoekstra, F. A. (2001) Biochim. Biophys. Acta 1544, 196-206[Medline] [Order article via Infotrieve] |
68. | Burke, M. J. (1986) in Membranes, Metabolism, and Dry Organisms (Leopold, A. C., ed) , pp. 358-364, Cornell University Press, New York |
69. | Sun, W. Q., and Leopold, A. C. (1997) Comp. Biochem. Physiol. 117B, 327-333[CrossRef] |
70. | Wolkers, W. F., van Kilsdonk, M. G., and Hoekstra, F. A. (1998) Biochim. Biophys. Acta 1425, 127-136[Medline] [Order article via Infotrieve] |
71. | Crowe, J. H., Oliver, A. E., Hoekstra, F. A., and Crowe, L. M. (1997) Cryobiology 3, 20-30[CrossRef] |
72. | Zimmerman, S. B., and Trach, S. O. (1991) J. Mol. Biol. 222, 599-620[Medline] [Order article via Infotrieve] |
73. | Swaminathan, R., Hwang, C. P., and Verkman, A. S. (1997) Biophys. J. 72, 1900-1907[Abstract] |
74. | Minton, A. P. (2000) Curr. Opin. Struct. Biol. 10, 34-39[CrossRef][Medline] [Order article via Infotrieve] |
75. |
Fuchs, E.,
and Cleveland, D. W.
(1998)
Science
279,
514-519 |
76. | Wolfe, J., Dowgert, M. F., Maier, B., and Steponkus, P. L. (1986) in Membranes, Metabolism, and Dry Organisms (Leopold, A. C., ed) , pp. 286-305, Cornell University Press, New York |
77. | Burgess, A. W., Ponnuswamy, P. K., and Sheraga, H. A. (1974) Isr. J. Chem. 12, 239-286 |
78. | Deléage, G., and Roux, B. (1987) Protein Eng. 4, 289-294 |
79. |
King, R. D.,
and Sternberg, M. J. E.
(1996)
Protein Sci.
5,
2298-2310 |
80. | Garnier, J., Gibrat, J. F., and Robson, B. (1996) Methods Enzymol. 266, 97-120 |
81. | Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120[Medline] [Order article via Infotrieve] |
82. | Holley, L. H., and Karplus, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 152-156[Abstract] |
83. | King, R. D., and Sternberg, M. J. E. (1990) J. Mol. Biol. 216, 441-457[Medline] [Order article via Infotrieve] |