Secondary Structure Changes and Thermal Stability of Plasma Membrane Proteins of Wheat Roots in Heat Stress

doi:10.4236/ajps.2011.26096

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American Journal of Plant Sciences, 2011, 2, 816-822

doi :1 0.4236/ aj ps.2011 .26096 Publ i s hed Online December 2011 (http://www.SciRP.org/journal/ajps)

Secondary Structure Changes and Thermal

Stability of Plasma Membrane Proteins of Wheat

Roots in Heat Stress

Xin Zhao1, Yong Shi1, Li Chen2, Fenlin Sheng3, Haiyan Zhou1

1Laboratory of Plant Stress Ecophysiology and Biotechnology, Cold and Arid Regions Environmental and Engineering Research

Institute, Chinese Academy of Sciences, Lanzhou, China; 2College of Life Science and Engineering, The Northwest University for

Nationalities, Lanzhou, China; 3Instrumental Analysis and Research Center, Lanzhou University, Lanzhou, China.

Email: [email protected]

Received July 21st, 2011; r e vised August 20th, 2011; accepted September 6th, 2011.

ABSTRACT

The wheat roots membrane separates the cell from the environment around it and encloses the cell contents. The pro-

tein secondary structure and thermal stability of the plasma membrane of wheat root have been characterized in D2O

buffer from 20˚C to 90˚C by Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Quantita-

tive analysis of the amide I band (1700 - 1600 cm–1) showed that the plasma membrane proteins contains 41% α-helix,

16% β-sheet, 18% turn, and 25% disorder structures at 20˚C. At elevated temperatures from 25˚C up to 90˚C, the

α-helix and the β-sheet structure unfold into turns and the disorder structure, with a major conformational transition

occurring at 50˚C. There is a rapid decline in H+-ATPase activity of plasma membrane from 35˚C to 55˚C and it re-

main very low level H+-ATPase activity of PM from 55˚C to 90˚C. Therefore the protein conformational transition was

one of reasons of loses H+-ATPase activity of plasma membrane.

Keywords: Plasma Membrane, Heat Stress, Protein Second Structure, ATR-FTIR

1. Introduction

The plant plasma membrane (PM) separates the cell from

the environment around it and encloses the cell contents.

It has four important functions: it acts as a semi-perme-

able barrier, it regulates transport in and out of the cell;

and it provides communication and adhesion. It is the

first line of cell defense against the stress from the out-

side environment and against harmful free radicals.

The heat sensitivity of the PM has both practical and

theoretical significance. The study of the heat denatura-

tion process can provide an insight into the structural

organization and individual roles of proteins in general.

More specifically, heat can induce functional change in

the plant PM [1]. A review by Arrondo [2] descripted the

applications of IR spectroscopy to the study of mem-

brane proteins. Shi [3] reported a study on FT-IR of the

secondary structure and conformational changes of a pho-

tosystem II (PS II) membrane and core complex during

heat denaturation. The core of the PS II denatured from

55˚C to 65˚C with transitions of the secondary structure

from co-helix and t urns to β-sheet and random coils. The

denaturation of light-harvesting chlorophyll protein (LHCP)

began at 65˚C and was characterized by a rapid increase

of the turns. However, the above studies were restricted

by a lack of information on the conformational changes

of proteins during heat denaturation; such information is

necessary for an understanding of the mechanism of the

heat inactivation of the PM.

Attenuated total reflection Fourier transform infrared

spectroscopy (ATR-FTIR) is one of the most powerful

tools for recording infrared spectra of biological materi-

als in general, and for biological membranes in particular.

It has fast speed, yields a strong signal with only a few

micrograms of sample, and most importantly it allows in-

formation about the orientation of various parts of the

molecule under study to be evaluated in an oriented sys-

tem. The environment of the molecules can be modulated

so that their conformation can be studied as a function of

temperature, pressure and pH, as well as in the presence

of species ligands. Because of the long IR wavelength,

light scattering problems are virtually non-existent and

highly aggregated materials or large membrane fragments

can be investigated. A unique advantage of infrared spec-

Secondary Structure Changes and Thermal Stability of Plasma Membrane Proteins of Wheat Roots in Heat Stress 817

troscopy is that it allows simultaneous study of the stru-

cture of lipids and proteins in intact biological mem-

branes without introduction of foreign perturbing probes

[4]. Some types of analysis of infrared data are claimed

to provide highly accurate quantitative estimates of the

content of secondary structures, with a standard devia-

tion as low as 2% - 3% with respect to the corresponding

X-ray structures. The advantage of FTIR is that protein

secondary structure is measured in the native environ-

ment of the proteins and that it is a noninvasive tech-

nique. FTIR spectroscopy is a unique possibility for the

simultaneous study of protein and lipid structures and

dynamics in biological membranes [5,6]. Differences in

the C-O stretching vibrations of the peptide groups (the

amide-I region between 1600 - 1700 cm–1) provide in-

formation on the type of secondary structure, such as

α-helix, β-strands, disorder and different kinds of turn

structures [7].

The processes controlling plant gro wth and survival in

a hostile environment might be clarified at molecular and

cellular level. Therefore, we examine the effect of heat

inactivation on the activity of PM H+-ATPase and the

PM protein, and investigate the changes in the protein

secondary structure of PM in response to high tempera-

tures by FTIR.

2. Material and Methods

2.1. Plan t Ma teria ls

Wheat seeds (Triticum aestivum var. Longchun No. 20,

purchased from Gansu Agriculture Sciences Academy,

China) were sterilized in 1% sodium hypochlorite (Na-

ClO) solution for 20 min, then soaked in water for 24 h

and germinated in the dark at 25˚C for 24 h., The seed-

lings were gro wn for 10 days at 19˚C in quartz sa nd and

irrigated with tap-water. They were illuminated for 14 h

each day with a light intensity of 100 µmol m–2·s–1. Since

the root plasma membrane is the site of absorption vari-

ous nutrition, we harvested the purification of plasma

membrane from wheat roots.

2.2. Wheat Roo ts Pla sma Mem brane Is olatio n

and Purification

Wheat roots plasma membrane was prepared by two-

phase partition [8]. Roots were cut into pieces and imme-

diately homogenized in the isolation medium containing

50 mM HEPES-tris, pH 7.5, 250 mM sucrose, 1 mM

EDTA, 0.6% PVP, 1 mM PMSF, and 1 mM DTT at 4˚C.

The homogenate was filtered through four layers of cot-

ton gauze and centrifuged (Beckman AJ-250) at 15,000 g

for 15 min. The supernatant was then centrifuged (Beck-

man AJ-250) at 80,000 g for 60 min in order to obtain a

microsomal pellet, which was resuspended in a buffer

containing 0.048% potassium phosphate (pH 7.8).

The suspension was then added to a phase mixture to

obtain a phase system consisting of 0.048% potassium

phosphate (pH 7.8), 6.2% PEG-3350, 6.2% dextran T-

500, 0.015% KCl, and 250 mM sucrose. After it was

centrifuged at 1000 g for 5 min for three times, the final

upper phases were collected, diluted at least twice with

50 mM HEPES-tris, pH 7.4, 250 mM sucrose, 1 mM

EDTA, 1 mM DTT, and 1 mM PMSF, and centrifuged

for 60 min at 80,000 g. The resulting pellet was resus-

pended in 50 mM Tris-HCl pH 7.4, 250 mM sucrose, 1

mM EDTA, 1 mM DTT , and 1 mM PMSF. All the above

steps were carried out at 4˚C. Plasma membrane pellets

were used immediately to obtain ATR-FTIR spectral.

2.3. PM H e at Trea tment

Polarized attenuated total internal reflection infrared spe-

ctroscopy is one of the best tools available for obtaining

information on the orientation of peptides interacting

with membranes. The method is simple (oriented multi-

layer systems are easily obtained by drying a membrane

suspension on the internal reflection element). It is sensi-

tive (less than 1 mg of peptide is required to form a sin-

gle monolayer) and it does not require specific labeling.

To remove the spectral interference from H2O absorption

bands (OH- bond strongly absorbed on amide I 1600 -

1700 cm–1), the PM samples were dissolved in an identi-

cal concentration but D2O-based buffer.

Heat inactivation was performed by incubating a puri-

fied PM sample on a CaF2 plate in a temperature bath

(Yamato-Komatsu Coolnics circulator CTE-24A) in steps

of 5˚C from 20˚C to 90˚C. After every heating step, the

PM sa mple was left to stab ilize for 5 min and then i m me-

diately subjected to ATR-FTIR measurement, and then

increased temperature in the step 5˚C.

The spectrum was obtained on an OMNI sampler of a

Fourier Transform Infrared spectrometer (Nexus 670;

Nicolet, Madison, WI) with germanium plane in a 2 mm2

sensing area. Attenuated total reflection infrared (ATR-

FTIR) spectra were recorded on an OMNIC sampling in-

strument. The internal reflection element was a germa-

nium ATR plate (50 × 20 × 2 mm) with an aperture angle

of 45˚. In total, 128 scans were accumulated for each

spectrum. Spectra were recorded at a nominal resolution

of 2 cm–1.

2.4. Assay of H+-ATPase A cti v it y

Heat inactivation was performed by incubating a purified

PM sa mple in tubes that p uted in Dri-Block Heater (DB -

2D Digital, United Kindom) in steps of 5˚C from 20˚C to

90˚C (three repeats). After every heating step, the PM

sample was left to stabilize for 5 min and then measure

its H+-ATPase activity as same treatment as the method

in 2.3. ATP hydrolysis assays were performed as de-

Secondary Structure Changes and Thermal Stability of Plasma Membrane Proteins of Wheat Roots in Heat Stress

818

scribed by Ohinishi [9]. Membrane proteins (10 - 15 µg)

were added to 0.5 ml of reaction medium containing 25

mM HEPES-Tris (pH 6.5), 3 mM ATP, 50 mM KCl, 1

mM Na3MoO4, and 0.015% (w/v) Triton X-100, in the

presence or absence of 400 µM Na3VO4. After 30 min

incubation at 37˚C, the reaction was quenched by the

addition of 10% (w/v) TCA. The H+-ATPase activity

was determined by measuring the release of Pi [9]. Pro-

tein in the plasma membranes was measured by the me-

thod of Bradford [10] with bovine serum albumin as the

standard.

2.5. FTIR Spectral Data Analysis

The IR Data Manager analytical software of OMNIC

(Version 6.0; Nicolet, USA) was used to obtain original

and Fourier self-deconvolution spectra and second-deri-

vati ve spec t ra.

The parameters for the Fourier self-deconvolution pro-

cedure were a smooth factor of 15.0 and a width factor of

10.0 cm–l, using the interactive OMNIC 6.0 tool for Fou-

rier self-deconvolution. The line width in the deconvo-

lved spectrum was chosen carefully to avoid introduction

of erroneous bands [11]. The second-derivative spectrum

was normalized, and the band position was calculated as

the average of the spectral positions at 80% of the total

peak height. They were smoothed over 15 data points.

The water spectra to be subtracted were collected un-

der the same conditions as the membrane spectra. Crite-

ria for the correctness of subtraction were removal of the

band near 2200 cm–1 and flat baseline between 1800 and

2000 cm–1 for samples in H2O and elimination of the

strong band at 1209 cm–1 for samples in D2O, avoiding

negative side lobes. Second-derivative and deconvolu-

tion spectra were used to determine the number and the

positions of the bands as starting parameters for the curve-

fitting procedure, assuming Pearson or Voigt band shapes.

The band-fitting used Origin 7.0 software with an ap-

pended PFM (Peak Fitting Maximum) function and for

protein studies the spectral region between 1800 and

1500 cm–1 was selected. This region contains the amide I

and amide II absorption bands of the protein backbones.

Band curve-fitting includes the decomposition of the

amide I band into its constituents and the assignment of

these components to protein structural features. To start

the quantification process, the number and position of the

bands and a rough estimation of band shapes, widths and

heights of the components is needed. The Gaussian peak

shape was used to fit the spectrum collected at each time

point (Chi-square < 10 - 5). Band curve-fitting is best

performed on original bands, although it is sometimes ac-

complished, less accurately, on more shapely spectra ob-

tained through self- deconvolution or s econd derivation .

3. Results and Discussion

3.1. High Temperatures Induced Inactivation of

PM H+-ATPase

An increase in temperature was accompanied by inhibit-

tion of the H+-ATPase activity of the PM vesicles iso-

lated from wheat roots (Figure 1), with a particularly

sharp reduction as the temperature rose from 35˚C to 50˚C,

and it was remaining at a very low level from 50˚C to

90˚C.

3.2. Relationship of Band positions of Amide I

with High Temperature Stress

The most studied infrared band of proteins is the amide I

band appearing between 1600 and 1700 cm–1, with a ma-

ximum for most proteins at around 1654 - 1674 cm–1.

This arises primarily from the stretch vibration of the pe-

ptide C=O group. Normal mode analysis reveals that the

C=O stretching couples slightly with CN stretching, CN

deformation and NH bending. Unfortunately, the HOH

bending motion of water almost coincides with the amide

I band and makes studies in proton aqueous solution dif-

ficult [12]. This problem is overcome by using D2O as

solvent. The D2O substitution leads to a shift of the am-

ide I band (amide I) between 2 and 9 cm–1 to lower fre-

quencies depending on the particular protein [13].

Because the C=O group is involved in different sec-

ondary structure elements via hydrogen bonding to the

peptide NH group, the experimentally observed amide I

band envelopes a multitude of single bands with different

frequencies which can be resolved as described above.

The large number o f infrared spectroscopic studie s in com-

20 2530 35 4045 5055 60 6570 75 8085 90

PM H+ -ATP as e A c t iv it

(um olP i/m g pr otei n h)

Temperature (˚C)

Figure 1. Inactivation of H+-ATPase in PM vesicl es isolate d

from wheat roots. The PM vesicles were heated from 20˚C

to 90˚C with 10 min at every 5˚C step, and then the H+-

ATPase activity was determined by measuring the release

of Pi. Values are mean ± SE of at least four replicates.

Secondary Structure Changes and Thermal Stability of Plasma Membrane Proteins of Wheat Roots in Heat Stress

819

were curve fitted with Origin 7.0 software with appended

PFM (Peak Fitting Maximum) function. The Ga ussian peak

peak shape was used to fit the spectrum collected at each

time point (Figure 3). The peaks belongs to the Amide I

bination with crystal st ructure and NMR anal yses on va-

rious proteins revealed some variability of the frequen-

cy of the assigned component amide I bands. However,

one can generally classify the components between 1658

cm–1 to α-helices [14,15], 1620 - 1635 cm–1 to intramo-

lecular β-sheets, and 1665 - 1690 cm–1 to turns [11,14,

15]. However, aggregated proteins show intermolecular

antiparallel β-sheets with infrared absorption around

1614 - 1624 cm–1 [11,14,15]. Some exceptions from this

general assignment have discussed in the recent review

by Arrondo and Goni [3] and by Heimburg [16]. Table 1

summarizes published assignments of amide I compo-

nents of the PM spectrum to secondary structure ele-

ments. Secondary structure composition can be estimated

from the relative area of the single bands assigned to the

different structures assuming that the extinction coeffi-

cient for the peptide CO stretch vibration is the same in

all hydrogen bonding structures.

Table 1. Frequencies and proposed structural assignments

of the bands for the decomposition between 1700 and 1580

cm–1 in the infrared spectrum of the PM.

Frequency (cm–1) Assignment References

1704 Ester C- O group Blume [17]

Menikh [18]

1693 β-Sheet He, WZ [19]

1665 Turns

Surewicz [20]

Dong [15]

1657 α-Helix He, WZ [19]

Dong [15]

1650 Rando m co il +

loops Surewicz [18]

Dong [15]

1638 β-Sheet He, WZ [19]

Surewicz [18]

Dong [14]

1628 β-Sheet (strands) He, WZ [16]

Surewicz [18]

Dong [15]

1610 Side-chain

(tyrosine) He, WZ [19]

MacDonald [21]

1582 Amide II band

He, WZ [19]

Haris [13]

3.3. Cha ng e s i n Prote in Second a ry

Conformation of PM Damaged under High

Temperature Stress

The original FTIR spectral band (Figure 2) often gener-

ated complex multi-component bands that overlap into a

broad unresolved absorption. To quantitatively analyze

the change in intensities of the amide I band, the IR spe-

ctra of the PM used Fourier self-deconvolution spectra

and second-derivative spectra. The IR spectra of the PM

1456.28

1650.96

0. 0

0. 2

0. 4

0. 6

0. 8

1. 0

1. 2

1. 4

1. 6

1. 8

2. 0

2. 2

2. 4

2. 6

2. 8

Abs

10 00 1100 1200 1300 1400 15 00 1600 1700 18 00

–1

Figure 2. Change in the band region from 1800 to 1000 cm–1 in the infrare d spectr um of t he plasma membrane i n D2O buffer

during the st epped heating process. Fro m upper to low er, the spectra are those at 2 0˚C to 90˚C wi th increasing te mperature

steps of 5˚C.

Secondary Structure Changes and Thermal Stability of Plasma Membrane Proteins of Wheat Roots in Heat Stress

820

(a)

Secondary Structure Changes and Thermal Stability of Plasma Membrane Proteins of Wheat Roots in Heat Stress

821

-0.4

-0.2

0.2

0.4

0.6

0.8

Absorbance

1710 1690 1670 1650 1630 1610 1590

Wavenumber (cm

-1

)

Temperature

(

(b)

Figure 3. (a) Curve-fitting result of Amide I from 20˚C - 80˚C. (b) Change of the amide I band in the infrared spectrum of the

plasma membrane during the stepped heating process. From inner to outer, the spectra recorded are those at increasing

temperature ste ps of 5˚C.

band, the five main peaks contained 41% α-helix(1657

cm–1), 16% β-sheet(1638 cm–1 and 1693 cm–1), 18% turn

(1665 cm–1), and 25% disorder(1650 cm–1) structures at

20˚C.

The amide I band has been widely used to reveal the

secondary structure of proteins. At temperatures from

25˚C to 90˚C, the α-helix and the β-sheet structure unfold

into the disorder structure and turns, with a major con-

formational transition occurring at 55˚C (Figure 4). This

leads to rapid decline of H+-ATPase activity from 35 to

10 20 30 40 5060 70 80 90100

Temperature (℃)

Relative band area (%)

radom coil+loops

а-helix

10 20 30 40 5060 70 80 90100

Temperature (℃)

Relative band area (%)

βsheet

turn

(a) (b)

Figure 4. Changes in the secondary structure content of the plasma membrane during heat denaturation obtained by

decomposition of the amide I band of the infrared spectrum. The β-sheet curve includes both β-sheet and extended chains

(β-strands). The random coils and loops represent unordered structures and loops. Turns usually mean β-turns. (a) α-helix

and random coil loops content of PM. (b) β sheet and turns content of PM.

Secondary Structure Changes and Thermal Stability of Plasma Membrane Proteins of Wheat Roots in Heat Stress

822

90˚C.

H+-ATPase activity was positively correlated (R2 =

0.6475) with the corresponding relative band area per-

centage of α-helix in the amide I region. Therefore the

protein conformational transition was one of reasons of

loses H+-ATPase activity of PM.

4. Acknowledgements

This work is financially supported by NSFC (30770343,

30870383 and 31070360) research foundation.

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