Ultramicroscopy 42-44 (1992) 1204-1213 North-Holland
Molecular images of cereal proteins by STM N.H. Thomson, M.J. Miles H.H. Wills Physics Laboratory, University of Bristol, Tyndall AL,enue, Bristol, BS8 1TL, UK
A.S. T a t h a m a n d P.R. S h e w r y AFRC Institute of Arable Crops Research, Department of Agricultural Sciences, Unit,ersity of Bristol, Long Ashton Research Station, Bristol, BS18 9AF, UK Received 12 August 1991
Scanning tunnelling microscopy (STM) has been used to study a seed storage protein of wheat known as y-gliadin. The protein was deposited onto highly oriented pyrolytic graphite ( H O P G ) from solutions of trifluoroethanol (TFE) and 1% acetic acid. Samples were dried down and then scanned in air. Transmission electron microscopy (TEM) was also used to visualise the distribution of protein on the substrate. Small-angle X-ray scattering (SAXS) was used to compare the molecular size and shape obtained with those from the STM images.
I. Introduction The seed storage proteins (prolamins) of wheat are the main determinants of the properties of wheat for various technological processes, including breadmaking. Gluten, the proteinaceous mass that remains when the starch is washed out from dough, has the unique properties (among the cereals) of elasticity and viscous flow. The bases for these properties are unknown [1]. The proteins that make up gluten can be divided into three groups: the high-molecularweight (HMW) subunits, which have been studied by STM [2], the sulphur-poor and sulphur-rich prolamins [3]. All of the prolamins contain sections of repetitive sequence; in the H M W and sulphur-poor prolamins this extends to most of the sequence and in the sulphur-rich for approximately 50%. The sulphur-rich prolamins are the major group of prolamins accounting for about 80% of the total fraction. Most have molecular weights between 35 000 and 45 000 and consist of 250-300 amino acid residues. They have a clear domain structure, a repetitive N-terminal domain
and a non-repetitive C-terminal domain, this latter domain contains the cysteine residues for cross-linking and most of the charged residues
[41. The y-type gliadins are a sub-family of the sulphur-rich gliadins and may closely correspond to the ancestral type of sulphur-rich prolamin [5,6], as they are present in meadow grasses as well as other cereals. They are an important group occurring as both intra-molecular disulphide bonded monomers and as inter-molecular disulphide bonded polymers. In the monomeric form they are thought to contribute to viscosity and in the polymeric form to elasticity. They consist of an N-terminal domain based on a repeat motif (Consensus Pro.Gln.Gln.Pro.Phe. Pro.Gin) and a C-terminal proline-poor, non-repetitive domain. Structural studies, using circular dichroism and secondary structure prediction, indicate that the two domains adopt different structures. The repetitive domain adopting a reverseturn rich conformation and the non-repetitive domain an a-helical rich globular conformation [7]. The molecule has been shown to be asymmet-
0304-3991/92/$05.00 © 1992 - Elsevier Science Publishers B.V. All rights reserved
N.H. Thomson et al. / Molecular images of cereal proteins by STM
ric, the repetitive domain may therefore have an extended conformation [8]. The y-type gliadins are related to the other sulphur-rich gliadins and their study should facilitate an understanding of this complex group of proteins in both their structure and functional properties. All these wheat storage proteins are exceptionally robust, maintaining their conformation at temperatures up to 80°C in both aqueous and non-aqueous solvents [9]. This means that solutions of them are easy to prepare for STM and that the proteins themselves are unlikely to be denatured during imaging. Other techniques have also been used to help in characterising these proteins. Transmission electron microscopy (TEM) was used to examine the distribution of the proteins on the STM substrates. Also, small-angle X-ray scattering has
1205
been used to investigate the size and shape of 7-gliadin.
2. Experiment The STM used for these studies was manufactured by WA Technology (Cambridge, UK). It was mounted on an anti-vibration table and operated in air. The protein solutions, of various concentrations, were deposited onto H O P G (Union Carbide, USA) in either 10 or 100/~l drops, and allowed to dry down. Since T F E is very volatile, these solutions dried almost immediately. For the acetic acid samples, excess solution was removed using filter paper. The tips used were either mechanically cut gold wire or electrochemically etched tungsten wire. Images were
Fig. 1. STM image (119.8x 119.8x 1.5 nm) of a monolayerof y-gliadindeposited from 1% acetic acid. Bias 800 mV; current 0.1 nA.
1206
N.H. Thomson et al. / Molecular images of cereal proteins by STM
recorded in the constant-current mode with typical tunnelling conditions in the range 0.01 to 0.1 nA and 100 to 2500 mV. Image acquisition times were in the range 44 s to 3 min 38 s. Replicas for T E M were prepared by evaporating p l a t i n u m / p a l l a d i u m alloy onto the H O P G substrates at a shadowing angle of 20 °. The small-angle X-ray scattering (SAXS) was carried out at the Synchrotron Radiation Source (SRS) at Daresbury, UK. The scattering from 7-gliadin was studied in 1% acetic acid at a concentration of 3 m g / m l .
3. Results and discussion
In initial STM studies 100 /xl of protein solution, with a concentration of 200 /xg/ml, was deposited. In many areas of the sample it was very difficult to achieve a stable tunnelling current. The tip tended to pick up protein and drag it across the substrate producing blurred images. This suggested that there was multilayer coverage of the protein on the substrate. However, in a few
areas stable conditions were reached and images were obtained. Figs. 1 and 2 are examples of these images, protein having been deposited from 1% acetic acid and TFE, respectively. They both indicate monolayer coverage of the protein on the H O P G substrates. The monolayers have holes in them revealing the bare substrate beneath. However, it is not possible to ascertain the true dimensions of the protein from these layers since they will not have the same conductivity as the substrate. The step feature in the bottom right of fig. 1 is associated with the H O P G substrate. To understand exactly how the protein was distributed on the substrates, replicas were taken from them and imaged in a transmission electron microscope. Fig. 3 shows two areas of a replica taken from a substrate on which protein had been deposited from 1% acetic acid. They indicate that multilayer coverage was widespread, but lighter regions suggest that monolayers were also present. Fig. 4 shows two areas of a replica (at different magnification) taken from a substrate on which protein had been deposited from TFE. These show multilayer coverage of protein with
Fig. 2. Three-dimensional representation of a STM image (65.5 x 65.5 × 2.0 nm) showing monolayer coverage of y-gliadin deposited from TFE. Bias 2500 mV; current 0.1 nA.
N.H. Thomson et al. / Molecular images of cereal proteins by STM
holes of various sizes. Again, the lighter regions suggest areas of monolayer coverage. The surface pressures and potentials of gliadin films have been studied at an a i r / w a t e r interface [10], and two-dimensional micelles have been shown to exist.
1207
In an attempt to image individual molecules, the amount of protein deposited on the substrates was reduced by a factor of one thousand. 10/~1 of protein solution, with a concentration of under 2 /xg/ml, was deposited. For these samples stable tunnelling currents were readily ob-
Fig. 3. TEM images of a replica taken from a STM substrate on which y-gliadin has been deposited from 1% acetic acid. Both images have the same magnification.Scale bars represent 3.4 p,m.
1208
N.H. Thomson et al. / Molecular images of cereal proteins by STM
tained. H o w e v e r , m a n y a r e a s o f t h e s e s u b s t r a t e s were devoid of p r o t e i n , b u t a r e a s o f p r o t e i n c o u l d be f o u n d a n d i m a g e d successfully. T h e distribution of the p r o t e i n a p p e a r e d to be very d i f f e r e n t d e p e n d i n g u p o n the solvent used. Figs. 5 a n d 6
show typical coverage. In fig. 5 T F E . It can still p r e s e n t ,
i m a g e s o b t a i n e d in a r e a s of p r o t e i n 7-gliadin has b e e n d e p o s i t e d from be seen that m o n o l a y e r c o v e r a g e is the p r o t e i n m o l e c u l e s a d s o r b e d with
Fig. 4. TEM images of a replica taken from a STM substrate on which y-gliadin has been deposited from TFE. (a) ScaLe bar represents 17.2/xm. (b) Scale bar represents 2.6/.tin.
N.H. Thomson et al. / Molecular images of cereal proteins by STM
their largest dimension perpendicular to the substrate. It is expected that the repetitive domain, which is the most hydrophobic of the two (since it contains few charged amino acid residues) will be adsorbed to the hydrophobic H O P G surface. Images very similar to these have also been obtained when monolayer coverage was found in the higher concentration samples (fig. 7). It can be seen that the packing density in these monolayers does not remain constant across the substrate. This could be due to the proteins moving under the influence of the tip. Monolayers of other proteins have been observed where loose packing allows lateral movement of the protein molecules under the tip [11]. It can be seen that the images in fig. 7 are quite different from those in figs. 1 and 2, even though they correspond to similar preparations and tunnelling conditions. This difference may be due to the packing arrangement of the proteins. In figs. 1 and 2 it is thought that the proteins are lying flat with their smallest axis perpendicular to the substrate, whereas in the other images the proteins are adsorbed with their
1209
longest axis perpendicular to the substrate. Although the heights of these images will not give the true dimensions of the protein the ratio of the heights should give the axial ratio for the molecules. For deposition from 1% acetic acid it is 2.73 and from T F E it is 2.35. Fig. 6 shows typical images obtained when 7-gliadin has been deposited from 1% acetic acid. Monolayers were not observed; only small aggregates or individual molecules (usually associated in two's or three's) were discovered. Fig. 6a contains several proteins lying with their longest axis parallel to the substrate. Their different shapes may arise from their orientation with respect to the scanning direction and the manner in which they adsorb to the surface. Fig. 6b shows two 7-gliadin molecules, aggregated head to tail, lying flat on the H O P G substrate. This image gives the dimensions of the protein molecule as 10 nm by 3 nm. The noisy appearance of the substrate in fig. 6b is attributed to salt contamination arising from solvent still present on the substrate. Subsequently, it has been found that such contamina-
Fig. 5. Three-dimensional representation of a STM image ('43.6x 43.6 x 2.7 nm) showing a monolayer of y-gliadin deposited from TFE. Bias 1000 mV; current 0.01 nA.
1210
N.H. Thomson et al. / Molecular images of cereal proteins by STM
tion can b e r e m o v e d by v a c u u m sublimation. H o w e v e r , this might not be d e s i r a b l e as it m a y c h a n g e t h e native c o n f o r m a t i o n of the p r o t e i n . T h e S A X S d a t a has given i n d e p e n d e n t confirm a t i o n o f the m o l e c u l a r d i m e n s i o n s of 7-gliadin. Figs. 8 a n d 9 show t h e G u i n i e r plot a n d cross-sec-
tion plot o b t a i n e d f r o m t h e c e n t r a l p a r t of t h e s c a t t e r i n g curve. T h e G u i n i e r a p p r o x i m a t i o n gives a r a d i u s of gyration ( R g ) o f 2.66 nm. If the p r o t e i n is a s s u m e d to b e rod-like, t h e r a d i u s of gyration of cross-section ( R c) is 1.22 nm. T h e n the m a x i m u m length of the p r o t e i n is 9.2 nm
Fig. 6. (a) STM image (52.2 × 52.2 x 1.5 nm) showing several y-gliadin molecules lying flat on HOPG with different orientations. Deposition was from 1% acetic acid. Bias 600 mV; current 0.01 nA. (b) Three-dimensional representation of a STM image (27.8 x 27.8 x 0.8 nm) of two 7-gliadin molecules on HOPG, deposited from 1% acetic acid. Bias 1000 mV; current 0.01 nA.
N.H. Thomson et al. / Molecular images of cereal proteins by STM
1211
(Rg x v/~ -) and its diameter is 3.4 nm (R c X 2V~-).
4. Conclusions
These values correspond very closely to the dimensions obtained from the STM images. Also, this gives the axial ratio of the protein as 2.70, which is almost the same as that determined from the monolayers of protein (deposited from 1% acetic acid) imaged in the STM.
STM has been used in conjunction with other techniques (TEM and SAXS) to determine the structure of the wheat storage protein y-gliadin. STM has shown that these proteins will form monolayers on H O P G if a suitable amount of
Fig. 7. Three-dimensional representations of STM images showingmonolayercoverage of y-gliadin. (a) Deposition from 1% acetic acid. Bias 2000 mV; current 0.1 nA. (b) Deposition from TFE. Bias 2000 mV; current 0.1 nA.
1212
N.H. Thomson et al. / Molecular images of cereal proteins by STM
protein is present. In these monolayers the proteins are adsorbed with their largest dimension perpendicular to the H O P G substrate. The repetitive domain is most likely to be bound to the surface, being more hydrophobic than the nonrepetitive domain. Imaging of individual protein molecules is possible, when a much smaller amount of protein is deposited from a solution of 1% acetic acid. The apparent size of the molecule from these images is 10 nm by 3 nm. These dimensions correspond to the values obtained from small-angle X-ray scattering of 7-gliadin in a solution of 1% acetic acid. These data have given dimensions of 10.1 nm by 3.1 nm. The heights of the monolayers also seem to relate to these dimensions. STM images of biological specimens tend to give one half to one third of the true height. The heights of the monolayers are between 2.7 and 4.1 nm. This will be determined by the solvent the protein was deposited from and the tunnelling conditions used
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(4sin20/:k~) x100000 Fig. 9. Cross-section plot o f SAXS data obtained from solution scattering o f 7-gliadin in 1% acetic acid.
12
in acquiring the image. When deposition and tunnelling conditions are almost identical, the ratio of the heights of these monolayers, which appear to have different packing orientations, gives the axial ratio of the protein. This closely corresponds to the axial ratio of the protein molecules determined from SAXS data.
11
vlO t-..J
Acknowledgements We thank the Agricultural Food Research Council for supporting this work and the Science and Engineering Research Council for providing financial support for N.H. Thomson. Thanks also go to Dr. Mary Hill for assistance in electron microscopy. I
I
1
[
5
10
15
20
25
(4sin2e/~) xlO0000 Fig. 8. Guinier plot of SAXS data obtained from solution scattering of 7-gliadin in 1% acetic acid.
References [1] B.J. Miflin, J.M. Field and P.R. Shewry, in: Seed Proteins, Eds. J. D.aussant, J. Mosse and Y. Vaughan (Academic Press, London, 1983) pp. 255-319.
N.H. Thomson et aL / Molecular images of cereal proteins by STM [2] M.J. Miles, H.J. Carr, T.J. McMaster, K.J. I'Anson, P.S. Belton, V.J. Morris, J.M. Field, P.R. Shewry and A.S. Tatham, Proc. Natl. Acad. Sci. USA 88 (1991) 68. [3] P.R. Shewry, A.S. Tatham, J. Forde, M. Kries and B.J. Miflin, J. Cereal Sci. 4 (1986) 97. [4] P.R. Shewry and A.S. Tatham, Biochem. J. 267 (1990) 1. [5] P.R. Shewry, S.J. Smith, E.J.L. Lew and D.D. Kasarda, J. Exp. Bot. 37 (1986) 633. [6] V. Cameron-Mills and A. Brandt, Plant. Mol. Biol. 11 (1988) 449.
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[7] A.S. Tatham, P. Masson and Y. Popineau, J. Cereal Sci. 11 (1990) 1. [8] Y. Popineau and F. Pineau, Lebensm. Wiss. Technol. 21 (1988) 113. [9] A.S. Tatham and P.R. Shewry, J. Cereal Sci. 3 (1985) 103. [10] E.G. Cockbain and J.H. Schulman, Trans. Faraday Soc. 35 (1939) 1266. [11] L. Haussling, B. Michel, H. Ringsdorf and H. Rohrer, Angew. Chem. Int. Ed. Engl. 30 No. 5 (1991) 569.
Molecular images of cereal proteins by STM N.H. Thomson, M.J. Miles H.H. Wills Physics Laboratory, University of Bristol, Tyndall AL,enue, Bristol, BS8 1TL, UK
A.S. T a t h a m a n d P.R. S h e w r y AFRC Institute of Arable Crops Research, Department of Agricultural Sciences, Unit,ersity of Bristol, Long Ashton Research Station, Bristol, BS18 9AF, UK Received 12 August 1991
Scanning tunnelling microscopy (STM) has been used to study a seed storage protein of wheat known as y-gliadin. The protein was deposited onto highly oriented pyrolytic graphite ( H O P G ) from solutions of trifluoroethanol (TFE) and 1% acetic acid. Samples were dried down and then scanned in air. Transmission electron microscopy (TEM) was also used to visualise the distribution of protein on the substrate. Small-angle X-ray scattering (SAXS) was used to compare the molecular size and shape obtained with those from the STM images.
I. Introduction The seed storage proteins (prolamins) of wheat are the main determinants of the properties of wheat for various technological processes, including breadmaking. Gluten, the proteinaceous mass that remains when the starch is washed out from dough, has the unique properties (among the cereals) of elasticity and viscous flow. The bases for these properties are unknown [1]. The proteins that make up gluten can be divided into three groups: the high-molecularweight (HMW) subunits, which have been studied by STM [2], the sulphur-poor and sulphur-rich prolamins [3]. All of the prolamins contain sections of repetitive sequence; in the H M W and sulphur-poor prolamins this extends to most of the sequence and in the sulphur-rich for approximately 50%. The sulphur-rich prolamins are the major group of prolamins accounting for about 80% of the total fraction. Most have molecular weights between 35 000 and 45 000 and consist of 250-300 amino acid residues. They have a clear domain structure, a repetitive N-terminal domain
and a non-repetitive C-terminal domain, this latter domain contains the cysteine residues for cross-linking and most of the charged residues
[41. The y-type gliadins are a sub-family of the sulphur-rich gliadins and may closely correspond to the ancestral type of sulphur-rich prolamin [5,6], as they are present in meadow grasses as well as other cereals. They are an important group occurring as both intra-molecular disulphide bonded monomers and as inter-molecular disulphide bonded polymers. In the monomeric form they are thought to contribute to viscosity and in the polymeric form to elasticity. They consist of an N-terminal domain based on a repeat motif (Consensus Pro.Gln.Gln.Pro.Phe. Pro.Gin) and a C-terminal proline-poor, non-repetitive domain. Structural studies, using circular dichroism and secondary structure prediction, indicate that the two domains adopt different structures. The repetitive domain adopting a reverseturn rich conformation and the non-repetitive domain an a-helical rich globular conformation [7]. The molecule has been shown to be asymmet-
0304-3991/92/$05.00 © 1992 - Elsevier Science Publishers B.V. All rights reserved
N.H. Thomson et al. / Molecular images of cereal proteins by STM
ric, the repetitive domain may therefore have an extended conformation [8]. The y-type gliadins are related to the other sulphur-rich gliadins and their study should facilitate an understanding of this complex group of proteins in both their structure and functional properties. All these wheat storage proteins are exceptionally robust, maintaining their conformation at temperatures up to 80°C in both aqueous and non-aqueous solvents [9]. This means that solutions of them are easy to prepare for STM and that the proteins themselves are unlikely to be denatured during imaging. Other techniques have also been used to help in characterising these proteins. Transmission electron microscopy (TEM) was used to examine the distribution of the proteins on the STM substrates. Also, small-angle X-ray scattering has
1205
been used to investigate the size and shape of 7-gliadin.
2. Experiment The STM used for these studies was manufactured by WA Technology (Cambridge, UK). It was mounted on an anti-vibration table and operated in air. The protein solutions, of various concentrations, were deposited onto H O P G (Union Carbide, USA) in either 10 or 100/~l drops, and allowed to dry down. Since T F E is very volatile, these solutions dried almost immediately. For the acetic acid samples, excess solution was removed using filter paper. The tips used were either mechanically cut gold wire or electrochemically etched tungsten wire. Images were
Fig. 1. STM image (119.8x 119.8x 1.5 nm) of a monolayerof y-gliadindeposited from 1% acetic acid. Bias 800 mV; current 0.1 nA.
1206
N.H. Thomson et al. / Molecular images of cereal proteins by STM
recorded in the constant-current mode with typical tunnelling conditions in the range 0.01 to 0.1 nA and 100 to 2500 mV. Image acquisition times were in the range 44 s to 3 min 38 s. Replicas for T E M were prepared by evaporating p l a t i n u m / p a l l a d i u m alloy onto the H O P G substrates at a shadowing angle of 20 °. The small-angle X-ray scattering (SAXS) was carried out at the Synchrotron Radiation Source (SRS) at Daresbury, UK. The scattering from 7-gliadin was studied in 1% acetic acid at a concentration of 3 m g / m l .
3. Results and discussion
In initial STM studies 100 /xl of protein solution, with a concentration of 200 /xg/ml, was deposited. In many areas of the sample it was very difficult to achieve a stable tunnelling current. The tip tended to pick up protein and drag it across the substrate producing blurred images. This suggested that there was multilayer coverage of the protein on the substrate. However, in a few
areas stable conditions were reached and images were obtained. Figs. 1 and 2 are examples of these images, protein having been deposited from 1% acetic acid and TFE, respectively. They both indicate monolayer coverage of the protein on the H O P G substrates. The monolayers have holes in them revealing the bare substrate beneath. However, it is not possible to ascertain the true dimensions of the protein from these layers since they will not have the same conductivity as the substrate. The step feature in the bottom right of fig. 1 is associated with the H O P G substrate. To understand exactly how the protein was distributed on the substrates, replicas were taken from them and imaged in a transmission electron microscope. Fig. 3 shows two areas of a replica taken from a substrate on which protein had been deposited from 1% acetic acid. They indicate that multilayer coverage was widespread, but lighter regions suggest that monolayers were also present. Fig. 4 shows two areas of a replica (at different magnification) taken from a substrate on which protein had been deposited from TFE. These show multilayer coverage of protein with
Fig. 2. Three-dimensional representation of a STM image (65.5 x 65.5 × 2.0 nm) showing monolayer coverage of y-gliadin deposited from TFE. Bias 2500 mV; current 0.1 nA.
N.H. Thomson et al. / Molecular images of cereal proteins by STM
holes of various sizes. Again, the lighter regions suggest areas of monolayer coverage. The surface pressures and potentials of gliadin films have been studied at an a i r / w a t e r interface [10], and two-dimensional micelles have been shown to exist.
1207
In an attempt to image individual molecules, the amount of protein deposited on the substrates was reduced by a factor of one thousand. 10/~1 of protein solution, with a concentration of under 2 /xg/ml, was deposited. For these samples stable tunnelling currents were readily ob-
Fig. 3. TEM images of a replica taken from a STM substrate on which y-gliadin has been deposited from 1% acetic acid. Both images have the same magnification.Scale bars represent 3.4 p,m.
1208
N.H. Thomson et al. / Molecular images of cereal proteins by STM
tained. H o w e v e r , m a n y a r e a s o f t h e s e s u b s t r a t e s were devoid of p r o t e i n , b u t a r e a s o f p r o t e i n c o u l d be f o u n d a n d i m a g e d successfully. T h e distribution of the p r o t e i n a p p e a r e d to be very d i f f e r e n t d e p e n d i n g u p o n the solvent used. Figs. 5 a n d 6
show typical coverage. In fig. 5 T F E . It can still p r e s e n t ,
i m a g e s o b t a i n e d in a r e a s of p r o t e i n 7-gliadin has b e e n d e p o s i t e d from be seen that m o n o l a y e r c o v e r a g e is the p r o t e i n m o l e c u l e s a d s o r b e d with
Fig. 4. TEM images of a replica taken from a STM substrate on which y-gliadin has been deposited from TFE. (a) ScaLe bar represents 17.2/xm. (b) Scale bar represents 2.6/.tin.
N.H. Thomson et al. / Molecular images of cereal proteins by STM
their largest dimension perpendicular to the substrate. It is expected that the repetitive domain, which is the most hydrophobic of the two (since it contains few charged amino acid residues) will be adsorbed to the hydrophobic H O P G surface. Images very similar to these have also been obtained when monolayer coverage was found in the higher concentration samples (fig. 7). It can be seen that the packing density in these monolayers does not remain constant across the substrate. This could be due to the proteins moving under the influence of the tip. Monolayers of other proteins have been observed where loose packing allows lateral movement of the protein molecules under the tip [11]. It can be seen that the images in fig. 7 are quite different from those in figs. 1 and 2, even though they correspond to similar preparations and tunnelling conditions. This difference may be due to the packing arrangement of the proteins. In figs. 1 and 2 it is thought that the proteins are lying flat with their smallest axis perpendicular to the substrate, whereas in the other images the proteins are adsorbed with their
1209
longest axis perpendicular to the substrate. Although the heights of these images will not give the true dimensions of the protein the ratio of the heights should give the axial ratio for the molecules. For deposition from 1% acetic acid it is 2.73 and from T F E it is 2.35. Fig. 6 shows typical images obtained when 7-gliadin has been deposited from 1% acetic acid. Monolayers were not observed; only small aggregates or individual molecules (usually associated in two's or three's) were discovered. Fig. 6a contains several proteins lying with their longest axis parallel to the substrate. Their different shapes may arise from their orientation with respect to the scanning direction and the manner in which they adsorb to the surface. Fig. 6b shows two 7-gliadin molecules, aggregated head to tail, lying flat on the H O P G substrate. This image gives the dimensions of the protein molecule as 10 nm by 3 nm. The noisy appearance of the substrate in fig. 6b is attributed to salt contamination arising from solvent still present on the substrate. Subsequently, it has been found that such contamina-
Fig. 5. Three-dimensional representation of a STM image ('43.6x 43.6 x 2.7 nm) showing a monolayer of y-gliadin deposited from TFE. Bias 1000 mV; current 0.01 nA.
1210
N.H. Thomson et al. / Molecular images of cereal proteins by STM
tion can b e r e m o v e d by v a c u u m sublimation. H o w e v e r , this might not be d e s i r a b l e as it m a y c h a n g e t h e native c o n f o r m a t i o n of the p r o t e i n . T h e S A X S d a t a has given i n d e p e n d e n t confirm a t i o n o f the m o l e c u l a r d i m e n s i o n s of 7-gliadin. Figs. 8 a n d 9 show t h e G u i n i e r plot a n d cross-sec-
tion plot o b t a i n e d f r o m t h e c e n t r a l p a r t of t h e s c a t t e r i n g curve. T h e G u i n i e r a p p r o x i m a t i o n gives a r a d i u s of gyration ( R g ) o f 2.66 nm. If the p r o t e i n is a s s u m e d to b e rod-like, t h e r a d i u s of gyration of cross-section ( R c) is 1.22 nm. T h e n the m a x i m u m length of the p r o t e i n is 9.2 nm
Fig. 6. (a) STM image (52.2 × 52.2 x 1.5 nm) showing several y-gliadin molecules lying flat on HOPG with different orientations. Deposition was from 1% acetic acid. Bias 600 mV; current 0.01 nA. (b) Three-dimensional representation of a STM image (27.8 x 27.8 x 0.8 nm) of two 7-gliadin molecules on HOPG, deposited from 1% acetic acid. Bias 1000 mV; current 0.01 nA.
N.H. Thomson et al. / Molecular images of cereal proteins by STM
1211
(Rg x v/~ -) and its diameter is 3.4 nm (R c X 2V~-).
4. Conclusions
These values correspond very closely to the dimensions obtained from the STM images. Also, this gives the axial ratio of the protein as 2.70, which is almost the same as that determined from the monolayers of protein (deposited from 1% acetic acid) imaged in the STM.
STM has been used in conjunction with other techniques (TEM and SAXS) to determine the structure of the wheat storage protein y-gliadin. STM has shown that these proteins will form monolayers on H O P G if a suitable amount of
Fig. 7. Three-dimensional representations of STM images showingmonolayercoverage of y-gliadin. (a) Deposition from 1% acetic acid. Bias 2000 mV; current 0.1 nA. (b) Deposition from TFE. Bias 2000 mV; current 0.1 nA.
1212
N.H. Thomson et al. / Molecular images of cereal proteins by STM
protein is present. In these monolayers the proteins are adsorbed with their largest dimension perpendicular to the H O P G substrate. The repetitive domain is most likely to be bound to the surface, being more hydrophobic than the nonrepetitive domain. Imaging of individual protein molecules is possible, when a much smaller amount of protein is deposited from a solution of 1% acetic acid. The apparent size of the molecule from these images is 10 nm by 3 nm. These dimensions correspond to the values obtained from small-angle X-ray scattering of 7-gliadin in a solution of 1% acetic acid. These data have given dimensions of 10.1 nm by 3.1 nm. The heights of the monolayers also seem to relate to these dimensions. STM images of biological specimens tend to give one half to one third of the true height. The heights of the monolayers are between 2.7 and 4.1 nm. This will be determined by the solvent the protein was deposited from and the tunnelling conditions used
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(4sin20/:k~) x100000 Fig. 9. Cross-section plot o f SAXS data obtained from solution scattering o f 7-gliadin in 1% acetic acid.
12
in acquiring the image. When deposition and tunnelling conditions are almost identical, the ratio of the heights of these monolayers, which appear to have different packing orientations, gives the axial ratio of the protein. This closely corresponds to the axial ratio of the protein molecules determined from SAXS data.
11
vlO t-..J
Acknowledgements We thank the Agricultural Food Research Council for supporting this work and the Science and Engineering Research Council for providing financial support for N.H. Thomson. Thanks also go to Dr. Mary Hill for assistance in electron microscopy. I
I
1
[
5
10
15
20
25
(4sin2e/~) xlO0000 Fig. 8. Guinier plot of SAXS data obtained from solution scattering of 7-gliadin in 1% acetic acid.
References [1] B.J. Miflin, J.M. Field and P.R. Shewry, in: Seed Proteins, Eds. J. D.aussant, J. Mosse and Y. Vaughan (Academic Press, London, 1983) pp. 255-319.
N.H. Thomson et aL / Molecular images of cereal proteins by STM [2] M.J. Miles, H.J. Carr, T.J. McMaster, K.J. I'Anson, P.S. Belton, V.J. Morris, J.M. Field, P.R. Shewry and A.S. Tatham, Proc. Natl. Acad. Sci. USA 88 (1991) 68. [3] P.R. Shewry, A.S. Tatham, J. Forde, M. Kries and B.J. Miflin, J. Cereal Sci. 4 (1986) 97. [4] P.R. Shewry and A.S. Tatham, Biochem. J. 267 (1990) 1. [5] P.R. Shewry, S.J. Smith, E.J.L. Lew and D.D. Kasarda, J. Exp. Bot. 37 (1986) 633. [6] V. Cameron-Mills and A. Brandt, Plant. Mol. Biol. 11 (1988) 449.
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[7] A.S. Tatham, P. Masson and Y. Popineau, J. Cereal Sci. 11 (1990) 1. [8] Y. Popineau and F. Pineau, Lebensm. Wiss. Technol. 21 (1988) 113. [9] A.S. Tatham and P.R. Shewry, J. Cereal Sci. 3 (1985) 103. [10] E.G. Cockbain and J.H. Schulman, Trans. Faraday Soc. 35 (1939) 1266. [11] L. Haussling, B. Michel, H. Ringsdorf and H. Rohrer, Angew. Chem. Int. Ed. Engl. 30 No. 5 (1991) 569.
