Abstract
Hydroxyproline-rich glycoproteins (HRGPs) are major protein components in dicot primary cell walls and generally account for more than 10% of the wall dry weight. As essential members of the HRGP superfamily, extensins (EXTs) presumably function in the cell wall by assembling into positively charged protein scaffolds (Cannon et al., 2008) that direct the proper deposition of other wall polysaccharides, especially pectins, to ensure correct cell wall assembly (Hall and Cannon, 2002; Lamport et al., 2011a). Extensins are recalcitrant to purification as they are rapidly cross-linked into a covalent network after entering the cell wall but there exists a short time window in which newly synthesized extensin monomers can be extracted (Smith et al., 1984; Smith et al., 1986) by salt elution. A detailed protocol for extraction of extensin and other wall structural proteins has been described earlier (Lamport et al., 2011b). The protocol elaborated here provides an approach to studying the self-assembly of extensins and potentially of other cell wall components in vitro using AFM.
Keywords: Plant cell wall, Structural glycoprotein, Extensin, Self-assembly, Atomic force microscope
Materials and Reagents
Equipment
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Representative data
Figure 1. Images of extensin self-assembly. (a) Monomeric extensin precursor 1 from tomato suspension culture (TOMP1). (b) Extensin analog YK8. (c) Extensin analog FK9. (d) BSA used as a control. YK8 and FK9 are extensin analogs that contain 8 repeats of SOOOOSOSOOOOYYYK and 9 repeats of SOOOOSOSOOOOFFFK in their protein sequences, respectively, and are purified from tobacco BY2 cells in culture (Held et al., 2004). One letter amino acids codes: Tyrosine (Y), Lysine (K), Phenylalanine (F), Serine (S), Hydroxyproline (O). All proteins were imaged at 10 μg/ml in pH 5.2 NaOAc buffer, following deposition for 1 min. The white scale bar: 500 nm. TOMP1 and the extensin analogs all showed a head to tail dendritic self-assembly similar to that of EXTENSIN 3 from Arabidopsis (Cannon et al., 2008), while BSA showed no such assembly but only protein aggregation. Red arrow in image (d) indicates the scattered single sphere shaped BSA molecules. Figure 2. Concentration and deposition time dependent self-assembly of TOMP1 in pH 5.2 NaOAc buffer. a. TOMP1 showed head to tail dendritic self-assembly after 10 min deposition on HOPG at 5 µg/ml. b. At a higher concentration (10 µg/ml) TOMP1 assembled into a porous network in the same time period, indicating the extent of self-assembly is concentration dependent. c. Similar assembly of TOMP1 as in (a), highlighted by the white circles, was observed when 10 µg/ml of TOMP1 was deposited for 1 min, indicating that the extent of TOMP1 self-assembly also followed a time dependent manner. Examples of measurements for TOMP1 self-assembly: (1) The average segment length, highlighted by white bars in the circles in image (c), is 78.2 ± 4.2 nm this is in agreement with previously electron microscopy measurements of the TOMP1 polypeptide length at about 79 nm (Heckman et al., 1988). This observation indicates one TOMP1 molecule occurs in each segment. (2) The average single molecule height, highlighted by red bars in image (c), is 2.7 ± 0.2 nm. This value corresponds to the diameter of single TOMP1 molecule. (3) The average height of the connecting points for three molecules, highlighted by green circles in image (c), is 5.2 ± 0.2 nm. This value indicates that the TOMP1 molecules form lateral and overlapping associations up to two molecules deep. The white scale bar: 500 nm
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Acknowledgments
This protocol was adapted with modification from Cannon et al. (2008). Funding of this work was from National Science Foundation (#IOS955569 to M.J.K and IOS0955805 to M.C.C).
References
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