8. Imaging
This section, while by no means a review, uses a few images to illustrate the methods outlined in previous sections. Referring to the color plates in the middle of this book, Plate Ia shows a high-resolution image of DNA tightly adsorbed onto a cationic bilayer and imaged by contact mode AFM (Mou, Czajkowsky et al. 1995). The helical repeat of the major groove of the double helix is clearly resolved at many places in the image. This is representative of the highest resolution images of DNA obtained at the time of writing.
Fig 19: Time lapse series of images showing transcription of a 1047 dsDNA template by an RNAp molecule. The first two images show that before the NTPs enter, the DNA is mobile on the surface. The six images after NTP addition from time 0:00 onwards are sequential and show that one arm of the DNA template becomes progressively shorter until it is released (2:38). The 1047 bp template has a terminating sequence but it appears that the RNAp read through the terminator to the end of the DNA. (From Kasas et al. (Kasas, Thomson et al. 1997) with permission.)
Crystal surfaces provide stable packing and proteins have been imaged in these surfaces at very high resolution by contact mode (Baker, Helbert et al. 1997). Insulin crystals have been images at somewhat lower resolution using acoustic DFM (Yip and Ward 1996; Yip, Brader et al. 1998). Another system in which close packing permits extremely high resolution imaging in contact mode is a two-dimensional crystalline array of proteins (Schabert and Engel 1994; Engel, Schoenberger et al. 1997). Such films are often made using densely packed arrays of trans-membrane proteins and these can retain functionality despite immobilization in a 2D film. They are thus ideal candidates for the study of important conformational changes in situ with the high resolution offered by contact mode AFM and a strongly-immobilized sample. The Engel group have studied changes in E. coli porin OmpF as a function of electric potential applied across the membrane, pH and buffer changes. Images at low applied potential show an open channel that closes at higher potential. Images of the open structure fail to resolve a soluble domain that presumably floats about in solution, invisible to the scanning probe. At higher potential, this domain is presumably pulled back into the channel to close it. The process can also be induced by a pH drop or a change of the imaging buffer. It is illustrated in Plate II which shows the extracellular surface imaged at pH 7 (a), pH 6 (b), pH 2.5 (c), and pH 4 (d). To produce the montage at the bottom, the different conformations have been averaged and merged (from pH 7, left to pH 2.5, right). Scale bars are 5 nm (Muller and Engel 1999). This work is probably the first study of channel-protein mechanism by direct imaging.
Plate Ib illustrates DFM imaging of isolated molecules at high resolution. It shows images of DNA microcircles imaged using magnetic DFM (Han, Dlakic et al. 1997; Han, Lindsay et al. 1997). These molecules are made by ligating intrinsically bent DNA oligomers to produce a closed circle of just 168 base-pairs (about 18nm diameter). The lower region shows a scan over many molecules imaged in a buffer containing MgCl2. The average width of the DNA in the images is a little over 3nm implying about 1nm of broadening. This is just adequate for resolution of the major groove as illustrated in the magnified gallery of images at the top (arrowheads point to major groove). The ‘circles’ change dramatically when imaged in the presence of Zn, becoming straight DNA connected by four kinks (gallery of images below top).
High resolution magnetic DFM imaging of protein molecules is shown in Plates I c and d. Ic shows an isolated microtubule imaged in microtubule buffer on ATES treated mica. The image was stable over a period of hours. The strings of protofilaments are clearly visible (13 in all corresponding to flattened sheet formed from a whole microtubule) and the tubilin a and tubulin b subunits (spaced by 4nm) are also clearly visible. When katatin, an enzyme that digests microtubles, is added, the image immediately disintegrates (Plate Id). These data were obtained by Judy Zhu of Molecular Imaging and Ron Vale of UCSF (unpublished work).
We end this section with a series of acoustic-DFM images of a process, the polymerization of cRNA from a double stranded DNA template by RNA polymerase (RNAP) (Kasas, Thomson et al. 1997). This work relies on pinning the DNA loosely to the mica substrate using a buffer containing zinc. The zinc was then washed away and the reagents needed to bind the RNAP and initiate transcription added. This resulted in dissociation of Zn from the mica surface with consequent loosening of the DNA, giving rise to a time window in which the DNA was bound loosly enough to permit sliding of the polymerase yet still bound strongly enough to enable SPM imaging. Stalled complexes were generated by omission of one the NTP’s (NTP=ATP, CTP, GTP or UTP) so that the RNAP stopped at the appropriate codon. The complex was imaged repeatedly to ensure that the RNA was stationary. The missing triphosphate was added to initiate transcription and subsequent motion of the RNAP was observed. Figure 19 shows a movie of the process. The first two images show the stalled RNAP The next six images show progress of the RNAP along the DNA. The RNA transcript is not observed, presumably because it is suspended in solution. The last two images show the discarded RNAP and DNA template, frozen by the addition of Zn.
