Hydrodynamic Analysis and EM Applied to Large Multisubunit Complexes
The text box above showed the application of the Siegel–Monte analysis to SMC protein from B. subtilis, which had only one type subunit and was found to be a dimer. Similar hydrodynamic analysis can be used to analyze multisubunit protein complexes. There are many examples in the literature; I will show here an elegant application to DASH/Dam1.
The protein complex called DASH or Dam1 is involved in attaching chromosomal kinetochores to microtubules in yeast. DASH/Dam1 is a complex of ten proteins that assemble into a particle containing one copy of each subunit. These complexes further assemble into rings that can form a sliding washer on the microtubule (24, 25). The basic ten-subunit complex has been purified from yeast and has also been expressed in Escherichia coli and purified (this required the heroic effort of expressing all ten proteins simultaneously (24)). Figure 5 shows the hydrodynamic characterization of the purified protein complex and illustrates several important features.
| • | For both the gel filtration (size exclusion chromatography, Fig. 5 a) and gradient sedimentation, Fig. 5 b, two calibration curves of known protein standards are shown, green and black. These are independent calibration runs. In this study, the gel filtration column was calibrated in terms of the reciprocal diffusion coefficient, 1/D, which is proportional to R s (Eq. 6.2 ). |
| • | The fractions were analyzed by Western blot for the location of two proteins of the complex, Spc34p and Hsk3p. Methods notes that 1 ml fractions from gel filtration were precipitated with perchloric acid and rinsed with acetone prior to SDS-PAGE, an essential amplification for the dilute samples of yeast cytoplasmic extract. These two proteins eluted together in both gel filtration and sedimentation, consistent with their being part of the same complex. |
| • | The profiles of the two proteins were identical when analyzed in their native form in yeast cytoplasmic extract and as the purified complex expressed in E. coli. This is strong evidence that the expression protein is correctly folded and assembled. |
| • | There is minimal trailing of any subunits. This means that there is no significant dissociation during the tens of minutes for the gel filtration, or the 12-h centrifugation. The complex is held together by very high affinity bonds, making it essentially irreversible. |
| • | Combining the R s = 7.6 nm (from 1/D = 0.35 × 10−7, and S = 7.4, Eq. 7.1b gives a mass of M = 236 kDa, close to the 204 kDa obtained from adding the mass of the ten subunits. S max is 12.6 giving Smax /S = 1.7, suggesting a moderately elongated protein. |
Fig. 5 Hydrodynamic analysis of the DASH/Dam1 complex. Gel filtration is shown in a and sucrose gradient sedimentation in b. Independent calibration curves using standard proteins are shown in black and green. Dark and light blue show Spc34p in yeast cytoplasmic extract and in the purified recombinant protein. Red and purple show Hsk3p. The proteins were identified and quantitated by Western blot of the fractions, shown in c. The four protein bands eluted together at 1/D = 0.35 × 107, corresponding to R s = 7.6 nm, and at 7.4 S. Reproduced from Miranda et al. (24) with permission of the authors.
Figure 6 shows EM images of DASH/DAM1 by rotary shadowing (a) and negative stain (b). Rotary shadowing showed irregular particles about 13 nm in diameter (24). The particles had variable and frequently elongated shapes, but internal structure could not be resolved. A later study used state of the art negative staining and sophisticated computer programs to sort images into classes and average them (26). These images resolved a complex internal structure. The negative stain showed most of the particles (80%) to be dimers, with 15% monomers and 5% trimers. This contradicts the hydrodynamic analysis of Miranda et al. (24) showing that the particles were monomers. The reason for this discrepancy is not known.
Fig. 6 EM of DASH/Dam1. a Rotary shadowing shows particles roughly 13 nm in size, with irregular shape. b State-of-the-art negative stain coupled with single particle averaging shows a complex internal structure of the elongated particles. The scale bar indicates 100 nm for the unprocessed images. The averaged images on the right show a monomer, dimer, and trimer. These panels are 14 nm wide. The dimer was the predominant species. Left panel (rotary shadowing) reprinted with permission of Miranda et al. (24). Right panels (negative stain) reprinted with permission of Wang et al. (26).
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