title: Visualization of recombinant DNA and protein complexes using atomic force microscopy.
abstract: Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1). Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation, such as Ni(2+) or Mg(2+), which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase and a repair enzyme, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes) and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.
authors.affiliation: College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, USA. murphyp@seattleu.edu
authors.forename: Patrick J M
authors.initials: PJ
authors.lastname: Murphy
authors.forename: Morgan
authors.initials: M
authors.lastname: Shannon
authors.forename: John
authors.initials: J
authors.lastname: Goertz
mesh_terms: D000538:Aluminum Silicates / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D000538:Aluminum Silicates; D011506:Proteins; D011994:Recombinant Proteins; D004247:DNA; C011934:mica