AFM volumetric methods for the characterization of proteins and nucleic acids

Abstract

The atomic force microscope overestimates lateral dimensions and underestimates heights of nanometer size objects such as proteins and nucleic acids. This has made researchers cautious of AFM measurements, even though there is no other technique capable of measuring topography with sub-nanometer precision. Nevertheless, several approaches for determining the stoichiometry of protein and protein-DNA complexes have been developed which show that, although the absolute values may be incorrect, the AFM volume is essentially proportional to the mass. This has allowed the determination of the mass of protein complexes with the help of a calibration curve. Here we review the main techniques for AFM volume measurements and detail a methodology that significantly reduces the associated errors. This method uses a fragment of DNA as a fiducial marker by which the volume of a protein is normalized. The use of fiducial markers co-adsorbed together with the protein of interest minimizes the contribution of tip-induced artifacts as they affect both the object of interest and the marker. Finally, we apply this method to the measurement of the length of single-stranded DNA. A linear relationship between length and volume was obtained, opening the door to studies of ssDNA intermediates formed during complex DNA transactions such as replication, recombination and repair.

Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

“…Statistical analysis of 231 proteins resulted in <h> = 2.5 ± 0.2 nm as the height and <d> = 14.4 ± 2.9 nm as the diameter of the globular structures. Next, we used these results to estimate the volume of the proteins by using a "spherical cap" model, which has been successfully used in previous AFM studies of proteins and protein-DNA complexes [76][77][78][79]. Again, the statistical histogram of those computed protein volumes, as shown in Figure 2E has a narrow distribution, suggesting that the stoichiometry of the protein is uniform.…”

Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates

“…1A and Methods). First, we evaluated the oligomeric state of SA2 using a previously established method that estimates the molecular mass of a protein based on the calibration curve correlating AFM volume and molecular weight of proteins (42)(43)(44). Based on this method, SA2 molecules (141 kDa) display AFM volumes (146 nm 3 ) consistent with being predominantly monomers ( Fig.…”

Silk Reconstitution Disrupts Fibroin Self-Assembly

“…37,53 We note that the lateral dimensions of the imaged protein structures are of the same order as the size of the AFM probes used, and consequently, the apparent widths are most likely overestimated and heights underestimated relative to their true values. 52,56 Therefore, precise determinations of dimensions and volumes are very challenging. Nevertheless, relative comparisons to other AFM data, as well as coarse general assessments are feasible and useful.…”