Imaging by atomic force microscopy (AFM) offers high-resolution descriptions of many biological systems; however, regardless of resolution, conclusions drawn from AFM images are only as robust as the analysis leading to those conclusions. Vital to the analysis of biomolecules in AFM imagery is the initial detection of individual particles from large-scale images. Threshold and watershed algorithms are conventional for automatic particle detection but demand manual image preprocessing and produce particle boundaries which deform as a function of user-defined parameters, producing imprecise results subject to bias. Here, we introduce the Hessian blob to address these shortcomings. Combining a scale-space framework with measures of local image curvature, the Hessian blob formally defines particle centers and their boundaries, both to subpixel precision. Resulting particle boundaries are independent of user defined parameters, with no image preprocessing required. We demonstrate through direct comparison that the Hessian blob algorithm more accurately detects biomolecules than conventional AFM particle detection techniques. Furthermore, the algorithm proves largely insensitive to common imaging artifacts and noise, delivering a stable framework for particle analysis in AFM.

We visualize ATP-driven domain dynamics of individual SecA molecules in a near-native setting using atomic force microscopy.

SUMMARY Skeletal development and invasion by tumor cells depends on proteolysis of collagen by the pericellular metalloproteinase MT1-MMP. Its hemopexin-like (HPX) domain binds to collagen substrates to facilitate their digestion. Spin labeling and paramagnetic NMR detection have revealed how the HPX domain docks to collagen I-derived triple-helix. Mutations impairing triple-helical peptidase activity corroborate the interface. Saturation transfer difference NMR suggests rotational averaging around the longitudinal axis of the triple-helical peptide. Part of the interface emerges as unique and potentially targetable for selective inhibition. The triple-helix crosses the junction of blades I and II at a 45° angle to the symmetry axis of the HPX domain, placing the scissile Gly~Ile bond near the HPX domain and shifted ~25 Å from MMP-1 complexes. This raises the question of the MT1-MMP catalytic domain folding over the triple-helix during catalysis, a possibility accommodated by the flexibility between domains suggested by AFM images.

Interactions between short protein segments and phospholipid bilayers dictate fundamental aspects of cellular activity and have important applications in biotechnology. Yet, the lack of a suitable methodology for directly probing these interactions has hindered the mechanistic understanding. We developed a precision atomic force microscopy-based single-molecule force spectroscopy assay and probed partitioning into lipid bilayers by measuring the mechanical force experienced by a peptide. Protein segments were constructed from the peripheral membrane protein SecA, a key ATPase in bacterial secretion. We focused on the first 10 amino-terminal residues of SecA (SecA2-11) that are lipophilic. In addition to the core SecA2-11 sequence, constructs with nearly identical chemical composition but with differing geometry were used: two copies of SecA2-11 linked in series and two copies SecA2-11 linked in parallel. Lipid bilayer partitioning interactions of peptides with differing structures were distinguished. To model the energetic landscape, a theory of diffusive barrier crossing was extended to incorporate a superposition of potential barriers with variable weights. Analysis revealed two dissociation pathways for the core SecA2-11 sequence with well-separated intrinsic dissociation rates. Molecular dynamics simulations showed that the three peptides had significant conformational differences in solution that correlated well with the measured variations in the propensity to partition into the bilayer. The methodology is generalizable and can be applied to other peptide and lipid species.