I. Using high-speed atomic force microscopy (HS-AFM) to understand biomolecules (Cytoskeletal proteins, pore forming proteins, membrane proteins, etc) self-assembly, dynamics and working mechanisms in native-like environment.

i) Septins are a family of cytoskeletal GTP-binding proteins, conserved from fungi through humans, which have key roles in cell division, cell polarity, and membrane remodeling. While recent structural studies have provided snapshots of the various septin assembly levels, mechanistic questions how septin filaments elongate and further assemble into higher hierarchical structures, and crucial structural determinants and environmental factors regulating septin assembly remain unanswered. We use HS-AFM and determine that assembly of septin filaments and filament pairing are diffusing driven processes, while septin assembly into higher order 3D structures is more complex and involves septin self-templating. Environmental pH and KCl concentration influences septin filament assembly, packing, pairing and multilayer formation. This first direct dynamic imaging of septins with single molecule resolution indicates that septin itself and environmental factors allow cells to fine-tune septin assembly in a task-specific way.

ii) Perforin-2 is an immune system toxin that provides front line defense against bacteria that cause serious disease in humans when they grow within or on human cells. This is achieved by perforin-2 targeting bacterial cell membranes by assembling into rings and forming pores. Using HS-AFM and other approaches, we characterize the function and transition of pre-pore and pore states. Surprisingly, in regions of the protein responsible for pore formation face away from the membrane to which the protein is bound in the pre-pore.

II. Using in-situ AFM to understand diblock copolymer/peptide self-assembly and crystallization mechanisms.

We succeeded in synthesizing a new class of 2D materials from three peptoids (Pep-2, Pep-3, and Pep-4) that have an amphiphilic structure akin to the lipids that form bilayer cell membranes. Using in-situ AFM to both dissect these membrane-like materials and image their subsequent behavior, we explore their ability to self-repair on a range of solid substrates.

III. Investigating intermolecular interactions by AFM and dynamic force spectroscopy.

Contractile injection systems, ubiquitous in bacteria, are complex macromolecular machines that are able to perforate the host cell envelope and deliver proteins and/or DNA. While the structures of contractile sheaths are now solved in both extended and contracted states, almost nothing is known about the physics of the system. Here we use HS-AFM imaging and force measurements to characterize R-type pyocin sheaths. The head-on pyocins can be segregated into two classes, one visibly capped the other one open, certainly representing the far and the tube-releasing ends of the empty sheath. To analyze the force and energy stored in the system for host cell envelope penetration, we use the HS-AFM tip to extend the length of the contracted sheath and determine its spring constant k(pyocin)~117 pN/nm, indicating that the force applied by a pyocin may reach ~6.7 nN and the energy stored in the extended sheath may amount to ~46,000 kBT.