ABSTRACT
The ferritin cage iron-storage protein assembly has been widely used as a template for preparing nanomaterials. This assembly has a unique pH-induced disassembly/reassembly mechanism that provides a means for encapsulating molecules such as nanoparticles and small enzymes for catalytic and biomaterial applications. Although several researchers have investigated the disassembly process of ferritin, the dynamics involved in the initiation of the process and its intermediate states have not been elucidated due to a lack of suitable methodology to track the process in real-time. We describe the use of high-speed atomic force microscopy (HS-AFM) to image the dynamic event in real-time with single-molecule level resolution. The HS-AFM movies produced in the present work enable direct visualization of the movements of single ferritin cages in solution and formation of a hole prior to disassembly into subunit fragments. Additional support for these observations was confirmed at the atomic level by the results of all-atom molecular dynamics (MD) simulations, which revealed that the initiation process includes the opening of 3-fold symmetric channels. Our findings provide an essential contribution to a fundamental understanding of the dynamics of protein assembly and disassembly, as well as efforts to redesign the apo-ferritin cage for extended applications.
Subject(s)
Apoferritins/chemistry , Animals , Horses , Hydrogen-Ion Concentration , Microscopy, Atomic Force , Molecular Dynamics Simulation , Potassium Chloride/chemistry , Protein Multimerization , Protein Subunits/chemistry , SolutionsABSTRACT
Bacteriophage T4 and other bacteriophages have a protein component known as a molecular needle which is used for the transmembrane reaction in the infection process. In this paper, the transmembrane reaction mechanisms of artificial protein needles (PNs) constructed by protein engineering of the component protein of bacteriophage T4 are elucidated by observation of single-molecules by high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations. The HS-AFM images indicate that the tip of the needle structure stabilizes the interaction of the needle with the membrane surface and is involved in controlling the contact angle and angular velocity with respect to the membrane. The MD simulations indicate that the dynamic behavior of PN is governed by hydrogen bonds between the membrane phosphate fragments and the tip. Moreover, quartz crystal microbalance (QCM) and electrophysiological experiments indicate that the tip structure of PN affects its kinetic behavior and membrane potential. These results demonstrate that protein assemblies derived from natural biosupramolecules can be used to create nanomaterials with rationally-designed functionality.
Subject(s)
Biomimetic Materials/metabolism , Cell Membrane/metabolism , Viral Proteins/metabolism , Bacteriophage T4/chemistry , Biomimetic Materials/chemistry , Electrophysiological Phenomena , Microscopy, Atomic Force , Molecular Dynamics Simulation , Nanostructures/chemistry , Protein Binding , Protein Multimerization , Quartz Crystal Microbalance Techniques , Viral Proteins/chemistryABSTRACT
Protein crystals are formed via ordered arrangements of proteins, which assemble to form supramolecular structures. Here, we show a method for the assembly of supramolecular protein cages within a crystalline environment. The cages are stabilized by covalent cross-linking allowing their release via dissolution of the crystal. The high stability of the desiccated protein crystals allows cages to be constructed.
