The synergistic mechanisms of apo-ferritin structural transitions and Au(iii) ion transportation: molecular dynamics simulations with the Markov state model.

Due to its unique structure, recent years have witnessed the use of apo-ferritin to accumulate various non-natural metal ions as a scaffold for nanomaterial synthesis. However, the transport mechanism of metal ions into the cavity of apo-ferritin is still unclear, limiting the rational design and controllable preparation of nanomaterials. Here, we conducted all-atom classical molecular dynamics (MD) simulations combined with Markov state models (MSMs) to explore the transportation behavior of Au(iii) ions. We exhibited the complete transportation paths of Au(iii) from solution into the apo-ferritin cage at the atomic level. We also revealed that the transportation of Au(iii) ions is accompanied by coupled protein structural changes. It is shown that the 3-fold axis channel serves as the only entrance with the longest residence time of Au(iii) ions. Besides, there are eight binding clusters and five 3-fold structural metastable states, which are important during Au(iii) transportation. The conformational changes of His118, Asp127, and Glu130, acting as doors, were observed to highly correlate with the Au(iii) ion's position. The MSM analysis and Potential Mean Force (PMF) calculation suggest a remarkable energy barrier near Glu130, making it the rate-limiting step of the whole process. The dominant transportation pathway is from cluster 3 in the 3-fold channel to the inner cavity to cluster 5 on the inner surface, and then to cluster 6. These findings provide inspiration and theoretical guidance for the further rational design and preparation of new nanomaterials using apo-ferritin.

Atomic-resolution protein structure determination by cryo-EM.

Single-particle electron cryo-microscopy (cryo-EM) is a powerful method for solving the three-dimensional structures of biological macromolecules. The technological development of transmission electron microscopes, detectors and automated procedures in combination with user-friendly image processing software and ever-increasing computational power have made cryo-EM a successful and expanding technology over the past decade1. At resolutions better than 4 Å, atomic model building starts to become possible, but the direct visualization of true atomic positions in protein structure determination requires much higher (better than 1.5 Å) resolution, which so far has not been attained by cryo-EM. The direct visualization of atom positions is essential for understanding the mechanisms of protein-catalysed chemical reactions, and for studying how drugs bind to and interfere with the function of proteins2. Here we report a 1.25 Å-resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope that provides, to our knowledge, unprecedented structural detail. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution3). We can visualize individual atoms in a protein, see density for hydrogen atoms and image single-atom chemical modifications. Beyond the nominal improvement in resolution, we also achieve a substantial improvement in the quality of the cryo-EM density map, which is highly relevant for using cryo-EM in structure-based drug design.

An optimized low-cost protocol for standardized production of iron-free apoferritin nanocages with high protein recovery and suitable conformation for nanotechnological applications.

Ferritin is a globular protein that consists of 24 subunits forming a hollow nanocage structure that naturally stores iron oxyhydroxides. Elimination of iron atoms to obtain the empty protein called apoferritin is the first step to use this organic shell as a nanoreactor for different nanotechnological applications. Different protocols have been reported for apoferritin formation, but some are time consuming, others are difficult to reproduce and protein recovery yields are seldom reported. Here we tested several protocols and performed a complete material characterization of the apoferritin products using size exclusion chromatography, UV-vis spectroscopy, inductively coupled plasma optical emission spectrometry and dynamic light scattering. Our best method removes more than 99% of the iron from loaded holoferritin, recovering 70-80% of the original protein as monomeric apoferritin nanocages. Our work shows that pH conditions of the reduction step and the presence and nature of chelating agents affect the efficiency of iron removal. Furthermore, process conditions also seem to have an influence on the monomer:aggregate proportion present in the product. We also demonstrate that iron contents markedly increase ferritin absorbance at 280nm. The influence of iron contents on absorbance at 280nm precludes using this simple spectrophotometric measure for protein determination in ferritin­iron complexes. Apoferritin produced following our protocol only requires readily-available, cheap and biocompatible reagents, which makes this process standardizable, scalable and applicable to be used for in vivo applications of ferritin derivatives as well as nanotechnological and biotechnological uses.

Electrochemical detection of individual single nucleotide polymorphisms using monobase-modified apoferritin-encapsulated nanoparticles.

An electrochemical approach for detection of individual single nucleotide polymorphisms (SNPs) based on nucleobase-conjugated apoferritin probe loaded with metal phosphate nanoparticles is reported. Coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA was induced by DNA polymerase I (Klenow fragment) to preserve Watson-Crick base-pairing rules. After sequential liquid hybridization of biotinylated DNA probes with mutant DNA and complementary DNA, the resulting duplex DNA helixes were captured to the surface of magnetic beads through a well known and specific biotin-streptavidin affinity binding. For signaling each of eight possible Single-nucleotide polymorphisms (SNPs), Pb, Cu, Cd and Zn phosphate-loaded apoferritin nanoparticle probes were linked to adenosine (A), cytidine (C), guanosine (G), and thymidine (T) mononucleotides, respectively. Monobase-conjugated apoferritin probes were coupled to the mutant sites of the formed duplex DNA in the presence of DNA polymerase. Electrochemical stripping analyses of the metals loaded in apoferritin nanoparticle probes provide a means for detection and quantification of mutant DNA. Each mutation captures different nucleotide-conjugated apoferritin probe and provide a distinct four-potential voltammogram, whose peak potentials reflect the identity of the mismatch. The method is sensitive enough to accurately determine AG mutation, as the most thermodynamically stable mismatch to detect, in the range of 50-600 pM. The proposed protocol provides a simple, fast, cost-effective, accurate and sensitive method for detection of SNPs.

A comparison of neighbor search algorithms for large rigid molecules.

Fast determination of neighboring atoms is an essential step in molecular dynamics simulations or Monte Carlo computations, and there exists a variety of algorithms to efficiently compute neighbor lists. However, most of these algorithms are general, and not specifically designed for a given type of application. As a result, although their average performance is satisfactory, they might be inappropriate in some specific application domains. In this article, we study the case of detecting neighbors between large rigid molecules, which has applications in, e.g., rigid body molecular docking, Monte Carlo simulations of molecular self-assembly or diffusion, and rigid body molecular dynamics simulations. More precisely, we compare the traditional grid-based algorithm to a series of hierarchy-based algorithms that use bounding volumes to rapidly eliminate large groups of irrelevant pairs of atoms during the neighbor search. We compare the performance of these algorithms based on several parameters: the size of the molecules, the average distance between them, the cutoff distance, as well as the type of bounding volume used in the culling hierarchy (AABB, OBB, wrapped, or layered spheres). We demonstrate that for relatively large systems (> 100,000 atoms) the algorithm based on the hierarchy of wrapped spheres shows the best results and the traditional grid-based algorithm gives the worst timings. For small systems, however, the grid-based algorithm and the one based on the wrapped sphere hierarchy are beneficial.

Random number generation by a two-dimensional crystal of protein molecules.

We discuss 2D and binary self-assemblies of protein molecules using apo-ferritin and holo-ferritin, which have identical outer-shell structures but different inner structures. The assemblies do not show any phase separation but form 2D monomolecular-layer crystals. Statistical analyses showed a random molecular distribution in the crystal where the molar ratio was conserved as it was in the solution. This molecular pattern is readily prepared, but it is neither reproducible nor predictable and hence can be used as a nanometer-scale cryptographic device or an identification tag.