Controlled Assembly of Bifunctional Chimeric Protein Cages and Composition Analysis Using Noncovalent Mass Spectrometry

Abstract

Protein cage architectures, such as viral capsids, ferritins, and heat shock proteins (Hsp), have been extensively used as model systems to study the self-assembly of macromolecular complexes [1][2][3][4][5] and as nanoreactors for materials synthesis. [6][7][8][9][10][11][12][13] However, it is still challenging to manipulate their self-assembly in a controlled way and to analyze their assembled products precisely at the molecular level. 14,15 In this study, we have generated two different individual mutants of a protein cage with functional groups either inside or outside of the cage ( Figure 1A). We chemically modified different cages and reconstructed chimeric cages with a controlled ratio of two subunit types ( Figure 1B). Using mass spectrometry, we were able to determine the compositions of the ensemble population and also of the individual chimeric cages within the population at the molecular level. A model based on a binomial distribution suggested chimeric cages are assembled by random incorporation of the two individual subunits.Mass spectrometry has been used to monitor multicomponent systems, because it can simultaneously resolve individual molecular masses present in a mixture. [16][17][18] Using a combination of electrospray ionization (ESI) 19 and a time-of-flight (TOF) mass analyzer, it is possible to determine the masses of individual protein components of a noncovalently associated macromolecular complex as well as the mass of the intact macromolecular complex without disturbing the structures. 16,[20][21][22][23] The Dps (DNA binding protein from starved cells) from the Gram-positive bacterium Listeria innocua (Li) is a member of the ferritin superfamily and prevents oxidative damage of DNA by accumulating iron atoms within its central cavity to produce an iron oxide core similar to that of ferritins. 24,25 The LiDps consists of 12 identical 18 kDa subunits that self-assemble into a hollow protein cage having tetrahedral 23 symmetry ( Figure 1A). 24 The LiDps has an outer diameter of 9 nm and an inner cavity diameter of 5 nm with 0.8 nm pores at the 3-fold axis where molecules can pass through to the interior ( Figure 1A). 24 The LiDps has been used as a template for nanomaterials synthesis of metal oxides of iron 26 and cobalt 27 as well as cadmium sulfide 28 and platinum 29 with or without modifications. The small number of subunits, robustness at high temperature, 26,27 and intrinsic biomineralizing capability 25 of the LiDps protein cage make it an attractive modifiable nanoreactor for nanomaterials syntheses. In addition, the defined small cavity size 24 allows synthesis of extremely small nanostructured materials. 29 Two individual cysteine mutants, one exposed on the interior surface and the other on the exterior surface, were generated to adapt the LiDps for selective chemical modifications. The serine residue at position 138 located in the middle of helix E where it is directed toward the inside cavity was substituted with cysteine (S138C) 29 ( Figure 1A, blue). Alternatively, for th...

Top-down design of magnonic crystals from bottom-up magnetic nanoparticles through protein arrays

“…[9] Our NP arrays are similar to colloidal crystals, except that each NP is located in the central cavity of an apoferritin molecule, and they are formed by specific proteinprotein interactions mediated by cadmium ions [9,27] rather than isotropic interactions between surfactant-coated NPs. Other cage-shaped proteins and viruses can also form spherical NPs in their central cavities [31][32][33][34][35] while tube-like viruses can form nanorods. [36] There are therefore numerous possible alternatives to apoferritin as a basis for micro-machined periodic NP arrays.…”

Role of H-1 and H-2 Subunits of Soybean Seed Ferritin in Oxidative Deposition of Iron in Protein

“…The concentrations of all types of ferritin were determined according to the Lowry method with BSA as a standard sample. Recombinant heteropolymer rH-1H-2 was constructed by mixing dissociated H-1 and H-2 subunits in a 1:1 ratio and reassembled by slowly raising the pH to 7 according to a reported method (21,22).…”

From Metal Binding to Nanoparticle Formation: Monitoring Biomimetic Iron Oxide Synthesis within Protein Cages using Mass Spectrometry

“…Mass spectrometry: Mass analyses of the intact and metal-treated LiDps were carried out on samples in 10 mm TEAA, pH 6.8 under the same mass spectrometry condition as described previously. [17,20] Charge states and masses were obtained from the spectral data using a home-built program [21] …”