The physics of protein self-assembly

Understanding protein self-assembly is important for many biological and industrial processes. Proteins can self-assemble into crystals, filaments, gels, and other amorphous aggregates. The final forms include virus capsids and condensed phases associated with diseases such as amyloid fibrils. Although seemingly different, these assemblies all originate from fundamental protein interactions and are driven by similar thermodynamic and kinetic factors. Here we review recent advances in understanding protein self-assembly through a soft condensed matter perspective with an emphasis on three specific systems: globular proteins, viruses, and amyloid fibrils. We conclude with a discussion of unanswered questions in the field.     

Molecular self-assembly of solid-supported protein crystals

Highly ordered protein arrays have been proposed as a means for templating the organization of nanomaterials. Toward this end, we investigate the ability of the protein streptavidin to self-assemble into various configurations on solid-supported phospholipids. We identify two genetic variants of streptavidin (comprising amino acids 14-136 and 13-139) and examine their molecular organization at the liquid-solid interface. Our results demonstrate that the structural differences between these two protein variants affect both crystalline lattice and domain morphology. In general, these results for the liquid-solid interface are similar and consistent with those at the air-water interface with a few notable differences. Analogous to crystallization at the air-water interface, both forms of streptavidin yield H-like domains with lattice parameters that have C222 symmetry at pH 7. At pH 4, the native, truncated form of streptavidin yields needle-like domains consisting of molecules arranged in P1 symmetry. Unlike crystalline domains grown at the air-water interface, however, the lattice parameters of this P1 crystal are unique and have not yet been reported. The presence of a solid substrate does not appear to dramatically alter streptavidin's two-dimensional crystallization behavior, suggesting that local intermolecular interactions between proteins are more significant than interactions between the interface and protein. Our results also demonstrate that screening the electrostatic repulsion between protein molecules by modulating ionic strength will increase growth rate while decreasing crystalline domain size and macroscopic defects. Finally, we show that these domains are indeed functional by attaching biotinylated gold nanoparticles to the crystals. The ability to modulate molecular configuration, crystalline defects, and domain size on a functional array supports the potential application of this system toward materials assembly.     

Chemically controlled self-assembly of protein nanorings

The exploitation of biological macromolecules, such as nucleic acids, for the fabrication of advanced materials is a promising area of research. Although a greater variety of structural and functional uses can be envisioned for protein-based materials, systematic approaches for their construction have yet to emerge. Consistent with theoretical models of polymer macrocyclization, we have demonstrated that, in the presence of dimeric methotrexate (bisMTX), wild-type Escherichia coli dihydrofolate reductase (DHFR) molecules tethered together by a flexible peptide linker (ecDHFR(2)) are capable of spontaneously forming highly stable cyclic structures with diameters ranging from 8 to 20 nm. The nanoring size is dependent on the length and composition of the peptide linker, on the affinity and conformational state of the dimerizer, and on induced protein-protein interactions. Delineation of these and other rules for the control of protein oligomer assembly by chemical induction provides an avenue to the future design of protein-based materials and nanostructures.     

Modeling the early stages of self-assembly in nanophase materials. II. Role of symmetry and dimensionality

We study the early stages of self-assembly of elementary building blocks of nanophase materials, considering explicitly their structure and the symmetry and the dimensionality of the reaction space. Previous work [Kozak et al., J. Chem. Phys. 134, 154701 (2007)] focused on characterizing self-assembly on small square-planar templates. Here we consider larger lattices of square-planar symmetry having N = 255 sites, and both hexagonal and triangular lattices of N = 256 sites. Furthermore, to assess the consequences of a depletion zone above a basal layer (λ = 1), we study self-assembly on an augmented diffusion space defined by λ = 2 and λ = 5 stacked layers having the same characteristics as the basal plane. The effective decrease in the efficiency of self-assembly of individual nanophase units when the diffusion space is expanded, by increasing the template size and/or by enlarging the depletion zone, is then quantified. The results obtained reinforce our earlier conclusion that the most significant factor influencing the kinetics of formation of a final self-assembled unit is the number of reaction pathways from one or more precursor states. We draw attention to the relevance of these results to zeolite synthesis and reactions within pillared clays.     

Coarse-graining the self-assembly of beta-helical protein building blocks

Nanotubular structures constructed using self-assembled beta-helical protein building blocks one atop the other have been coarsened to develop a mesoscopic potential that reproduces the intermolecular interaction energies provided by atomistic force-fields. The resulting potential consists of an analytical expression that depends exclusively on the distance and the relative orientation between the two interacting entities. In spite of its complexity, this coarse-grained potential reproduces satisfactorily the energetic properties of two interacting building blocks. The coarse-grained potential has been used to predict that the interaction between building blocks formed by residues 131-165 of E. coli galactoside acteyltransferase becomes repulsive when the size of the nanotube is larger than a threshold, that is, about 45 self-assembled building blocks.     

DNA-templated self-assembly of protein and nanoparticle linear arrays

Self-assembling DNA tiling lattices represent a versatile system for nanoscale construction. Self-assembled DNA arrays provide an excellent template for spatially positioning other molecules with increased relative precision and programmability. Here we report an experiment using a linear array of DNA triple crossover tiles to controllably template the self-assembly of single-layer or double-layer linear arrays of streptavidin molecules and streptavidin-conjugated nanogold particles through biotin-streptavidin interaction. The organization of streptavidin and its conjugated gold nanoparticles into periodic arrays was visualized by atomic force microscopy and scanning electron microscopy.     

Transition metal vinylidene complexes as supramolecular building blocks: nucleobase-mediated self-assembly of crystals with hexagonal symmetry

Reaction of the ruthenium half sandwich compound RuCl(eta(5)-C(5)H(5))(PPh(3))(2) with the uracil (Ur) substituted alkyne HC[triple bond, length as m-dash]CUr in the presence of halide scavengers NH(4)X (X = PF(6), BF(4), OTf) results in the formation of the vinylidene complexes [Ru([double bond, length as m-dash]C[double bond, length as m-dash]CHUr)(eta(5)-C(5)H(5))(PPh(3))(2)][X] which crystallize in the hexagonal space group P6(3)/m. The hexagonal symmetry inherent to the system is due to the formation of a hydrogen bonded array mediated by the two sets of donor-acceptor units on the uracil, resulting in the formation of a cyclic "rosette" containing six ruthenium cations. In solution the (1)H and (31)P{(1)H} NMR spectra of the vinylidene complexes are both concentration and temperature dependent, in accord with the presence of monomer-dimer equilibria in which the rate of rotation of the vinylidene group is fast on the NMR timescale in the monomeric species, but slow in the dimers. The isoelectronic molybdenum-containing vinylidene complex [Mo(eta(7)-C(7)H(7))(dppe)([double bond, length as m-dash]C[double bond, length as m-dash]CHUr)][BF(4)] (dppe = 1,2-bis(diphenylphosphino)ethane) has also been prepared, but forms symmetric dimers in the solid state.     

A new paradigm for protein design and biological self-assembly

Very few molecules with biological origins contain the element fluorine. Nature's inability to incorporate fluorine into biomolecules is related to the low concentration of free fluoride in sea and surface water. However, judicious introduction of fluorine into proteins, nucleic acids, lipids and carbohydrates has allowed mechanistic scrutiny of enzyme catalysis, control of protein oligomerization in membranes, clustered display of ligands on surfaces of living cells, and in increasing the protease stability of protein and peptide therapeutics.     

Quantum dot self-assembly for protein detection with sub-picomolar sensitivity

A novel approach to sensitive and rapid antigen detection is described. In the presence of a specific antigen, quantum dot-antibody conjugates rapidly self-assemble into agglomerates that are typically more than 1 order of magnitude larger than their individual components. The size distribution of the agglomerated colloids depends on, among other things, the relative concentration of quantum dot conjugates and antigen molecules. Quantum dot agglomerates mediated by antigen recognition were characterized by measuring their light scattering and fluorescence characteristics in an unmodified flow cytometer. Protein antigens angiopoietin-2 and mouse IgG were detected to sub-picomolar concentrations using this method. This simple technique enables the potential simultaneous detection of multiple antigenic biomarkers directly from physiological media and could be used for early detection and frequent screening of cancers and other diseases.     

Physical mechanisms and biological significance of supramolecular protein self-assembly

In living cells, chemical reactions of metabolism, information processing, growth and development are organized in a complex network of interactions. At least in part, the organization of this network is accomplished as a result of physical assembly by supramolecular scaffolds. Indeed, most proteins function in cells within the context of multimeric or supramolecular assemblies. With the increasing availability of atomic structures and molecular thermodynamics, it is possible to recast the problem of non-covalent molecular self-assembly from a unified perspective of structural thermodynamics and kinetics. Here, we present a generalized theory of self-assembly based on Wegner's kinetic model and use it to delineate three physical mechanisms of self-assembly: as limited by association of assembly units (nucleation), by association of monomers (isodesmic), and by conformational reorganization of monomers that is coupled to assembly (conformational). Thus, we discuss actin, tubulin, clathrin, and the capsid of icosahedral cowpea chlorotic mottle virus with respect to assembly of architectural scaffolds that perform largely mechanical functions, and pyruvate dehydrogenase, and RING domain proteins PML, arenaviral Z, and BRCA1:BARD1 with regard to assembly of supramolecular enzymes with metabolic and chemically directive functions. In addition to the biological functions made possible by supramolecular self-assembly, such as mesoscale mechanics of architectural scaffolds and metabolic coupling of supramolecular enzymes, we show that the physical mechanisms of self-assembly and their structural bases are biologically significant as well, having regulatory roles in both formation and function of the assembled structures in health and disease.     

Application of self-assembly techniques in the design of biocompatible protein microarray surfaces

This review focuses on the application of novel technologies for generating biocompatible surfaces for high-throughput screening (HTS) of proteins. Various methods of coupling and spotting proteins on self-assembled monolayer (SAM) surfaces will be described along with the protein chip challenges pertaining to spot homogeneity, morphology, biocompatibility and reproducibility.     

7-Azaindolyl- and 2,2'-dipyridylamino-functionalized molecular stars with sixfold symmetry: self-assembly, luminescence, and coordination compounds

Two novel star molecules functionalized with 7-azaindolyl and 2,2'-dipyridylamino groups have been synthesized. Both molecules possess a sixfold rotation symmetry. Molecule L1 is based on the hexaphenylbenzene core with the formula of hexa[p-(7-azaindolyl)phenyl]benzene, while molecule L2 is based on the hexakis(biphenyl)benzene core with the formula of hexa[p-(2,2'-dipyridylamino)biphenyl]benzene. Both compounds have been characterized by single-crystal X-ray diffraction analyses. Molecule L1 forms extended two-dimensional layered structure, while L2 forms interpenetrating columnar stacks in the solid state, as revealed by X-ray diffraction analyses. Nanowire structures based on columnar stacks through self-assembly of L2 on a graphite surface were revealed by an STM study. Molecules L1 and L2 are capable of binding to metal ions, resulting in unusual structural motifs. Two Ag(I) complexes with the formulae of [(AgNO(3))(2)(L1)] (1) and [(AgNO(3))(3)(L1)] (2) were isolated from the reactions of AgNO(3) with L1. Compound 1 displays extended intermolecular pi-pi stacking interactions that are responsible for its extended two-dimensional structure in the crystal lattice. Complex 2 has a "bowl" shape and forms polar stacks in the crystal lattice. A Cu(II) complex with the formula of [{Cu(NO(3))(2)}(6)(L 2)] (3) was isolated from the reaction of Cu(NO(3))(2) with compound L2. The six Cu(II) ions in 3 are chelated by the 2,2'-dipyridylamino groups of the star ligand L2. Intermolecular Cu-O (nitrate) bonds lead to the formation of an extended two-dimensional coordination network of 3. Both L1 and L2 are blue luminescent. Their interactions with Ag(I) or Cu(II) cause drastic quenching of emission. In addition, the luminescence of L1 and L2 is sensitive to the presence of protons, which cause a reduction of emission intensity and a red shift of the emission energy.     

Programmable self-assembly of antibody-oligonucleotide conjugates as small molecule and protein carriers

Dihydrofolate reductase single-chain variable fragment (scFv) fusion proteins can be used for the targeted cellular delivery of oligonucleotides, conjugated small molecules, and proteins via labeling of oligonucleotides by bis-methotrexate.     

Hierarchical and helical self-assembly of ADP-ribosyl cyclase into large-scale protein microtubes

Proteins are macromolecules with characteristic structures and biological functions. It is extremely challenging to obtain protein microtube structures through self-assembly as proteins are very complex and flexible. Here we present a strategy showing how a specific protein, ADP-ribosyl cyclase, helically self-assembles from monomers into hexagonal nanochains and further to highly ordered crystalline microtubes. The structures of protein nanochains and consequently self-assembled superlattice were determined by X-ray crystallography at 4.5 A resolution and imaged by scanning electron microscopy. The protein initially forms into dimers that have a fixed size of 5.6 nm, and then, helically self-assembles into 35.6 nm long hexagonal nanochains. One such nanochain consists of six dimers (12 monomers) that stack in order by a pseudo P6(1) screw axis. Seven nanochains produce a series of large-scale assemblies, nanorods, forming the building blocks for microrods. A proposed aging process of microrods results in the formation of hollow microstructures. Synthesis and characterization of large scale self-assembled protein microtubes may pave a new pathway, capable of not only understanding the self-assembly dynamics of biological materials, but also directing design and fabrication of multifunctional nanobuilding blocks with particular applications in biomedical engineering.     

From surface self-assembly to crystallization: prediction of protein crystallization conditions

A new criterion based on surface and volume diffusion kinetics was established to predict protein crystallization. Similar to the layer-by-layer crystal growth process of protein, the kinetics of the two-dimensional self-assembly of protein at the aqueous solution surface provides a convenient and reliable way to estimate the surface integration and the volume transport during protein crystallization. Both the surface and diffusion kinetics were estimated based on the protein self-assembly at the air/solution interface, which can be obtained by measuring the surface tension. A crystallization coefficient is found to provide an effective and reliable criterion to predict protein crystallization conditions. This criterion has been applied to lysozyme, concanavalin A and BSA crystallization, and it turns out to be very successful and more reliable than the second virial coefficient criterion.     

Metal-mediated self-assembly of protein superstructures: influence of secondary interactions on protein oligomerization and aggregation

We have previously demonstrated that non-self-associating protein building blocks can oligomerize to form discrete supramolecular assemblies under the control of metal coordination. We show here that secondary interactions (salt bridges and hydrogen bonds) can be critical in guiding the metal-induced self-assembly of proteins. Crystallographic and hydrodynamic measurements on appropriately engineered cytochrome cb562 variants pinpoint the importance of a single salt-bridging arginine side chain in determining whether the protein monomers form a discrete Zn-induced tetrameric complex or heterogeneous aggregates. The combined ability to direct PPIs through metal coordination and secondary interactions should provide the specificity required for the construction of complex protein superstructures and the selective control of cellular processes that involve protein-protein association reactions.     

Dendronized protein polymers: synthesis and self-assembly of monodisperse cylindrical macromolecules

Monodisperse dendronized protein polymers (DPPs), cylindrical dendrimers containing protein core, can be efficiently produced through a combined modular biosynthetic strategy. These DPP materials possess predictable size, shape, and solubility. In organic solutions, the DPPs self-assemble to form highly ordered liquid crystalline structures with nanoscale order controlled by their exact molecular dimensions.     

Magnetic field induced synthesis and self-assembly of super paramagnetic particles in a protein matrix

Aqueous solution of a globular protein named bovine serum albumin was homogeneously mixed with ferrous and ferric ions and allowed to gel at ambient conditions. Gels were then oxidized using sodium hydroxide, in the presence of magnetic field of magnitude 0.13 T. The effect of magnetic field on the above biomimetic synthesis was a reduction in particle size and a directional assembly of synthesized super paramagnetic particles into a regular pattern in the protein film. The microstructural revelation was complimentary to Mossbauer results and magnetic measurement studies, i.e., an interesting variation in the magnetic behaviour of self-assembled super paramagnetic particles as a function of dc magnetic field induced ordering.