Biochemistry and Protein Interactions of the CYREN Microprotein.

Over the past decade, advances in genomics have identified thousands of additional protein-coding small open reading frames (smORFs) missed by traditional gene finding approaches. These smORFs encode peptides and small proteins, commonly termed micropeptides or microproteins. Several of these newly discovered microproteins have biological functions and operate through interactions with proteins and protein complexes within the cell. CYREN1 is a characterized microprotein that regulates double-strand break repair in mammalian cells through interaction with Ku70/80 heterodimer. Ku70/80 binds to and stabilizes double-strand breaks and recruits the machinery needed for nonhomologous end join repair. In this study, we examined the biochemical properties of CYREN1 to better understand and explain its cellular protein interactions. Our findings support that CYREN1 is an intrinsically disordered microprotein and this disordered structure allows it to enriches several proteins, including a newly discovered interaction with SF3B1 via a distinct short linear motif (SLiMs) on CYREN1. Since many microproteins are predicted to be disordered, CYREN1 is an exemplar of how microproteins interact with other proteins and reveals an unknown scaffolding function of this microprotein that may link NHEJ and splicing.

A Structure-Based Assembly Screen of Protein Cage Libraries in Living Cells: Experimentally Repacking a Protein-Protein Interface To Recover Cage Formation in an Assembly-Frustrated Mutant.

Cage proteins, which assemble into often highly symmetric hollow nanoscale capsules, have great potential in applications as far reaching as drug delivery, hybrid nanomaterial engineering, and catalysis. In addition, they are promising model systems for understanding how cellular nanostructures are constructed through protein-protein interactions, and they are beginning to be used as scaffolds for synthetic biology approaches. Recently, there has been renewed interest in the engineering of protein cages, and in support of these strategies, we have recently described a fluorescence-based assay for protein cage assembly that is specific for certain oligomerization states and symmetry-related protein-protein interfaces. In this work, we expand this assay to living cells and a high-throughput assay for screening protein cage libraries using flow cytometry. As a proof of principle, we apply this technique to the screening of libraries of a double-alanine mutant of the mini-ferritin, DNA-binding protein from starved cells (Dps). This mutant, due to disruption of key protein-protein interactions, is unable to assemble into a cage. Randomization of residues surrounding the double mutation afforded a repacked interface and proteins with recovered cage formation, demonstrating the strength and utility of this approach.

Using Cooperatively Folded Peptides To Measure Interaction Energies and Conformational Propensities.

The rates and equilibria of the folding of biopolymers are determined by the conformational preferences of the subunits that make up the sequence of the biopolymer and by the interactions that are formed in the folded state in aqueous solution. Because of the centrality of these processes to life, quantifying conformational propensities and interaction strengths is vitally important to understanding biology. In this Account, we describe our use of peptide model systems that fold cooperatively yet are small enough to be chemically synthesized to measure such quantities. The necessary measurements are made by perturbing an interaction or conformation of interest by mutation and measuring the difference between the folding free energies of the wild type (in which the interaction or conformation is undisturbed) and the mutant model peptides (in which the interaction has been eliminated or the conformational propensities modified). With the proper controls and provided that the peptide model system in question folds via a two-state process, these folding free energy differences can be accurate measures of interaction strengths or conformational propensities. This method has the advantage of having high sensitivity and high dynamic range because the energies of interest are coupled to folding free energies, which can be measured with precisions on the order of a few tenths of a kilocalorie by well-established biophysical methods, like chaotrope or thermal denaturation studies monitored by fluorescence or circular dichroism. In addition, because the model peptides can be chemically synthesized, the full arsenal of natural and unnatural amino acids can be used to tune perturbations to be as drastic or subtle as desired. This feature is particularly noteworthy because it enables the use of analytical tools developed for physical organic chemistry, especially linear free energy relationships, to decompose interaction energies into their component parts to obtain a deeper understanding of the forces that drive interactions in biopolymers. We have used this approach, primarily with the WW domain derived from the human Pin1 protein as our model system, to assess hydrogen bond strengths (especially those formed by backbone amides), the dependence of hydrogen bond strengths on the environment in which they form, ß-turn propensities of both natural sequences and small molecule ß-turn mimics, and the energetics of carbohydrate-protein interactions. In each case, the combination of synthetic accessibility, the ease of measuring folding energies, and the robustness of the structure of the Pin1 WW domain to mutation enabled us to obtain incisive measurements of quantities that have been challenging to measure by other methods.

Designability of Aromatic Interaction Networks at E. coli Bacterioferritin B-Type Channels.

The bacterioferritin from E. coli (BFR), a maxi-ferritin made of 24 subunits, has been utilized as a model to study the fundamentals of protein folding and self-assembly. Through structural and computational analyses, two amino acid residues at the B-site interface of BFR were chosen to investigate the role they play in the self-assembly of nano-cage formation, and the possibility of building aromatic interaction networks at B-type protein-protein interfaces. Three mutants were designed, expressed, purified, and characterized using transmission electron microscopy, size exclusion chromatography, native gel electrophoresis, and temperature-dependent circular dichroism spectroscopy. All of the mutants fold into α-helical structures and possess lowered thermostability. The double mutant D132W/N34W was 12 °C less stable than the wild type, and was also the only mutant for which cage-like nanostructures could not be detected in the dried, surface-immobilized conditions of transmission electron microscopy. Two mutants-N34W and D132W/N34W-only formed dimers in solution, while mutant D132W favored the 24-mer even more robustly than the wild type, suggesting that we were successful in designing proteins with enhanced assembly properties. This investigation into the structure of this important class of proteins could help to understand the self-assembly of proteins in general.

Stabilization of a protein nanocage through the plugging of a protein-protein interfacial water pocket.

The unique structural properties of the ferritin protein cages have provided impetus to focus on the methodical study of these self-assembling nanosystems. Among these proteins, Escherichia coli bacterioferritin (EcBfr), although architecturally very similar to other members of the family, shows structural instability and an incomplete self-assembly behavior by populating two oligomerization states. Through computational analysis and comparison to its homologues, we have found that this protein has a smaller than average dimeric interface on its 2-fold symmetry axis mainly because of the existence of an interfacial water pocket centered around two water-bridged asparagine residues. To investigate the possibility of engineering EcBfr for modified structural stability, we have used a semiempirical computational method to virtually explore the energy differences of the 480 possible mutants at the dimeric interface relative to that of wild-type EcBfr. This computational study also converged on the water-bridged asparagines. Replacing these two asparagines with hydrophobic amino acids resulted in proteins that folded into α-helical monomers and assembled into cages as evidenced by circular dichroism and transmission electron microscopy. Both thermal and chemical denaturation confirmed that, in all cases, these proteins, in agreement with the calculations, possessed increased stability. One of the three mutations shifts the population in favor of the higher-order oligomerization state in solution as evidenced by both size exclusion chromatography and native gel electrophoresis. These results taken together suggest that our low-level design was successful and that it may be possible to apply the strategy of targeting water pockets at protein--protein interfaces to other protein cage and self-assembling systems. More generally, this study further demonstrates the power of jointly employing in silico and in vitro techniques to understand and enhance biostructural energetics.

Stereoelectronic effects in stabilizing protein-N-glycan interactions revealed by experiment and machine learning.

The energetics of protein-carbohydrate interactions, central to many life processes, cannot yet be manipulated predictably. This is mostly due to an incomplete quantitative understanding of the enthalpic and entropic basis of these interactions in aqueous solution. Here, we show that stereoelectronic effects contribute to stabilizing protein-N-glycan interactions in the context of a cooperatively folding protein. Double-mutant cycle analyses of the folding data from 52 electronically varied N-glycoproteins demonstrate an enthalpy-entropy compensation depending on the electronics of the interacting side chains. Linear and nonlinear models obtained using quantum mechanical calculations and machine learning explain up to 79% and 97% of the experimental interaction energy variability, as inferred from the R2 value of the respective models. Notably, the protein-carbohydrate interaction energies strongly correlate with the molecular orbital energy gaps of the interacting substructures. This suggests that stereoelectronic effects must be given a greater weight than previously thought for accurately modelling the short-range dispersive van der Waals interactions between the N-glycan and the protein.

Design and Applications of Protein-Cage-Based Nanomaterials.

Materials science is beginning to focus on biotemplation, and in support of that trend, it is realized that protein cages-proteins that assemble from multiple monomers into architectures with hollow interiors-can instill a number of unique advantages to nanomaterials. In addition, the structural and functional plasticity of many protein-cage systems permits their engineering for specific applications. In this review, the most commonly used viral and non-viral protein cages, which exhibit a wide diversity of size, functionality, and chemical and thermal stabilities, are described. Moreover, how they have been exploited for nanomaterial and nanotechnology applications is summarized.

Computationally assisted engineering of protein cages.

A hybrid computational method incorporating topographic analysis of protein surfaces and free-energy calculations of protein-protein interactions in protein nanocages is described. This design strategy can be used to engineer protein cages for enhanced structural stability and assembly.

Differential scanning calorimetry to quantify the stability of protein cages.

Differential scanning calorimetry (DSC) is an experimental technique through which the differences in amount of heat required to maintain equal temperature between a sample and a reference cell are measured at various temperatures. The quantified heat relates to the differences in apparent heat capacity of the analytes. The data from DSC studies will thereby provide direct information about the energetics of thermally induced processes in the sample. Here we present a detailed protocol to quantify the thermostability of protein cage, bacterioferritin (BFR), using differential scanning calorimetry.

Mutagenesis study to disrupt electrostatic interactions on the twofold symmetry interface of Escherichia coli bacterioferritin.

Ferritins and other cage proteins have been utilized as models to understand the fundamentals of protein folding and self-assembly. The bacterioferritin (BFR) from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for a mutagenesis study to investigate the role of electrostatic intermolecular interactions mediated through charged amino acids. Through structural and computational analyses, three charged amino acids R30, D56 and E60 which involved in an electrostatic interaction network were mutated to the opposite charge. Four mutants, R30D, D56R, E60H and D56R-E60H, were expressed, purified and characterized. All of the mutants fold into α-helical structures. Consistent with the computational prediction, they all show a lowered thermostability; double mutant D56R-E60H was found to be 16°C less stable than the wild type. Except for the mutant E60H, all the other mutations completely shut down the formation of protein cages to favour the dimer state in solution. The mutants, however, retain their ability to form cage-like nanostructures in the dried, surface immobilized conditions of transmission electron microscopy. Our findings confirm that even a single charge-inversion mutation at the 2-fold interface of BFR can affect the quaternary structure of its dimers and their ability to self-assemble into cage structures.

Complete shift of ferritin oligomerization toward nanocage assembly via engineered protein-protein interactions.

Computational redesign of a dimorphic protein nano-cage at the C3-symmetrical interfaces forces it to assemble into the monomorphic cage. These monodisperse assemblies are at least 20 °C more stable than the parent. This approach adds to the toolkit of bottom-up molecular design with applications in protein engineering and hybrid nano-materials.