The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential ...applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C₃-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C₄-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C₄ and C₃ symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.
Capturing the dynamic interplay between proteins and their myriad interaction partners is critically important for advancing our understanding of almost every biochemical process and human disease. ...The importance of this general area has spawned many measurement methods capable of assaying such protein complexes, and the mass spectrometry-based structural biology methods described in this review form an important part of that analytical arsenal. Here, we survey the basic principles of such measurements, cover recent applications of the technology that have focused on protein-small-molecule complexes, and discuss the bright future awaiting this group of technologies.
The organization of proteins into new hierarchical forms is an important challenge in synthetic biology. However, engineering new interactions between protein subunits is technically challenging and ...typically requires extensive redesign of protein–protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiled‐coil domains to assemble proteins into higher‐order structures. Here, we demonstrate the assembly of a trimeric enzyme into a well‐defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiled‐coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiled‐coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.
Geometry lesson: A modular approach for assembling proteins into large‐scale geometric structures was developed in which coiled‐coil domains acted as “twist ties” to facilitate assembly. The geometry of the cage was specified primarily by the rotational symmetries of the coiled coil and building block protein and was largely independent of protein structural details.
Ion mobility-mass spectrometry (IM-MS) is a technology of growing importance for structural biology, providing complementary 3D structure information for biomolecules within samples that are ...difficult to analyze using conventional analytical tools through the near-simultaneous acquisition of ion collision cross sections (CCSs) and masses. Despite recent advances in IM-MS instrumentation, the resolution of closely related protein conformations remains challenging. Collision induced unfolding (CIU) has been demonstrated as a useful tool for resolving isocrossectional protein ions, as they often follow distinct unfolding pathways when subjected to collisional heating in the gas phase. CIU has been used for a variety of applications, from differentiating binding modes of activation state-selective kinase inhibitors to characterizing the domain structure of multidomain proteins. With the growing utilization of CIU as a tool for structural biology, significant challenges have emerged in data analysis and interpretation, specifically the normalization and comparison of CIU data sets. Here, we present CIUSuite, a suite of software modules designed for the rapid processing, analysis, comparison, and classification of CIU data. We demonstrate these tools as part of a series of workflows for applications in comparative structural biology, biotherapeutic analysis, and high throughput screening of kinase inhibitors. These examples illustrate both the potential for CIU in general protein analysis as well as a demonstration of best practices in the interpretation of CIU data.
Despite the growing application of gas-phase measurements in structural biology and drug discovery, the factors that govern protein stabilities and structures in a solvent-free environment are still ...poorly understood. Here, we examine the solvent-free unfolding pathway for a group of homologous serum albumins. Utilizing a combination of chemical probes and noncovalent reconstructions, we draw new specific conclusions regarding the unfolding of albumins in the gas phase, as well as more general inferences regarding the sensitivity of collision induced unfolding to changes in protein primary and tertiary structure. Our findings suggest that the general unfolding pathway of low charge state albumin ions is largely unaffected by changes in primary structure; however, the stabilities of intermediates along these pathways vary widely as sequences diverge. Additionally, we find that human albumin follows a domain associated unfolding pathway, and we are able to assign each unfolded form observed in our gas-phase data set to the disruption of specific domains within the protein. The totality of our data informs the first detailed mechanism for multidomain protein unfolding in the gas phase, and highlights key similarities and differences from the known solution-phase pathway.
Multiprotein complexes are central to our understanding of cellular biology, as they play critical roles in nearly every biological process. Despite many impressive advances associated with ...structural characterization techniques, large and highly-dynamic protein complexes are too often refractory to analysis by conventional, high-resolution approaches. To fill this gap, ion mobility-mass spectrometry (IM-MS) methods have emerged as a promising approach for characterizing the structures of challenging assemblies due in large part to the ability of these methods to characterize the composition, connectivity, and topology of large, labile complexes. In this Critical Insight, we present a series of bioinformatics studies aimed at assessing the information content of IM-MS datasets for building models of multiprotein structure. Our computational data highlights the limits of current coarse-graining approaches, and compelled us to develop an improved workflow for multiprotein topology modeling, which we benchmark against a subset of the multiprotein complexes within the PDB. This improved workflow has allowed us to ascertain both the minimal experimental restraint sets required for generation of high-confidence multiprotein topologies, and quantify the ambiguity in models where insufficient IM-MS information is available. We conclude by projecting the future of IM-MS in the context of protein quaternary structure assignment, where we predict that a more complete knowledge of the ultimate information content and ambiguity within such models will undoubtedly lead to applications for a broader array of challenging biomolecular assemblies.
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Collision-induced unfolding (CIU) of protein ions and their noncovalent complexes offers relatively rapid access to a rich portfolio of biophysical information, without the need to tag or purify ...proteins prior to analysis. Such assays have been characterized extensively for a range of therapeutic proteins, proving exquisitely sensitive to alterations in protein sequence, structure, and post-translational modification state. Despite advantages over traditional probes of protein stability, improving the throughput and information content of gas-phase protein unfolding assays remains a challenge for current instrument platforms. In this report, we describe modifications to an Agilent 6560 drift tube ion mobility-mass spectrometer in order to perform robust, simultaneous CIU across all precursor ions detected. This approach dramatically increases the speed associated with typical CIU assays, which typically involve mass selection of narrow m/z regions prior to collisional activation, and thus their development requires a comprehensive assessment of charge-stripping reactions that can unintentionally pollute CIU data with chemical noise when more than one precursor ion is allowed to undergo simultaneous activation. By studying the unfolding and dissociation of intact antibody ions, a key analyte class associated with biotherapeutics, we reveal a predictive relationship between the precursor charge state, the amount of buffer components bound to the ions of interest, and the amount of charge stripping detected. We then utilize our knowledge of antibody charge stripping to rapidly capture CIU data for a range of antibody subclasses and subtypes across all charge states simultaneously, demonstrating a strong charge state dependence on the information content of CIU. Finally, we demonstrate that CIU data collection times can be further reduced by scanning fewer voltage steps, enabling us to optimize the throughput of our improved CIU methods and confidently differentiate antibody variant ions using ∼20% of the data typically collected during CIU. Taken together, our results characterize a new instrument platform for biotherapeutic stability measurements with dramatically improved throughput and information content.
Stability is a key critical quality attribute monitored throughout the development of monoclonal antibody (mAb) therapeutics. Minor changes in their higher order structure (HOS) caused by stress or ...environment may alter mAb aggregation, immunogenicity, and efficacy. In addition, the structures of the resulting mAb aggregates are largely unknown, as are their dependencies on conditions under which they are created. In this report, we investigate the HOS of mAb monomers and dimers under a variety of forced degradation conditions with ion mobility–mass spectrometry (IM–MS) and collision-induced unfolding (CIU) technologies. We evaluate two model IgG1 antibodies that differ significantly only in their complementarity-determinant regions: IgG1α and IgG1β. Our data covering both heat- and pH-based forced degradation conditions, aquired on two different IM–MS platforms, show that these mAbs undergo global HOS changes at both monomer and dimer levels upon degradation, but shifts in collision cross section (CCS) differ under pH or heat degradation conditions. In addition, the level of CCS change detected is different between IgG1α and IgG1β, suggesting that differences in the CDR drive differential responses to degradation that influence the antibody HOS. Dramatically different CIU fingerprints are obtained for IgG1α and IgG1β monomers and dimers for both degradation conditions. Finally, we constructed a series of computational models of mAb dimers for comparison with experimental CCS values and found evidence for a compact, overlapped dimer structure under native and heat degradation conditions, possibly adopting an inverted or nonoverlapped quaternary structure when produced through pH degredation. We conclude by discussing the potential impact of our findings on ongoing biotherapeutic discovery and development efforts.
Activity-based protein profiling (ABPP) has revolutionized the discovery and optimization of active-site ligands across distinct enzyme families, providing a robust platform for in-class selectivity ...profiling. Nonetheless, this approach is less straightforward for profiling reversible inhibitors and does not access proteins outside the ABPP probe’s target profile. While the active-site competitive acyl protein thioesterase 2 inhibitor ML349 (K i = 120 nM) is highly selective within the serine hydrolase enzyme family, it could still interact with other cellular targets. Here we present a chemoproteomic workflow to enrich and profile candidate ML349-binding proteins. In human cell lysates, biotinylated-ML349 enriches a recurring set of proteins, including metabolite kinases and flavin-dependent oxidoreductases that are potentially enhanced by avidity-driven multimeric interactions. Confirmatory assays by native mass spectrometry and fluorescence polarization quickly rank-ordered these weak off-targets, providing justification to explore ligand interactions and stoichiometry beyond ABPP.
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