Fabrication of Pascal-triangle Lattice of Proteins by Inducing Ligand Strategy

A protein Pascal triangle has been constructed as new type of supramolecular architecture by using the inducing ligand strategy that we previously developed for protein assemblies. Although mathematical studies on this famous geometry have a long history, no work on such Pascal triangles fabricated from native proteins has been reported so far due to their structural complexity. In this work, by carefully tuning the specific interactions between the native protein building block WGA and the inducing ligand R-SL , a 2D Pascal-triangle lattice with three types of triangular voids has been assembled. Moreover, a 3D crystal structure was obtained based on the 2D Pascal triangles. The distinctive carbohydrate binding sites of WGA and the intralayer as well as interlayer dimerization of RhB was the key to facilitate nanofabrication in solution. This strategy may be applied to prepare and explore various sophisticated assemblies based on native proteins.     

Conversion of the Native 24‐mer Ferritin Nanocage into Its Non‐Native 16‐mer Analogue by Insertion of Extra Amino Acid Residues

Protein assemblies with high symmetry are widely distributed in nature. Most efforts so far have focused on repurposing these protein assemblies, a strategy that is ultimately limited by the structures available. To overcome this limitation, methods for fabricating novel self‐assembling proteins have received intensive interest. Herein, by reengineering the key subunit interfaces of native 24‐mer protein cage with octahedral symmetry through amino acid residues insertion, we fabricated a 16‐mer lenticular nanocage whose structure is unique among all known protein cages. This newly non‐native protein can be used for encapsulation of bioactive compounds and exhibits high uptake efficiency by cancer cells. More importantly, the above strategy could be applied to other naturally occurring protein assemblies with high symmetry, leading to the generation of new proteins with unexplored functions.     

Cross‐Linking/Mass Spectrometry for Studying Protein Structures and Protein–Protein Interactions: Where Are We Now and Where Should We Go from Here?

Structural mass spectrometry (MS) is gaining increasing importance for deriving valuable three‐dimensional structural information on proteins and protein complexes, and it complements existing techniques, such as NMR spectroscopy and X‐ray crystallography. Structural MS unites different MS‐based techniques, such as hydrogen/deuterium exchange, native MS, ion‐mobility MS, protein footprinting, and chemical cross‐linking/MS, and it allows fundamental questions in structural biology to be addressed. In this Minireview, I will focus on the cross‐linking/MS strategy. This method not only delivers tertiary structural information on proteins, but is also increasingly being used to decipher protein interaction networks, both in vitro and in vivo. Cross‐linking/MS is currently one of the most promising MS‐based approaches to derive structural information on very large and transient protein assemblies and intrinsically disordered proteins.     

Artificial Multienzyme Supramolecular Device: Highly Ordered Self‐Assembly of Oligomeric Enzymes In Vitro and In Vivo

A strategy for scaffold‐free self‐assembly of multiple oligomeric enzymes was developed by exploiting enzyme oligomerization and protein–protein interaction properties, and was tested both in vitro and in vivo. Octameric leucine dehydrogenase and dimeric formate dehydrogenase were fused to a PDZ (PSD95/Dlg1/zo‐1) domain and its ligand, respectively. The fusion proteins self‐assembled into extended supramolecular interaction networks. Scanning‐electron and atomic‐force microscopy showed that the assemblies assumed two‐dimensional layer‐like structures. A fluorescence complementation assay indicated that the assemblies were localized to the poles of cells. Moreover, both in vitro and in vivo assemblies showed higher NAD(H) recycling efficiency and structural stability than did unassembled structures when applied to a coenzyme recycling system. This work provides a novel method for developing artificial multienzyme supramolecular devices and for compartmentalizing metabolic enzyme cascades in living cells.     

An Integrated Mass Spectrometry Based Approach to Probe the Structure of the Full‐Length Wild‐Type Tetrameric p53 Tumor Suppressor

We present an integrated approach for investigating the topology of proteins through native mass spectrometry (MS) and cross‐linking/MS, which we applied to the full‐length wild‐type p53 tetramer. For the first time, the two techniques were combined in one workflow to obtain not only structural insight in the p53 tetramer, but also information on the cross‐linking efficiency and the impact of cross‐linker modification on the conformation of an intrinsically disordered protein (IDP). P53 cross‐linking was monitored by native MS and as such, our strategy serves as a quality control for different cross‐linking reagents. Our approach can be applied to the structural investigation of various protein systems, including IDPs and large protein assemblies, which are challenging to study by the conventional methods used for protein structure characterization.     

Self‐Assembly of Proteins: Towards Supramolecular Materials

The study of protein self‐assembly has attracted great interest over the decades, due to the important role that proteins play in life. In contrast to the major achievements that have been made in the fields of DNA origami, RNA, and synthetic peptides, methods for the design of self‐assembling proteins have progressed more slowly. This Concept article provides a brief overview of studies on native protein and artificial scaffold assemblies and highlights advances in designing self‐assembling proteins. The discussions are focused on design strategies for self‐assembling proteins, including protein fusion, chemical conjugation, supramolecular, and computational‐aided de novo design.     

Strategies for the construction of supramolecular assemblies from poly-NHC ligand precursors

The field of supramolecular assemblies has developed rapidly in the last few decades, thanks in a large part to their diverse applications. These assemblies have been mostly based on Werner-type coordination motifs in which metal centres are coordinated by nitrogen or oxygen donors. Recently, N-heterocyclic carbene(NHC) ligands have been employed as carbon donors not only because of their appealing structures but also due to the extensive applications in catalysis, biomedicine and material science of the resulting assemblies. During the last decade, NHC-based supramolecular assemblies have witnessed rapid growth and extensive application in molecular recognition, luminescent materials and catalysis. For different topological systems, a diverse selection of poly-NHC precursors and synthetic strategies is crucial to precisely control the synthesis of supramolecular architectures. Several synthetic strategies have been developed to synthesise two-dimensional(2D) molecular metallacycles and three-dimensional(3D) metallacages from a wide range of poly-NHC precursors, including a straightforward one-pot strategy,supramolecular transmetalation, stepwise synthesis, an improved one-pot strategy involving self-sorting behaviour of 3D metallacages and a subtle variation strategy of poly-NHC ligand precursors. This review offers a summary of the synthetic strategies applied for the construction of different poly-NHC-based supramolecular assemblies, particularly emphasizes recent progress in the synthesis of large and complex supramolecular assemblies from poly-NHC precursors, and further attention is given to their application in postsynthetic modifications(PSMs), host-guest chemistry, luminescent properties and biomedical applications.     

Allosterically Activated Protein Self‐Assembly for the Construction of Helical Microfilaments with Tunable Helicity

Protein allostery, a chemical‐to‐mechanical effect that can precisely regulate protein structure, exists in many proteins. Herein, we demonstrate that protein allostery can be used to drive self‐assembly for the construction of tunable protein architectures. Calmodulin (CaM) was chosen as a model allosteric protein. Ca²⁺‐mediated contraction of CaM to a closed state can activate CaM and its ligand to self‐assemble into a 1D protein helical microfilament. Conversely, relaxation of CaM to the open state can unwind and further dissociate the helical assemblies. Fine regulation of the protein conformation by tuning the external Ca²⁺ level allows us to obtain various protein helical nanostructures with tunable helicity. This study offers a new approach toward chemomechanically controlled protein self‐assembly.     

Synthesis,Characterizations, Magnetism and Thermal Degradation of a 2‐Fold Interpenetrated 3D Cobalt‐Organic Framework

A new coordination polymer, [Co₂(L)₂(4,4′‐bipy)]_n·3nH₂O ( 1 ) based on 5‐(3‐methyl‐5‐phenyl‐4H‐1,2,4‐triazol‐4‐yl)isophthalic acid (H₂ L ) and 4,4′‐bipyridine (4,4′‐bipy) has been hydrothermally synthesized and characterized by single‐crystal X‐ray diffraction, XRPD, IR, and elemental analysis. Temperature‐dependent magnetic susceptibility and thermal degradation for 1 were also studied. The asymmetric unit of compound 1 consists of two crystallographically independent Co(II) ion, two L ^2? ligand, one 4,4′‐bipy ligand, and three lattice water molecules. The 2D triangle networks were linked by the bridging 4,4′‐bipy ligand to give rise to a 2‐fold interpenetrated 3D architecture. The simplest cyclic motif of the 2D networks is a triangle ring consisting of three Co(II) cations and three L ^2? ligands. So we can define Co(II) ions as 4‐connected nodes and the L ^2? ligands as 3‐connected nodes. Thus, the 3D structure can be described as a 2‐fold parallel interpenetrated ins InS 3,4‐conn topology.     

Site‐Specific Investigation of the Steady‐State Kinetics and Dynamics of the Multistep Binding of Bile Acid Molecules to a Lipid Carrier Protein

The investigation of multi‐site ligand–protein binding and multi‐step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D ¹H–¹⁵N line‐shape analysis, which allows a reliable investigation of ligand binding occurring on micro‐ to millisecond timescales, have been extended to model a two‐step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule–ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi‐step events could be modelled, for several residues, with a two‐step binding mechanism. The protein in the ligand‐bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed.     

Noncovalent Synthesis of Hierarchical Zinc Phosphates from a Single Zn4O12P4 Double‐Four‐Ring Building Block: Dimensionality Control through the Choice of Auxiliary Ligands

In contrast to the well‐known reaction of phosphonic acids RP(O)(OH)₂ with divalent transition‐metal ions that yields layered metal phosphonates [RPO₃M(H₂O)]_n, the 2,6‐diisopropylphenyl ester of phosphoric acid, dippH₂, reacts with zinc acetate in methanol under ambient conditions to afford tetrameric zinc phosphate [(ArO)PO₃Zn(MeOH)]₄ ( 1 ). The coordinated methanol in 1 can be readily exchanged by stronger Lewis basic ligands at room temperature. This strategy opens up a new avenue for building double‐four‐ring (D4R) cubane‐based supramolecular assemblies through strong intercubane hydrogen‐bonding interactions. Seventeen pyridinic ligands have been used to synthesize as many D4R cubanes [(ArO)PO₃Zn(L)]₄ ( 2 – 18 ) from 1 . The ligands have been chosen in such a way that the majority of them contain an additional functional group that could be used for noncovalent synthesis of extended structures. When the ligand does not contain any other hydrogen‐bonding donor–acceptor sites (e.g., 2,4,6‐trimethylpyridine (collidine)), zero‐dimensional D4R cubanes have been obtained. The use of pyridine, lutidine, 2‐aminopyridine, and 2,6‐diaminopyridine, however, results in the formation of linear or zigzag one‐dimensional assemblies of D4R cubanes through strong intermolecular C? H???O or N? H???O interactions. Construction of two‐dimensional assemblies of zinc phosphates has been achieved by employing 2‐hydroxypyridine or 2‐methylimidazole as the exo‐cubane ligand on zinc centers. The introduction of an alcohol side chain on the pyridinic ligand in such a way that the ? CH₂OH group cannot participate in intracubane hydrogen bonding (e.g., pyridine‐3‐methanol, pyridine‐4‐methanol, and 3,5‐dimethylpyrazole‐N‐ethanol) leads to the facile noncovalent synthesis of three‐dimensional framework structures. Apart from being useful as building blocks for noncovalent synthesis of zeolite‐like materials, compounds 1 – 18 can also be thermolyzed at approximately 500 °C to yield high‐purity zinc pyrophosphate (Zn₂P₂O₇) ceramic material.     

Mechanistic Insight into Nanomolar Binding of Multivalent Neoglycopeptides to Wheat Germ Agglutinin

Multivalent carbohydrate–protein interactions are frequently involved in essential biological recognition processes. Accordingly, multivalency is often also exploited for the design of high‐affinity lectin ligands aimed at the inhibition of such processes. In a previous study (D. Schwefel et al., J. Am. Chem. Soc. 2010 , 132, 8704–8719) we identified a tetravalent cyclopeptide‐based ligand with nanomolar affinity to the model lectin wheat germ agglutinin (WGA). To unravel the structural features of this ligand required for high‐affinity binding to WGA, we synthesized a series of cyclic and linear neoglycopeptides that differ in their conformational freedom as well as the number of GlcNAc residues. Combined evidence from isothermal titration calorimetry (ITC), enzyme‐linked lectin assays (ELLA), and dynamic light scattering (DLS) revealed different binding modes of tetra‐ and divalent ligands and that conformational preorganization of the ligands by cyclization is not a prerequisite for achieving high binding affinities. The high affinities of the tetravalent ligands rather stem from their ability to form crosslinks between several WGA molecules. The results illustrate that binding affinities and mechanisms are strongly dependent on the used multivalent system which offers opportunities to tune and control binding processes.     

Design of Bioinorganic Materials at the Interface of Coordination and Biosupramolecular Chemistry

Protein assemblies have recently become known as potential molecular scaffolds for applications in materials science and bio‐nanotechnology. Efforts to design protein assemblies for construction of protein‐based hybrid materials with metal ions, metal complexes, nanomaterials and proteins now represent a growing field with a common aim of providing novel functions and mimicking natural functions. However, the important roles of protein assemblies in coordination and biosupramolecular chemistry have not been systematically investigated and characterized. In this personal account, we focus on our recent progress in rational design of protein assemblies using bioinorganic chemistry for (1) exploration of unnatural reactions, (2) construction of functional protein architectures, and (3) in vivo applications.     

An in situ Dynamic Continuum of Supramolecular Phosphoglycopeptides Enables Formation of 3D Cell Spheroids

Higher‐order assemblies of proteins, with a structural and dynamic continuum, is an important concept in biology, but these insights have yet to be applied in designing biomaterials. Dynamic assemblies of supramolecular phosphoglycopeptides (sPGPs) transform a 2D cell sheet into 3D cell spheroids. A ligand–receptor interaction between a glycopeptide and a phosphopeptide produces sPGPs that form nanoparticles, which transform into nanofibrils upon partial enzymatic dephosphorylation. The assemblies form dynamically and hierarchically in situ on the cell surface, and interact with the extracellular matrix molecules and effectively abolish contact inhibition of locomotion (CIL) of the cells. Integrating molecular recognition, catalysis, and assembly, these active assemblies act as a dynamic continuum to disrupt CIL, thus illustrating a new kind of biomaterial for regulating cell behavior.     

Recrystallization‐Induced Self‐Assembly for the Growth of Cu2O Superstructures

The assembly of inorganic nanoparticles (NPs) into 3D superstructures with defined morphologies is of particular interest. A novel strategy that is based on recrystallization‐induced self‐assembly (RISA) for the construction of 3D Cu₂O superstructures and employs Cu₂O mesoporous spheres with diameters of approximately 300 nm as the building blocks has now been developed. Balancing the hydrolysis and recrystallization rates of the CuCl precursors through precisely adjusting the experimental parameters was key to success. Furthermore, the geometry of the superstructures can be tuned to obtain either cubes or tetrahedra and was shown to be dependent on the growth behavior of bulk CuCl. The overall strategy extends the applicability of recrystallization‐based processes for the guided construction of assemblies and offers unique insights for assembling larger particles into complicated 3D superstructures.     

Repeat Module‐Based Rational Design of a Photoswitchable Protein for Light‐Driven Control of Biological Processes

Light‐driven control of biological processes using photoswitchable proteins allows high spatiotemporal interrogation or manipulation of such processes, assisting in understanding their functions. Despite considerable advances, however, the wide spread use of optical control has been hampered by a limited repertoire of photoswitchable proteins and a lack of generalized design strategy. Herein, we present a repeat module‐based rational design of a photoswitchable protein composed of LRR (Leucine‐rich repeat) modules using azobenzene as a photochromic ligand. Our design approach involves the rational selection of a C_β pair between two nearby modules within a convex region and subsequent cross‐linking with a photochromic ligand. We demonstrate the general utility and potential of our strategy by showing the design of three target‐specific photoswitchable proteins and a light‐driven modulation of the cell signaling. With an abundance of LRR proteins in nature, our approach can expand the repertoire of photoswitchable proteins for light‐driven control of biological processes.     

Characterization and purification of supramolecular metal complexes using gel-permeation chromatography

Gel-permeation chromatography (GPC) has been used to analyze transition-metal-based squares, triangles, and related supramolecular complexes. Using rhenium-containing molecular squares of different sizes, a linear calibration curve has been established, which was used for confirming the relative sizes of other assemblies. GPC can also discriminate cyclic trimers and tetramers of a dirhodium building block. Preparative GPC has been used to resolve macroscopic samples of a rhenium-based supramolecular mixture into pure syn and anti isomers. A mixture of "triangle" and "square" has also been successfully separated.     

Native mass spectrometry—A valuable tool in structural biology

Proteins and the complexes they form with their ligands are the players of cellular action. Their function is directly linked with their structure making the structural analysis of protein‐ligand complexes essential. Classical techniques of structural biology include X‐ray crystallography, nuclear magnetic resonance spectroscopy and recently distinguished cryo‐electron microscopy. However, protein‐ligand complexes are often dynamic and heterogeneous and consequently challenging for these techniques. Alternative approaches are therefore needed and gained importance during the last decades. One alternative is native mass spectrometry, which is the analysis of intact protein complexes in the gas phase. To achieve this, sample preparation and instrument conditions have to be optimised. Native mass spectrometry then reveals stoichiometry, protein interactions and topology of protein assemblies. Advanced techniques such as ion mobility and high‐resolution mass spectrometry further add to the range of applications and deliver information on shape and microheterogeneity of the complexes. In this tutorial, we explain the basics of native mass spectrometry including sample requirements, instrument modifications and interpretation of native mass spectra. We further discuss the developments of native mass spectrometry and provide example spectra and applications.     

Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane‐mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co‐polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high‐resolution solid‐state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD.     

Metal‐Directed Self‐Assembly of a Polyoxometalate‐Based Molecular Triangle: Using Powerful Analytical Tools to Probe the Chemical Structure of Complex Supramolecular Assemblies

A polyoxometalate‐based molecular triangle has been synthesized through the metal‐driven self‐assembly of covalent organic/inorganic hybrid oxo‐clusters with remote pyridyl binding sites. The new metallomacrocycle was unambiguously characterized by using a combination of ¹H NMR spectroscopy, 2D diffusion NMR spectroscopy (DOSY), electrospray ionization travelling wave ion mobility mass spectrometry (ESI‐TWIM‐MS), small‐angle X‐ray scattering (SAXS) and molecular modelling. The collision cross‐sections obtained from TWIM‐MS and the hydrodynamic radii derived from DOSY are in good agreement with the geometry‐optimized structures obtained by using theoretical calculations. Furthermore, SAXS was successfully employed and proved to be a powerful technique for characterizing such large supramolecular assemblies.