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We describe the capture and structuring of disordered N-terminal regions by the macrocycle sulfonato-calix[4]arene ( sclx 4 ). Using the trimeric β-propeller Ralstonia solanacearum lectin (RSL) as a scaffold, we generated a series of mutants with extended and dynamic N-termini. Three of the mutants feature an N-terminal methionine-lysine motif. The fourth mutant contains the disordered 8-residue N-terminus of Histone 3, a component of the nucleosome. X-ray crystallography and NMR spectroscopy provide evidence for sclx 4 binding to the flexible N-terminal regions. Three crystal structures reveal that the calixarene recognizes the N-terminal Met-Lys motif, capturing either residue. We provide crystallographic proof for sclx 4 encapsulation of N-terminal methionine. Calixarene capture of intrinsically disordered regions may have applications in regulating protein secondary (and tertiary) structure.

Engineered protein cages are of great interest considering their diverse applications in delivery and catalysis. Here, we describe macrocycle-triggered icosahedral cage assembly of a designed β-propeller. Cage assembly was evidenced by small-angle X-ray scattering and X-ray crystallography.

Supramolecular synthons, defined as reproducible intermolecular structural units, have greatly aided small molecule crystal engineering. In this paper, we propose that supramolecular synthons guide ligand-mediated protein crystallization. The protein RSL and the macrocycle sulfonato-calix[8]arene cocrystallize in at least four ways. One of these cocrystals is a highly porous cube comprising protein nodes connected by calixarene dimers. We show that mutating an aspartic acid to an asparagine results in two new cubic assemblies that depend also on the crystallization method. One of the new cubic arrangements is mediated by calixarene trimers and has a ∼30% increased cell volume relative to the original crystal with calixarene dimers. Crystals of the sulfonato-calix[8]arene sodium salt were obtained from buffered conditions similar to those used to grow the protein-calix[8]arene cocrystals. X-ray analysis reveals a coordination polymer of the anionic calix[8]arene and sodium cation in which the macrocycle is arranged as staggered stacks of the pleated loop conformation. Remarkably, the calixarene packing arrangement is the same in the simple salt as in the protein cocrystal. With the pleated loop conformation, the calixarene presents an extended surface for binding other calixarenes (oligomerization) as well as binding to a protein patch (biomolecular complexation). Small-angle X-ray scattering data suggest pH-dependent calixarene assembly in solution. Therefore, the calix[8]arene-calix[8]arene structural unit may be regarded as a supramolecular synthon that directs at least two types of protein assembly, suggesting applications in protein crystal engineering., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

We describe complex formation between a designed pentameric β-propeller and the anionic macrocycle sulfonato-calix[8]arene ( sclx 8 ), as characterized by X-ray crystallography and NMR spectroscopy. Two crystal structures and 15 N HSQC experiments reveal a single calixarene binding site in the concave pocket of the β-propeller toroid. Despite the symmetry mismatch between the pentameric protein and the octameric macrocycle, they form a high affinity multivalent complex, with the largest protein-calixarene interface observed to date. This system provides a platform for investigating multivalency.

Controlled protein assembly and crystallization is necessary as a means of generating diffraction-quality crystals as well as providing a basis for new types of biomaterials. Water-soluble calixarenes are useful mediators of protein crystallization. Recently, it was demonstrated that Ralstonia solanacearum lectin (RSL) co-crystallizes with anionic sulfonato-calix[8]arene (sclx 8 ) in three space groups. Two of these co-crystals only grow at pH ≤ 4 where the protein is cationic, and the crystal packing is dominated by the calixarene. This paper describes a fourth RSL-sclx 8 co-crystal, which was discovered while working with a cation-enriched mutant. Crystal form IV grows at high ionic strength in the pH range 5-6. While possessing some features in common with the previous forms, the new structure reveals alternative calixarene binding modes. The occurrence of C 2 -symmetric assemblies, with the calixarene at special positions, appears to be an important result for framework fabrication. Questions arise regarding crystal screening and exhaustive searching for polymorphs., (open access.)

The donut-shaped cucurbit[n]urils (Qn) are a class of rigid macrocyclic receptor with protein recognition capabilities. Qn encapsulation of amino acid side chains can enable protein assembly. Recently, cucurbit[7]uril (Q7) has been applied as a molecular glue for organizing protein building blocks into crystalline architectures. Q7 co-crystallization with dimethylated Ralstonia solanacearum lectin (RSL*) has yielded novel crystalline architectures. Co-crystallization of RSL* and Q7 yields either cage- or sheet-like architectures which may be modulated via protein engineering. However, questions remain as to the factors dictating the formation of one architecture over another (cage versus sheet). Here, we make use of an engineered RSL*-Q7 system which co-crystallizes as the cage or sheet assembly with easily-distinguished crystal morphologies. Using this model system, we probe how the crystallization conditions dictate which crystalline architecture is adopted. Protein-ligand ratios and the sodium concentration were identified as key determinants for the growth of the cage versus sheet assemblies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein frameworks are an emerging class of biomaterial with medical and technological applications. Frameworks are studied mainly by X-ray diffraction or scattering techniques. Complementary strategies are required. Here, we report solid-state NMR analyses of a microcrystalline protein-macrocycle framework and the rehydrated freeze-dried protein. This methodology may aid the characterization of low-crystallinity frameworks.

We have identified a novel shell protein, accripin11, as a major soluble component of the calcitic prisms of the fan mussel Pinna nobilis. Initially retrieved from a cDNA library, its full sequence is confirmed here by transcriptomic and proteomic approaches. The sequence of the mature protein is 103 residues with a theoretical molecular weight of 11 kDa and is moderately acidic (pI 6.74) except for its C-terminus which is highly enriched in aspartic acid. The protein exhibits a peculiar cysteine pattern in its central domain. The full sequence shares similarity with six other uncharacterized molluscan shell proteins from the orders Ostreida, Pteriida and Mytilida, all of which are pteriomorphids and produce a phylogenetically restricted pattern of nacro-prismatic shell microstructures. This suggests that accripin11 is a member of a family of clade-specific shell proteins. A 3D model of accripin11 was predicted with AlphaFold2, indicating that it possesses three short alpha helices and a disordered C-terminus. Recombinant accripin11 was tested in vitro for its ability to influence the crystallization of CaCO 3 , while a polyclonal antibody was able to locate accripin11 to prismatic extracts, particularly in the acetic acid-soluble matrix. The putative functions of accripin11 are further discussed in relation to shell biomineralization., (© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Owing to their remarkable features, calix[n]arenes are being exploited to study different aspects of molecular recognition, including protein complexation. Different complexation modes have been described, depending on the moieties that complement the aromatic cavity, allowing for function regulation and/or controlled assembly of the protein target. Here, a rigid cone calix[4]arene, bearing four anionic alanine units at the upper rim, was tested as a ligand for cytochrome c . Cocrystallization attempts were unfruitful, preventing a solid-state study of the system. Next, the complex was studied using NMR spectroscopy, which revealed the presence of two binding sites at lysine residues with dissociation constants ( K d ) in the millimolar range.

This Account summarizes the progress in protein-calixarene complexation, tracing the developments from binary recognition to the glue activity of calixarenes and beyond to macrocycle-mediated frameworks. During the past 10 years, we have been tackling the question of protein-calixarene complexation in several ways, mainly by cocrystallization and X-ray structure determination as well as by solution state methods, NMR spectroscopy, isothermal titration calorimetry (ITC), and light scattering. Much of this work benefitted from collaboration, highlighted here. Our first breakthrough was the cocrystallization of cationic cytochrome c with sulfonato-calix[4]arene leading to a crystal structure defining three binding sites. Together with NMR studies, a dynamic complexation was deduced in which the calixarene explores the protein surface. Other cationic proteins were similarly amenable to cocrystallization with sulfonato-calix[4]arene, confirming calixarene-arginine/lysine encapsulation and consequent protein assembly. Calixarenes bearing anionic substituents such as sulfonate or phosphonate, but not carboxylate, have proven useful.Studies with larger calix[ n ]arenes ( n = 6, 8) demonstrated the bigger better binder phenomenon with increased affinities and more interesting assemblies, including solution-state oligomerization and porous frameworks. While the calix[4]arene cavity accommodates a single cationic side chain, the larger macrocycles adopt different conformations, molding to the protein surface and accommodating several residues (hydrophobic, polar, and/or charged) in small cavities. In addition to accommodating protein features, the calixarene can bind exogenous components such as polyethylene glycol (PEG), metal ions, buffer, and additives. Ternary cocrystallization of cytochrome c , sulfonato-calix[8]arene, and spermine resulted in altered framework fabrication due to calixarene encapsulation of the tetraamine. Besides host-guest chemistry with exogenous components, the calixarene can also self-assemble, with numerous instances of macrocycle dimers.Calixarene complexation enables protein encapsulation, not merely side chain encapsulation. Cocrystal structures of sulfonato-calix[8]arene with cytochrome c or Ralstonia solanacearum lectin (RSL) provide evidence of encapsulation, with multiple calixarenes masking the same protein. NMR studies of cytochrome c and sulfonato-calix[8]arene are also consistent with multisite binding. In the case of RSL, a C 3 symmetric trimer, up to six calixarenes bind the protein yielding a cubic framework mediated by calixarene dimers. Biomolecular calixarene complexation has evolved from molecular recognition to framework construction. This latter development contributes to the challenge in design and preparation of porous molecular materials. Cytochrome c and sulfonato-calix[8]arene form frameworks with >60% solvent in which the degree of porosity depends on the protein:calixarene ratio and the crystallization conditions. Recent developments with RSL led to three frameworks with varying porosity depending on the crystallization conditions, particularly the pH. NMR studies indicate a pH-triggered assembly in which two acidic residues appear to play key roles. The field of supramolecular protein chemistry is growing, and this Account aims to encourage new developments at the interface between biomolecular and synthetic/supramolecular chemistry.

Controlled protein assembly provides a means to generate biomaterials. Synthetic macrocycles such as the water-soluble sulfonato-calix[n]arenes are useful mediators of protein assembly. Sulfonato-thiacalix[4]arene ( tsclx 4 ), with its metal-binding capacity, affords the potential for simultaneous macrocycle- and metal-mediated protein assembly. Here, we describe the tsclx 4 -/Zn-directed assembly of two proteins: cationic α-helical cytochrome c (cyt c ) and neutral β-propeller Ralstonia solanacearum lectin (RSL). Two co-crystal forms were obtained with cyt c , each involving multinuclear zinc sites supported by the cone conformation of tsclx 4 . The tsclx 4 /Zn cluster acted as an assembly node via both lysine encapsulation and metal-mediated protein-protein contacts. In the case of RSL, tsclx 4 adopted the 1,2-alternate conformation and supported a dinuclear zinc site with concomitant encapsulation and metal-binding of two histidine side chains. These results, together with the knowledge of thiacalixarene/metal nanoclusters, suggest promising applications for thiacalixarenes in biomaterials and MOF fabrication., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

One approach to protein assembly involves water-soluble supramolecular receptors that act like glues. Bionanoarchitectures directed by these scaffolds are often system-specific, with few studies investigating their customization. Herein, the modulation of cucurbituril-mediated protein assemblies through the inclusion of peptide tectons is described. Three peptides of varying length and structural order were N-terminally appended to RSL, a β-propeller building block. Each fusion protein was incorporated into crystalline architectures mediated by cucurbit[7]uril (Q7). A trimeric coiled-coil served as a spacer within a Q7-directed sheet assembly of RSL, giving rise to a layered material of varying porosity. Within the spacer layers, the coiled-coils were dynamic. This result prompted consideration of intrinsically disordered peptides (IDPs) as modulatory tectons. Similar to the coiled-coil, a mussel adhesion peptide (Mefp) also acted as a spacer between protein-Q7 sheets. In contrast, the fusion of a nucleoporin peptide (Nup) to RSL did not recapitulate the sheet assembly. Instead, a Q7-directed cage was adopted, within which disordered Nup peptides were partially "captured" by Q7 receptors. IDP capture occurred by macrocycle recognition of an intrapeptide Phe-Gly motif in which the benzyl group was encapsulated by Q7. The modularity of these protein-cucurbituril architectures adds a new dimension to macrocycle-mediated protein assembly. Segregated protein crystals, with alternating layers of high and low porosity, could provide a basis for new types of materials., (© 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

53BP1 is recruited to chromatin in the vicinity of DNA double-strand breaks (DSBs). We identify the nuclear kinesin, KIF18B, as a 53BP1-interacting protein and define its role in 53BP1-mediated DSB repair. KIF18B is a molecular motor protein involved in destabilizing astral microtubules during mitosis. It is primarily nuclear throughout the interphase and is constitutively chromatin bound. Our observations indicate a nuclear function during the interphase for a kinesin previously implicated in mitosis. We identify a central motif in KIF18B, which we term the Tudor-interacting motif (TIM), because of its interaction with the Tudor domain of 53BP1. TIM enhances the interaction between the 53BP1 Tudor domain and dimethylated lysine 20 of histone H4. TIM and the motor function of KIF18B are both required for efficient 53BP1 focal recruitment in response to damage and for fusion of dysfunctional telomeres. Our data suggest a role for KIF18B in efficient 53BP1-mediated end-joining of DSBs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Controlled protein assembly holds great potential in the fabrication of biohybrid materials. However, the structural diversity and complexity of proteins present an obstacle to controlled assembly. Supramolecular chemistry is a possible solution as it offers tools to mediate self-assembly with molecular precision. This paper deals with the calixarene- and zinc-mediated assembly and crystallization of the histidine-rich Penicillium chrysogenum antifungal protein B (PAFB). We report crystal structures of pure PAFB, PAFB in complex with Zn 2+ , and the ternary complex of PAFB, Zn 2+ and sulfonato-calix[8]arene (sclx 8 ). A comparison of the three crystal structures revealed the structural plasticity of PAFB. While the flexible and highly anionic sclx 8 resulted in large molecular weight aggregates of PAFB in solution, diffraction-quality crystals of PAFB-sclx 8 were not obtained. We report crystals of PAFB-Zn 2+ -sclx 8 in which a trinuclear zinc cluster occurred adjacent to a calixarene binding site. Interestingly, the combination of sclx 8 complexation and zinc coordination resulted in a porous framework with channels of circa 2 nm diameter. Detailed analysis of the crystal structure highlighted novel molecular recognition features. This research enriches the set of supramolecular interactions available to promote protein assembly., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Water-soluble, anionic calix[ n ]arenes are useful receptors for protein recognition and assembly. For example, sulfonato-calix[8]arene ( sclx 8 ) can encapsulate proteins and direct their assembly into porous frameworks. In this work, we turned our attention to an "extended arm" calixarene with 16 phenyl rings. We hypothesized that this larger receptor would have increased capacity for protein masking/encapsulation. A cocrystal structure of p -benzyl-sulfonato-calix[8]arene ( b-sclx 8 ) and cytochrome c (cyt c ) revealed a surprising assembly. A pseudorotaxane comprising a stack of three b-sclx 8 molecules threaded by polyethylene glycol (PEG) was bound to the protein. The trimeric b-sclx 8 stack, a tubelike structure with a highly charged surface, mediated assembly via a new mode of protein recognition. The calixarene stack presents four hydrophobic grooves, each of which binds to one cyt c by accommodating the N-terminal α-helix. This unprecedented binding mode suggests new possibilities for supramolecular protein chemistry., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Precisely defined protein aggregates, as exemplified by crystals, have applications in functional materials. Consequently, engineered protein assembly is a rapidly growing field. Anionic calix[n]arenes are useful scaffolds that can mold to cationic proteins and induce oligomerization and assembly. Here, we describe protein-calixarene composites obtained via cocrystallization of commercially available sulfonato-calix[8]arene ( sclx 8 ) with the symmetric and "neutral" protein RSL. Cocrystallization occurred across a wide range of conditions and protein charge states, from pH 2.2-9.5, resulting in three crystal forms. Cationization of the protein surface at pH ∼ 4 drives calixarene complexation and yielded two types of porous frameworks with pore diameters >3 nm. Both types of framework provide evidence of protein encapsulation by the calixarene. Calixarene-masked proteins act as nodes within the frameworks, displaying octahedral-type coordination in one case. The other framework formed millimeter-scale crystals within hours, without the need for precipitants or specialized equipment. NMR experiments revealed macrocycle - modulated side chain p K a values and suggested a mechanism for pH-triggered assembly. The same low pH framework was generated at high pH with a permanently cationic arginine-enriched RSL variant. Finally, in addition to protein framework fabrication, sclx 8 enables de novo structure determination.

The donut-shaped cucurbit[n]urils (Qn, n = 6-8) are rigid macrocyclic receptors with widespread use in protein recognition. To date, most applications have centred on the encapsulation of N-terminal aromatic residues by Q7 or Q8. Less attention has been placed on Q6, which can recognize lysine side chains due to its high affinity for alkylamines. In this work, we investigated protein-Q6 complexation by using NMR spectroscopy. Attempts to crystallize protein-Q6 complexes were thwarted by the crystallization of Q6. We studied four proteins that vary in size, net charge, and lysine content. In addition to Q6 interactions with specific Lys or dimethylated Lys residues, we report striking evidence for N-terminal recognition. High affinity (micromolar) binding occurred with the N-terminal Met-Lys motif present in one of the four model proteins. Engineering this feature into another model protein yielded a similar high affinity site. We also present evidence for Q8 binding at this N-terminal feature. These data expand the cucurbituril toolkit for protein sensing.

Sulfonato-calix[n]arenes (sclxn) are promising tools to generate crystalline protein frameworks. We report, for the first time, a lower rim functionalised octa-anionic calix[4]arene (sclx4mc) in complex with proteins. Two crystal structures of sclx4mc bound to yeast or horse heart cytochrome c (cytc) are described. Highly porous honeycomb or tubular assemblies were obtained with yeast or horse cytc, respectively. Related frameworks were obtained previously with sclx8 and sclx6 but not with sclx4, suggesting that the ligand charge is a determining factor.

A crystalline biohybrid with a 4 : 1 protein : cucurbituril mass ratio is presented. This result was achieved by engineering additional cucurbit[7]uril (Q7) binding sites into a β-propeller protein. In contrast to the parent protein, Q7-controlled assembly of the engineered variant occurred in solution, as evidenced by NMR and SAXS measurements.

Protein crystals with their precise, periodic array of functional building blocks have potential applications in biomaterials, sensing, and catalysis. This paper describes how a highly porous crystalline framework of a cationic redox protein and an anionic macrocycle can be modulated by a small cationic effector. Ternary composites of protein (∼13 kDa), calix[8]arene (∼1.5 kDa), and effector (∼0.2 kDa) formed distinct crystalline architectures, dependent on the effector concentration and the crystallization technique. A combination of X-ray crystallography and density functional theory (DFT) calculations was used to decipher the framework variations, which appear to be dependent on a calixarene conformation change mediated by the effector. This "switch" calixarene was observed in three states, each of which is associated with a different interaction network. Two structures obtained by co-crystallization with the effector contained an additional protein "pillar", resulting in framework duplication and decreased porosity. These results suggest how protein assembly can be engineered by supramolecular host-guest interactions.

Co-crystallization of a 2 kDa tether-free sulfonated foldamer and the 13 kDa lysine-rich cytochrome c yielded a remarkable biohybrid assembly with chiral resolution of the foldamer helix handedness. In the crystal a ∼5 nm foldamer stack was surrounded by eight molecules of protein. NMR and CD experiments suggest interesting differences in the solution state recognition processes.

Supramolecular receptors such as water-soluble calixarenes are in development as 'molecular glues' for protein assembly. Here, we obtained cocrystals of sulfonato-calix[6]arene (sclx 6 ) and yeast cytochrome c (cytc) in the presence of imidazole. A crystal structure at 2.65 Å resolution reveals major structural rearrangement and disorder in imidazole-bound cytc. The largest protein-calixarene interface involves 440 Å 2 of the protein surface with key contacts at Arg13, Lys73, and Lys79. These lysines participate in alkaline transitions of cytc and are part of Ω-loop D, which is substantially restructured in the complex with sclx 6 . The structural modification also includes Ω-loop C, which is disordered (residues 41-55 inclusive). These results suggest the possibility of using supramolecular scaffolds to trap partially disordered proteins., (© 2019 Federation of European Biochemical Societies.)

PEGylation is the most widely used half-life extension strategy for protein therapeutics. While it imparts a range of attractive attributes PEGylation can impede protein binding and reduce efficacy. A model system to probe the effects of PEGylation on protein binding has practical applications. Here, we present a system based on complex formation between a hexavalent lectin (RSL) and the globular polysaccharide Ficoll PM70 (a type of glycocluster). Mutants of the lectin were used to generate conjugates with 3, 6, or 12 PEG (1 kDa) chains. Using NMR spectroscopy we monitored how the degree of PEGylation impacted the lectin-Ficoll interaction. The binding propensity was observed to decrease with increasing polymer density. Apparently, the extended PEG chains sterically impede the lectin-Ficoll binding. This deduction was supported by molecular dynamics simulations of the protein-polymer conjugates. The implications for protein-surface interactions are discussed.

Ligand-mediated regulation of protein assembly occurs frequently in different cellular contexts. Auto-regulated assembly, where a ligand acts as its own competitive inhibitor, provides a mechanism for exquisite control of assembly. Unlike simple protein-ligand systems a quantification of the binding thermodynamics is not straightforward. Here, we characterize the interactions of a recently identified model system in which the oligomerization of cytochrome c is controlled by sulfonato-calix[8]arene, an anionic supramolecular scaffold. Isothermal titration calorimetry and thermodynamic modelling, in combination with Bayesian fitting, were used to quantify the ligand binding and assembly equilibria for this system. The approach and variations of this model may prove useful for the analysis of auto-regulated protein assembly in general., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Synthetic macrocycles such as calixarenes and cucurbiturils are increasingly applied as mediators of protein assembly and crystallization. The macrocycle can facilitate assembly by providing a surface on which two or more proteins bind simultaneously. This work explores the capacity of the sulfonato-calix[ n ]arene (sclx n ) series to effect crystallization of PAF, a small, cationic antifungal protein. Co-crystallization with sclx 4 , sclx 6 or sclx 8 led to high-resolution crystal structures. In the absence of sclx n , diffraction-quality crystals of PAF were not obtained. Interestingly, all three sclx n were bound to a similar patch on PAF. The largest and most flexible variant, sclx 8 , yielded a dimer of PAF. Complex formation was evident in solution via NMR and ITC experiments, showing more pronounced effects with increasing macrocycle size. In agreement with the crystal structure, the ITC data suggested that sclx 8 acts as a bidentate ligand. The contributions of calixarene size/conformation to protein recognition and assembly are discussed. Finally, it is suggested that the conserved binding site for anionic calixarenes implicates this region of PAF in membrane binding, which is a prerequisite for antifungal activity.

Noncovalent or supramolecular PEGylation, in combination with the site of administration, has great potential to increase the half-life of therapeutic proteins. To date, a variety of noncovalent PEGylation strategies have been devised. However, questions remain concerning the nature of the protein-PEG interaction. Here, we report structural analyses of a model system that comprised the lysine-rich cytochrome c and two PEGylated variants of sulfonatocalix[4]arene. Complex formation was characterized in solution by NMR spectroscopy. It was found that mono- or di-PEGylated sulfonatocalix[4]arene bound the protein similar to the parent calixarene. X-ray crystal structures at <2.7 Å resolution of the PEGylated derivatives in complex with cytochrome c revealed that the PEG chains were mostly disordered or encapsulated within the calixarene cavity. These results suggest that there was minimal interaction between the PEG and the protein surface, providing further evidence in favor of PEG maintaining a random coil conformation.

Controlled protein assembly provides a means to regulate function. Supramolecular building blocks, including rigid macrocycles, are versatile triggers of protein assembly. Now it is shown that sulfonato-calix[8]arene (sclx 8 ) mediates the formation of cytochrome c tetramers in solution. This tetramer spontaneously disassembles at ≥2 equivalents of sclx 8 , providing a remarkable example of auto-regulation. Using X-ray crystallography the sclx 8 binding sites on cytochrome c were characterized. Crystal structures at different protein-ligand ratios reveal varying degrees (up to 35 %) of protein surface coverage by the flexible calixarene and suggest a mechanism for oligomer disassembly. The solution structure of the oligomer was characterized by small-angle X-ray scattering. Overall, the data indicate calixarene-controlled protein assembly and disassembly without the requirement for a competitive inhibitor, and point to protein encapsulation by a flexible macrocycle., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Here, we provide the first structural characterization of host-guest complexation between cucurbit[7]uril (Q7) and dimethyllysine (KMe 2 ) in a model protein. Binding was dominated by complete encapsulation of the dimethylammonium functional group. While selectivity for the most sterically accessible dimethyllysine was observed both in solution and in the solid state, three different modes of Q7-KMe 2 complexation were revealed by X-ray crystallography. The crystal structures revealed also entrapped water molecules that solvated the ammonium group within the Q7 cavity. Remarkable Q7-protein assemblies, including inter-locked octahedral cages that comprise 24 protein trimers, occurred in the solid state. Cucurbituril clusters appear to be responsible for these assemblies, suggesting a strategy to generate controlled protein architectures., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The interactions of two mono-functionalized sulfonatocalix[4]arenes with cytochrome c were investigated by structural and thermodynamic methods. The replacement of a single sulfonate with either a bromo or a phenyl substituent resulted in altered recognition of cytochrome c as evidenced by X-ray crystallography. The bromo-substituted ligand yielded a new binding mode in which a self-encapsulated calixarene dimer contributed to crystal packing. This ligand also formed a weak halogen bond with the protein. The phenyl-substituted ligand was bound to Lys4 of cytochrome c, in a 1.7 Å resolution crystal structure. A dimeric packing arrangement mediated by ligand-ligand contacts in the crystal suggested a possible assembly mechanism. The different protein recognition properties of these calixarenes are discussed., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although essential to numerous biotech applications, knowledge of molecular recognition by arginine-rich motifs in live cells remains limited. 1 H, 15 N HSQC and 19 F NMR spectroscopies were used to investigate the effects of C-terminal -GR n (n = 1-5) motifs on GB1 interactions in Escherichia coli cells and cell extracts. While the "biologically inert" GB1 yields high-quality in-cell spectra, the -GR n fusions with n = 4 or 5 were undetectable. This result suggests that a tetra-arginine motif is sufficient to drive interactions between a test protein and macromolecules in the E. coli cytoplasm. The inclusion of a 12 residue flexible linker between GB1 and the -GR 5 motif did not improve detection of the "inert" domain. In contrast, all of the constructs were detectable in cell lysates and extracts, suggesting that the arginine-mediated complexes were weak. Together these data reveal the significance of weak interactions between short arginine-rich motifs and the E. coli cytoplasm and demonstrate the potential of such motifs to modify protein interactions in living cells. These interactions must be considered in the design of (in vivo) nanoscale assemblies that rely on arginine-rich sequences.

Complex formation between cationic cytochrome c and the water-soluble, poly-anionic p-phosphonatocalix[6]arene (pclx 6 ) was investigated. A crystal structure (at 1.8 Å resolution) revealed a remarkable dimeric disc of pclx 6 that acts like glue to mediate a symmetric (C 2 ) protein dimer. The calixarene disc has a diameter of about 1.5 nm and masks about 360 Å 2 of protein surface. The key protein-calixarene contacts occur via two linchpin lysines, with additional contacts provided by a small hydrophobic patch. The protein-calixarene supramolecular assemblies were observed in solution by size-exclusion chromatography with multi-angle light scattering and NMR spectroscopy. Using isothermal titration calorimetry and NMR data, an apparent K d in the low micromolar range was determined for the charge-rich protein-calixarene complex. In contrast to p-sulfonatocalix[4]arene, the larger pclx 6 has a single, well-defined binding site that mediates the assembly of cytochrome c in solution., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Decades of dilute-solution studies have revealed the influence of charged residues on protein stability, solubility and stickiness. Similar characterizations are now required in physiological solutions to understand the effect of charge on protein behavior under native conditions. Toward this end, we used free boundary and native gel electrophoresis to explore the charge of cytochrome c in buffer and in Escherichia coli extracts. We find that the charge of cytochrome c was ∼2-fold lower than predicted from primary structure analysis. Cytochrome c charge was tuned by sulfate binding and was rendered anionic in E. coli extracts due to interactions with macroanions. Mutants in which three or four cationic residues were replaced with glutamate were charge-neutral and "inert" in extracts. A comparison of the interaction propensities of cytochrome c and the mutants emphasizes the role of negative charge in stabilizing physiological environments. Charge-charge repulsion and preferential hydration appear to prevent aggregation. The implications for molecular organization in vivo are discussed., (© 2016 The Protein Society.)

PEGylation, the covalent modification of proteins with polyethylene glycol, is an abundantly used technique to improve the pharmacokinetics of therapeutic proteins. The drawback with this methodology is that the covalently attached PEG can impede the biological activity (e.g., reduced receptor-binding capacity). Protein therapeutics with "disposable" PEG modifiers have potential advantages over the current technology. Here, we show that a protein-polymer "Medusa complex" is formed by the combination of a hexavalent lectin with a glycopolymer. Using NMR spectroscopy, small-angle X-ray scattering (SAXS), size exclusion chromatography, and native gel electrophoresis it was demonstrated that the fucose-binding lectin RSL and a fucose-capped polyethylene glycol (Fuc-PEG) form a multimeric assembly. All of the experimental methods provided evidence of noncovalent PEGylation with a concomitant increase in molecular mass and hydrodynamic radius. The affinity of the protein-polymer complex was determined by ITC and competition experiments to be in the micromolar range, suggesting that such systems have potential biomedical applications.

Current models of the cell interior emphasise its crowded, chemically complex and dynamically organised structure. Although the chemical composition of cells is known, the cooperative intermolecular interactions that govern cell ultrastructure are poorly understood. A major goal of biochemistry is to capture these myriad interactions in vivo. We consider the landmark discoveries that have shaped this objective, starting from the vitalist framework established by early natural philosophers. Through this historical revisionism, we extract important lessons for the bioinspired chemists of today. Scientific specialisation tends to insulate seminal ideas and hamper the unification of paradigms across biology. Therefore, we call for interdisciplinary collaboration in grappling with the complex cell interior. Recent successes in integrative structural biology and chemical biology demonstrate the power of hybrid approaches. The future roles of the (bio)chemist and model systems are also discussed as starting points for in vivo explorations., (© 2016 Federation of European Biochemical Societies.)

Progress in the field of bio-supramolecular chemistry, the bottom-up assembly of protein-ligand systems, relies on a detailed knowledge of molecular recognition. To address this issue, we have characterised complex formation between human ubiquitin (HUb) and four supramolecular anions. The ligands were: pyrenetetrasulfonic acid (4PSA), p-sulfonato-calix[4]arene (SCLX4), bisphosphate tweezers (CLR01) and meso-tetrakis (4-sulfonatophenyl)porphyrin (TPPS), which vary in net charge, size, shape and hydrophobicity. All four ligands induced significant changes in the HSQC spectrum of HUb. Chemical shift perturbations and line-broadening effects were used to identify binding sites and to quantify affinities. Supporting data were obtained from docking simulations. It was found that these weakly interacting ligands bind to extensive surface patches on HUb. A comparison of the data suggests some general indicators for the protein-binding specificity of supramolecular anions. Differences in binding were observed between the cavity-containing and planar ligands. The former had a preference for the arginine-rich, flexible C terminus of HUb., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sugar binding by a cell surface ∼29 kDa lectin (RSL) from the bacterium Ralstonia solanacearum was characterized by NMR spectroscopy. The complexes formed with four monosaccharides and four fucosides were studied. Complete resonance assignments and backbone dynamics were determined for RSL in the sugar-free form and when bound to l-fucose or d-mannose. RSL was found to interact with both the α- and the β-anomer of l-fucose and the "fucose like" sugars d-arabinose and l-galactose. Peak splitting was observed for some resonances of the binding site residues. The assignment of the split signals to the α- or β-anomer was confirmed by comparison with the spectra of RSL bound to methyl-α-l-fucoside or methyl-β-l-fucoside. The backbone dynamics of RSL were sensitive to the presence of ligand, with the protein adopting a more compact structure upon binding to l-fucose. Taking advantage of tryptophan residues in the binding sites, we show that the indole resonance is an excellent reporter on ligand binding. Each sugar resulted in a distinct signature of chemical shift perturbations, suggesting that tryptophan signals are a sufficient probe of sugar binding.

PEGylated proteins are a mainstay of the biopharmaceutical industry. Although the use of poly(ethylene glycol) (PEG) to increase particle size, stability and solubility is well-established, questions remain as to the structure of PEG-protein conjugates. Here we report the structural characterization of a model β-sheet protein (plastocyanin, 11.5 kDa) modified with a single PEG 5,000. An NMR spectroscopy study of the PEGylated conjugate indicated that the protein and PEG behaved as independent domains. A crystal structure revealed an extraordinary double-helical assembly of the conjugate, with the helices arranged orthogonally to yield a highly porous architecture. Electron density was not observed for the PEG chain, which indicates that it was disordered. The volume available per PEG chain in the crystal was within 10% of the calculated random coil volume. Together, these data support a minimal interaction between the protein and the synthetic polymer. Our work provides new possibilities for understanding this important class of protein-polymer hybrids and suggests a novel approach to engineering protein assemblies.

The assembly of redox proteins on electrodes is an important step in biosensor development. Recently, p-sulfonato-calix[4]arene was shown to act as "molecular glue" for the assembly and crystallization of cytochrome c (cyt c). Electrochemical data are presented for microscale cyt c-calixarene crystals grown on self-assembled monolayers (SAM)-modified Au electrodes. The crystals were characterized by cyclic voltammetry and exceptionally high concentrations of electroactive cyt c were obtained. The peak currents were found to increase linearly with the square root of the scan rate, thus allowing an evaluation of the rate constant for electron self-exchange. This study revealed high electroactivity accompanied by fast interprotein electron transfer in crystals, which may have implications for the construction of novel bioelectronic devices., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein characterization in situ remains a major challenge for protein science. Here, the interactions of ΔTat-GB1 in Escherichia coli cell extracts were investigated by NMR spectroscopy and size exclusion chromatography (SEC). ΔTat-GB1 was found to participate in high molecular weight complexes that remain intact at physiologically-relevant ionic strength. This observation helps to explain why ΔTat-GB1 was not detected by in-cell NMR spectroscopy. Extracts pre-treated with RNase A had a different SEC elution profile indicating that ΔTat-GB1 predominantly interacted with RNA. The roles of biological and laboratory ions in mediating macromolecular interactions were studied. Interestingly, the interactions of ΔTat-GB1 could be disrupted by biologically-relevant multivalent ions. The most effective shielding of interactions occurred in Mg(2+) -containing buffers. Moreover, a combination of RNA digestion and Mg(2+) greatly enhanced the NMR detection of ΔTat-GB1 in cell extracts., (© 2014 The Protein Society.)

Lysine is a ubiquitous residue on protein surfaces. Post translational modifications of lysine, including methylation to the mono-, di- or trimethylated amine result in chemical and structural alterations that have major consequences for protein interactions and signalling pathways. Small molecules that bind to methylated lysines are potential tools to modify such pathways. To make progress in this direction, detailed structural data of ligands in complex with methylated lysine is required. Here, we report a crystal structure of p -sulfonatocalix[4]arene (sclx 4 ) bound to methylated lysozyme in which the lysine residues were chemically modified from Lys-NH 3 + to Lys-NH(Me 2 ) + . Of the six possible dimethyllysine sites, sclx 4 selected Lys116-Me 2 and the dimethylamino substituent was deeply buried in the calixarene cavity. This complex confirms the tendency for Lys-Me 2 residues to form cation-π interactions, which have been shown to be important in protein recognition of histone tails bearing methylated lysines. Supporting data from NMR spectroscopy and MD simulations confirm the selectivity for Lys116-Me 2 in solution. The structure presented here may serve as a stepping stone to the development of new biochemical reagents that target methylated lysines.

Artificial assemblies consisting of the cationic cytochrome c (cyt c) and double-stranded DNA are interesting for the field of biohybrid systems because of the high electro-activity of the incorporated redox protein. However, little is known about the interactions between these two biomolecules. Here, the complex of reduced cyt c and a 41 base pair oligonucleotide was characterized in solution as a function of pH and ionic strength. Persistent cyt c-DNA agglomerates were observed by UV-vis and DLS (dynamic light scattering) at pH 5.0 and low ionic strength. The strength of the interaction was attenuated by raising the pH or the ionic strength. At pH 7.0 agglomerates were not formed, allowing interaction analysis by NMR spectroscopy. Using TROSY (transverse relaxation-optimized spectroscopy)-HSQC (heteronuclear single quantum coherence) experiments it was possible to identify the DNA binding site on the cyt c surface. Numerous residues surrounding the exposed heme edge of cyt c were involved in transient binding to DNA under these conditions. This result was supported by SEC (size exclusion chromatography) experiments at pH 7.0 showing that the interaction is sufficient for co-elution of cyt c and DNA.

A crystal structure of lysozyme in complex with p-sulfonato-calix[4]arene (sclx4) reveals a linear assembly of protein tetramers glued together by protein-calixarene interactions. One interaction involves encapsulation of the highly exposed C-terminal Arg128. The other involves an intricate protein-bound complex of sclx4, Mg(2+) and a fragment of polyethylene glycol.

Fluorine-containing amino acids are valuable probes for the biophysical characterization of proteins. Current methods for (19)F-labeled protein production involve time-consuming genetic manipulation, compromised expression systems and expensive reagents. We show that Escherichia coli BL21, the workhorse of protein production, can utilise fluoroindole for the biosynthesis of proteins containing (19)F-tryptophan.

Small molecules that recognize protein surfaces are important tools for modifying protein interaction properties. Since the 1980s, several thousand studies concerning calixarenes and host-guest interactions have been published. Although there is growing interest in protein-calixarene interactions, only limited structural information has been available to date. We now report the crystal structure of a protein-calixarene complex. The water-soluble p-sulfonatocalix[4]arene is shown to bind the lysine-rich cytochrome c at three different sites. Binding curves obtained from NMR titrations reveal an interaction process that involves two or more binding sites. Together, the data indicate a dynamic complex in which the calixarene explores the surface of cytochrome c. In addition to providing valuable information on protein recognition, the data also indicate that the calixarene is a mediator of protein-protein interactions, with potential applications in generating assemblies and promoting crystallization.

Protein science is shifting towards experiments performed under native or native-like conditions. In-cell NMR spectroscopy for instance has the potential to reveal protein structure and dynamics inside cells. However, not all proteins can be studied by this technique. (15)N-labelled cytochrome c (cyt c) over-expressed in Escherichia coli was undetectable by in-cell NMR spectroscopy. When whole-cell lysates were subjected to size-exclusion chromatography (SEC) cyt c was found to elute with an apparent molecular weight of >150 kDa. The presence of high molecular weight species is indicative of complex formation between cyt c and E. coli cytosolic proteins. These interactions were disrupted by charge-inverted mutants in cyt c and by elevated concentrations of NaCl. The physiologically relevant salt, KGlu, was less efficient at disrupting complex formation. Notably, a triple mutant of cyt c could be detected in cell lysates by NMR spectroscopy. The protein, GB1, yields high quality in-cell spectra and SEC analysis of lysates containing GB1 revealed a lack of interaction between GB1 and E. coli proteins. Together these data suggest that protein "stickiness" is a limiting factor in the application of in-cell NMR spectroscopy., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

NMR-monitored pH titration experiments are routinely used to measure site-specific protein pKa values. Accurate experimental pKa values are essential in dissecting enzyme catalysis, in studying the pH-dependence of protein stability and ligand binding, in benchmarking pKa prediction algorithms, and ultimately in understanding electrostatic effects in proteins. However, due to the complex ways in which pH-dependent electrostatic and structural changes manifest themselves in NMR spectra, reported apparent pKa values are often dependent on the way that NMR pH-titration curves are analyzed. It is therefore important to retain the raw NMR spectroscopic data to allow for documentation and possible re-interpretation. We have constructed a database of primary NMR pH-titration data, which is accessible via a web interface. Here, we report statistics of the database contents and analyze the data with a global perspective to provide guidelines on best practice for fitting NMR titration curves. Titration&#95;DB is available at http://enzyme.ucd.ie/Titration&#95;DB. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

The beta-sandwich cupredoxin Plastocyanin (Pc) was found to self-assemble in the presence of Zn(2+), a known mediator of protein-protein interfaces. Diffraction-quality crystals of Pc grew from solutions containing zinc acetate as the sole precipitant. Di- and trinuclear zinc sites contribute to the crystal contacts in this structure. A different crystal form, also involving numerous zinc bridging ions, was obtained in the presence of poly(ethylene glycol) 8 000. Comparison of the two crystal forms reveals the effect of macromolecular crowding on self-assembly. Solution-state structural characterisation of the Zn(2+)-mediated Pc oligomers was performed by using a combination of chemical shift perturbation mapping and small-angle X-ray scattering. The data indicate the formation of dimers in solution. The implications for metal-mediated assembly and crystallisation are discussed.

The influence of pi-interactions with a His ligand have been investigated in a family of copper-containing redox metalloproteins. The Met16Phe and Met16Trp pseudoazurin, and Leu12Phe spinach and Leu14Phe Phormidium laminosum plastocyanin variants possess active-site pi-contacts between the introduced residue and His81 and His87/92 respectively. The striking overlap of the side chain of Phe16 in the Met16Phe variant and that of Met16 in wild type pseudoazurin identifies that this position provides an important second coordination sphere interaction in both cases. His-ligand protonation and dissociation from Cu(I) occurs in the wild type proteins resulting in diminished redox activity, providing a [H(+)]-driven switch for regulating electron transfer. The introduced pi-interaction has opposing effects on the pKa for the His ligand in pseudoazurin and plastocyanin due to subtle differences in the pi-contact, stabilizing the coordinated form of pseudoazurin whereas in plastocyanin protonation and dissociation is favored. Replacement of Pro36, a residue that has been suggested to facilitate structural changes upon His ligand protonation, with a Gly, has little effect on the pKa of His87 in spinach plastocyanin. The mutations at Met16 have a significant influence on the reduction potential of pseudoazurin. Electron self-exchange is enhanced, whereas association with the physiological partner, nitrite reductase, is only affected by the Met16Phe mutation, but kcat is halved in both the Met16Phe and Met16Trp variants. Protonation of the His ligand is the feature most affected by the introduction of a pi-interaction.