Variable Linkers Research Articles

Cyclic peptide conjugated photosensitizer for targeted phototheranostics of gram-negative bacterial infection

Antimicrobial photodynamic therapy (PDT) is a promising alternative to antibiotics for eradicating pathogenic bacterial infections. It holds advantage of not inducing antimicrobial resistance but is limited for the treatment of gram-negative bacterial infection due to the lack of photosensitizer (PS) capable of targeted permeating the outer membrane (OM) of gram-negative bacteria. To facilitate the targeted permeability of PS, cyclic polymyxin b nonapeptide that can specifically bind to the lipopolysaccharide on OM, is conjugated to an FDA approved PS chlorin e6 via variable linkers. Based on structure to activity study, C6pCe6 with aminohexanoic linker and P2pCe6 with amino-3, 6-dioxaoctanoic linker are identified to preferentially image gram-negative bacteria. These two conjugates also exhibit improved aqueous dispersity and enhanced ROS generation, consequently enabled their selective bactericidal activities against gram-negative bacteria upon 660 nm light irradiation. The effective photobactericidal ability of P2pCe6 is further validated on P. aeruginosa infected G. mellonella. Moreover, it is demonstrated to effectively treat the P. aeruginosa infection and accelerate the healing process at the wound site of mouse. Owing to the light irradiation triggered targeted imaging and enhanced bactericidal capacities, P2pCe6 hold great potential to serve as a potent PS for mediating the phototheranostics of gram-negative bacterial infection.

The Chan-Lam-type synthesis of thioimidazolium salts for thiol-(hetero)arene conjugation.

The design of stable and variable aryl linkers for conjugating drug moieties to the metabolism-related thiols is of importance in drug discovery. We disclosed that thioimidazolium groups are unique scaffolds for the thiol-(hetero)arene conjugation under mild conditions. The drug bound thioimidazolium salts, which are easily accessible via a copper-mediated Chan-Lam process in gram-scale, could be successfully applied to the late-stage coupling of bioactive thiols to construct a broad array of drug-like molecules.

Cyclodextrin Polymers as a Promising Drug Carriers for Stabilization of Meropenem Solutions

Here we report the development of new drug carriers for meropenem based on the hydroxypropyl-β-cyclodextrin (HPCD) polymers with variable linkers, namely, 1,6-hexamethylenediisocyanate (HMD), citric acid (CA), succinic anhydride (SA). The structures of obtained polyesters and polyurethanes nanoparticles (120–200 nm) were investigated by NMR and FTIR-spectroscopy. The PXRD pattern demonstrated that HPCD polymers form complexes with meropenem (MP), and the majority of MP molecules are encapsulated into a complex. MP’s imprinting in the HPCD-HMD polymer matrix lead to an encapsulation efficiency of up to 82%. HPCD-HMD and HPCD-SA polymers increase MP’s stability during the storage of its aqueous solution (in 1.4 and 1.2 times, respectively). In contrast, HPCD-CA polymer negatively affects MP’s stability. In prospect, the HPCD-HMD polymer may be promising for the development of a highly efficient drug delivery system for MP.

Anaerobic demethylation of guaiacyl-derived monolignols enabled by a designed artificial cobalamin methyltransferase fusion enzyme.

Lignin-derived aryl methyl ethers (e.g. coniferyl alcohol, ferulic acid) are expected to be a future carbon source for chemistry. The well-known P450 dependent biocatalytic O-demethylation of these aryl methyl ethers is prone to side product formation especially for the oxidation sensitive catechol products which get easily oxidized in the presence of O2. Alternatively, biocatalytic demethylation using cobalamin dependent enzymes may be used under anaerobic conditions, whereby two proteins, namely a methyltransferase and a carrier protein are required. To make this approach applicable for preparative transformations, fusion proteins were designed connecting the cobalamin-dependent methyltransferase (MT) with the corrinoid-binding protein (CP) from Desulfitobacterium hafniense by variable glycine linkers. From the proteins created, the fusion enzyme MT-L5-CP with the shortest linker performed best of all fusion enzymes investigated showing comparable and, in some aspects, even better performance than the separated proteins. The fusion enzymes provided several advantages like that the cobalamin cofactor loading step required originally for the CP could be skipped enabling a significantly simpler protocol. Consequently, the biocatalytic demethylation was performed using Schlenk conditions allowing the O-demethylation e.g. of the monolignol coniferyl alcohol on a 25 mL scale leading to 75% conversion. The fusion enzyme represents a promising starting point to be evolved for alternative demethylation reactions to diversify natural products and to valorize lignin.

Bis-Boronic Acid Liposomes for Carbohydrate Recognition and Cellular Delivery.

Liposomes are effective therapeutic nanocarriers due to their ability to encapsulate and enhance the pharmacokinetic properties of a wide range of drugs and diagnostic agents. A primary area in which improvement is needed for liposomal drug delivery is to maximize the delivery of these nanocarriers to cells. Cell membrane glycans provide exciting targets for liposomal delivery since they are often densely clustered on cell membranes and glycan overabundance and aberrant glycosylation patterns are a common feature of diseased cells. Herein, we report a liposome platform incorporating bis-boronic acid lipids (BBALs) to increase valency in order to achieve selective saccharide sensing and enhance cell surface recognition based on carbohydrate binding interactions. In order to vary properties, multiple BBALs (1 a-d) with variable linkers in between the binding units were designed and synthesized. Fluorescence-based microplate screening of carbohydrate binding showed that these compounds exhibit varying binding properties depending on their structures. Additionally, fluorescence microscopy experiments indicated enhancements in cellular association when BBALs were incorporated within liposomes. These results demonstrate that multivalent BBALs serve as an exciting glycan binding liposome system for targeted delivery.

Accelerating PROTAC drug discovery: Establishing a relationship between ubiquitination and target protein degradation

PROTACs have emerged as a new class of drugs that can target the “undruggable” proteome by hijacking the ubiquitin proteasome system. Despite PROTACs’ success, most current PROTACs interface with a limited number of E3 ligases, hindering their expansion to many challenging therapeutic uses. Currently, PROTAC drug discovery relies heavily on traditional Western blotting and reporter gene assays which are insensitive and prone to artifacts, respectively. New reliable methods to monitor true PROTAC function (i.e., ubiquitination and subsequent degradation of targets at physiological expression levels) without external tags are essential to accelerate the PROTAC discovery process and to address many unmet therapeutic areas. In this study, we developed a new high-throughput screening technology using “TUBEs” as ubiquitin-binding entities to monitor PROTAC-mediated poly-ubiquitination of native target proteins with exceptional sensitivity. As a proof of concept, targets including BRD3, Aurora A Kinase, and KRAS were used to demonstrate that ubiquitination kinetics can reliably establish the rank order potencies of PROTAC with variable ligands and linkers. PROTAC-treated cell lysates with the highest levels of endogenous target protein ubiquitination - termed “UbMax” - display excellent correlations with DC50 values obtained from traditional Western blots with the added benefits of being high throughput, providing improved sensitivity, and reducing technical errors.

The design, synthesis and cellular imaging of a tumor-anchored, potent and cell-permeable BRD4-targeted fluorescent ligands

Bromodomain 4 (BRD4) proteins play an important role in histone post-translational modifications and facilitate several important physiological and pathological processes, including cancers. The inhibition of BRD4 by small molecule inhibitors shows promise as a therapeutic strategy for cancer treatment. However, their clinical applications were limited, which is largely hampered by off-target effects-induced toxicity. We herein report the design, synthesis, and cellular imaging of a set of tumor-anchored and BRD4-targeted fluorescent ligands by introducing selective and potent BRD4 inhibitor into different fluorophores via variable linkers. One of the fluorescent conjugates (compound 6) was demonstrated to be cell-permeable and low cytotoxic, preferentially accumulated in cancer cells, and display pronounced fluorescent signal. More importantly, 6 was identified to show specific BRD4 engagement in the cellular content. Collectively, this study provides a pathway for developing labeled BRD4 ligands and highlights that compound 6 may represent a valuable tool for explorative learning and target delivery study of BRD4.

A conjugated plier-linked nano-spacing graphite network for sodium-ion battery

A sodium-ion battery to use natural graphite is challenging due to the hard accessibility of sodium ions into graphite. Here, we report a plier-linked graphite network (PGN) with plier-like, conjugated active-molecule for sodium-ion battery anode. The newly designed PGN with conjugated linkers provides additional sodium-ion storage sites and sufficient nano-meter level wide two-dimensional spaces for sodium-ion access. The plier-linked graphite network (PDA-PGN) with 9,9-dihexylfluorene-2,7-diboronic acid (PDA) among variable linkers delivers the highest sodiation/desodiation capacity (298/245 mAh g−1), at approximately 10 times the level of graphite, while maintaining acceptable rate capability and stability, realizing 1.52 mAh cm−2 areal capacity with high electrode thickness (282 µm) and mass loading (7.8 mg cm−2). We revealed the knowledge of a major synergistic phenomenon between the conjugated PDA molecules and the nano-sized interlayer distance. Our PDA-PGN design added the new understanding to the alkali-metal ion storage and offers the production potential for low-cost and versatile electrodes.

Synthesis of σ Receptor Ligands with a Spirocyclic System Connected with a Tetrahydroisoquinoline Moiety via Different Linkers.

With the aim to develop new σ2 receptor ligands, spirocyclic piperidines or cyclohexanamines with 2‐benzopyran and 2‐benzofuran scaffolds were connected to the 6,7‐dimethoxy‐1,2,3,4‐tetrahydroisoquinoline moiety by variable linkers. In addition to flexible alkyl chains, linkers containing an amide as functional group were synthesized. The 2‐benzopyran and 2‐benzofuran scaffold of the spirocyclic compounds were synthesized from 2‐bromobenzaldehyde. The amide linkers were constructed by acylation of amines with chloroacetyl chloride and subsequent nucleophilic substitution, the alkyl linkers were obtained by LiAlH4 reduction of the corresponding amides. For the development of σ2 receptor ligands, the spirocyclic 2‐benzopyran scaffold is more favorable than the ring‐contracted 2‐benzofuran system. Compounds bearing an alkyl chain as linker generally show higher σ affinity than acyl linkers containing an amide as functional group. A higher σ1 affinity for the cis‐configured cyclohexanamines than for the trans‐configured derivatives was found. The highest σ2 affinity was observed for cis‐configured spiro[[2]benzopyran‐1,1′‐cyclohexan]‐4′‐amine connected to the tetrahydroisoquinoline system by an ethylene spacer (cis‐31, K i (σ2)=200 nM; the highest σ1 affinity was recorded for the corresponding 2‐benzofuran derivative with a CH2C=O linker (cis‐29, K i (σ1)=129 nM).

Dinuclear manganese(iii) complexes with bioinspired coordination and variable linkers showing weak exchange effects: a synthetic, structural, spectroscopic and computation study.

Three dimanganese(iii) complexes have been synthesised and fully characterised by standard spectroscopic methods and spectroelectrochemistry. Each MnIII ion is chelated by a salen type ligand (H2L), but there is variation in the bridging group: LMn(OOCCH[double bond, length as m-dash]CHCOO)MnL, LMn(OOCC6H4COO)MnL, and LMn(OOCC6H4C6H4COO)MnL. X-ray diffraction revealed an axial compression of each six-coordinate high-spin d4 MnIII ion, which is a Jahn-Teller-active ion. Temperature dependent magnetic susceptibility and variable temperature-variable field (VTVH) magnetisation measurements, as well as high-frequency and -field EPR (HFEPR) spectroscopy were used to accurately describe the magnetic properties of the complexes, not only the single-ion spin Hamiltonian parameters: g-values and zero-field splitting (ZFS) parameters D and E, but also the exchange interaction constant J between the two ions, which has been seldom determined for a di-MnIII complex, particularly when there is more than a single bridging atom. Quantum chemical calculations reproduced well the electronic and geometric structure of these unusual complexes, and, in particular, their electronic absorption spectra along with the spin Hamiltonian and exchange parameters.

Crowding Effects on Energy-Transfer Efficiencies of Hetero-FRET Probes As Measured Using Time-Resolved Fluorescence Anisotropy.

Macromolecular crowding is prevalent in all living cells due to the presence of large biomolecules and organelles. Cellular crowding is heterogeneous and is known to influence biomolecular transport, biochemical reactions, and protein folding. Emerging evidence suggests that some cell pathologies may be correlated with compartmentalized crowding. As a result, there is a need for robust biosensors that are sensitive to crowding as well as quantitative, noninvasive fluorescence methods that are compatible with living cells studies. Here, we have developed a model that describes the rotational dynamics of hetero-Förster resonance energy transfer (FRET) biosensors as a means to determine the energy-transfer efficiency and donor-acceptor distance. The model was tested on wavelength-dependent time-resolved fluorescence anisotropy of hetero-FRET probes (mCerulean3-linker-mCitrine) with variable linkers in both crowded (Ficoll-70) and viscous (glycerol) solutions at room temperature. Our results indicate that the energy-transfer efficiencies of these FRET probes increase as the linker becomes shorter and more flexible in pure buffer at room temperature. In addition, the FRET probes favor compact structures with enhanced energy-transfer efficiencies and a shorter donor-acceptor distance in the heterogeneous, polymer-crowded environment due to steric hindrance. In contrast, the extended conformation of these FRET probes is more favorable in viscous, homogeneous environments with a reduced energy-transfer efficiency compared to those in pure buffer, which we attribute to reduced structural fluctuations of the mCerulean3-mCitrine FRET pair in the glycerol-enriched buffer. Our results represent an important step toward the application of quantitative and noninvasive time-resolved fluorescence anisotropy of hetero-FRET probes to investigate compartmentalized macromolecular crowding and protein-protein interactions in living cells as well as in controlled environments.

ProtaTETHER - a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc-GFP fusion protein.

Protein fusions are of fundamental importance in the study of cellular biology and the elucidation of cell signaling pathways, and the importance of linkers for the proper function of protein fusions is well documented in the literature. However, there are few convenient methods available to experimentalists for the systematic implementation of linkers in protein fusions. In this work, we describe a universal approach to the creation and insertion of focused linker libraries into protein fusions. This process, deemed protaTETHER, utilizes reiterative oligomer design, PCR‐mediated linker library generation, and restriction enzyme‐free cloning methods in a straightforward, three‐step cloning process. We utilize a fusion between the catalytic subunit of cAMP‐dependent protein kinase A (PKAc) and green fluorescent protein (GFP) for the development of the protaTETHER method, implementing small linker libraries that vary by length, sequence, and predicted secondary structural elements. We analyze the impact of linker length and sequence on the expression, activity, and subcellular localization of the PKAc‐GFP fusions, and use these results to select a PKAc‐GFP fusion construct with robust expression and enzymatic activity. Based upon the results of both biochemical experiments and molecular modeling, we determine that linker flexibility is more important than linker length for optimal kinase activity and expression.

Epitope-Targeted Macrocyclic Peptide Ligand with Picomolar Cooperative Binding to Interleukin-17F.

The IL-17 cytokine family is associated with multiple immune and autoimmune diseases and comprises important diagnostic and therapeutic targets. This work reports the development of epitope-targeted ligands designed for differential detection of human IL-17F and its closest homologue IL-17A. Non-overlapping and unique epitopes on IL-17F and IL-17A were identified by comparative sequence analysis of the two proteins. Synthetic variants of these epitopes were utilized as targets for in situ click screens against a comprehensive library of synthetic peptide macrocycles with 5-mer variable regions. Single generation screens yielded selective binders for IL-17F and IL-17A with low cross-reactivity. Macrocyclic peptide binders against two distinct IL-17F epitopes were coupled using variable length chemical linkers to explore the physical chemistry of cooperative binding. The optimized linker length yielded a picomolar affinity binder, while retaining high selectivity. The presented method provides a rational approach towards targeting discontinuous epitopes, similar to what is naturally achieved by many B cell receptors.

Design and Synthesis of Cyclic Mismatch-Binding Ligands (CMBLs) with Variable Linkers by Ring-Closing Metathesis and their Photophysical and DNA Repeat Binding Properties.

Cyclophane-containing bis(2-amino-1,8-naphthyridine) moieties attached to variable linkers at the C2-position (linker B) were synthesized as cyclic mismatch-binding ligands (CMBLs). Ring-closing metathesis (RCM) is used as a key step for the introduction of double bonds at the linker B. Decreasing the size of the linker of the substrate, formation of the RCM products with an increasing trans/cis (E/Z) ratio was observed with moderate to high overall yields. Concentration-dependent fluorescence spectra were observed for CMBLs with longer linkers (n=3), whereas concentration-independent spectra were observed for CMBLs with shorter linkers (n=2 and/or 1) with a marked exception of the E-alkene 6 a. Concomitant changes in the absorption as well as in the fluorescence spectra were also observed for the CMBLs with an increasing hydrophobicity of the solvent. Absorption and fluorescence spectra of the CMBLs in solutions containing 99-100 % methanol resembled to that of the monomer. The binding behavior of these CMBLs with repeat DNA structures was investigated by using a surface plasmon resonance (SPR) assay and circular dichroism (CD) spectra. The cyclic E-alkenes 1 a (n=3) and 3 a (n=2) show an orthogonal binding relationship with d(CCTG)9 and d(CAG)9 . However, the selectivity for the cyclic Z-alkenes increased with decreasing the length of the linker from compound 2 b (n=3) to compound 7 b (n=1). These compounds display a large molecular diversity, which allowed the tuning of the binding affinity and selectivity of the CMBLs by varying the linkers towards various biologically significant repeat DNA structures.

Structural Insight into the Recognition of r(UAG) by Musashi-1 RBD2, and Construction of a Model of Musashi-1 RBD1-2 Bound to the Minimum Target RNA.

Musashi-1 (Msi1) controls the maintenance of stem cells and tumorigenesis through binding to its target mRNAs and subsequent translational regulation. Msi1 has two RNA-binding domains (RBDs), RBD1 and RBD2, which recognize r(GUAG) and r(UAG), respectively. These minimal recognition sequences are connected by variable linkers in the Msi1 target mRNAs, however, the molecular mechanism by which Msi1 recognizes its targets is not yet understood. We previously determined the solution structure of the Msi1 RBD1:r(GUAGU) complex. Here, we determined the first structure of the RBD2:r(GUAGU) complex. The structure revealed that the central trinucleotide, r(UAG), is specifically recognized by the intermolecular hydrogen-bonding and aromatic stacking interactions. Importantly, the C-terminal region, which is disordered in the free form, took a certain conformation, resembling a helix. The observation of chemical shift perturbation and intermolecular NOEs, together with increases in the heteronuclear steady-state {1H}-15N NOE values on complex formation, indicated the involvement of the C-terminal region in RNA binding. On the basis of the two complex structures, we built a structural model of consecutive RBDs with r(UAGGUAG) containing both minimal recognition sequences, which resulted in no steric hindrance. The model suggests recognition of variable lengths (n) of the linker up to n = 50 may be possible.

Rare Earth pcu Metal–Organic Framework Platform Based on RE4(μ3-OH)4(COO)62+ Clusters: Rational Design, Directed Synthesis, and Deliberate Tuning of Excitation Wavelengths

The Td point group symmetry of rare earth (RE3+) metal clusters RE4(μ3-OH)4(COO)62+ makes them attractive building blocks for creating metal-organic frameworks (MOFs) with controllable topologies. Herein, we describe the design and synthesis of a series of isoreticular MOFs featuring pcu topology [MOF-1114(RE) and MOF-1115(RE)] with variable rare earth metal ions (RE3+ = Y3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, Yb3+) and linear amino-functionalized dicarboxylate linkers of different lengths. In total, we report 22 MOFs that vary in both composition and structure yet share the same RE4(μ3-OH)4 cluster motif. We demonstrate that these pcu MOFs are cationic and that anion exchange can be used to affect the MOF properties. We also investigate the luminescence properties of a representative member of this MOF series [MOF-1114(Yb)] that exhibits near-infrared emission. We show that the excitation energy for Yb3+ sensitization can be carefully adjusted to lower energy via covalent postsynthetic modification at the amino group sites within the MOF.

Sending Innate Immune Signals Across the Membrane: A Multiscale Simulation Approach to Toll-Like Receptor Assembly

Toll-like receptors (TLRs) are single transmembrane-spanning proteins that sense pathogenic molecular patterns within the innate immune system. Upon activation, TLRs form homodimers or heterodimers and initiate immune response pathways. The ability of TLRs to dimerize is therefore critical to their function in responding to invading pathogens. Crystallographic structures have been solved for the ectodomains of various TLR homodimers, but high-resolution structural information is not available for the full-length proteins, or for the transmembrane (TM) regions. We performed ab-initio modelling of the TM regions of all ten human TLRs, based on secondary structural predictions and spectroscopic data. Subsequently, coarse grained (CG) molecular dynamics (MD) simulations were performed to follow assembly and homo/hetero-dimerization within a phospholipid membrane environment. Our results have been used to evaluate the stability of TLR dimers, and to identify key sequence motifs that stabilize TM interactions, helping to rationalize in vitro data. Using acceptor photobleaching FRET on live cells, it has been demonstrated that TM domains including TLR2-TLR1 and TLR2-TLR6 interact within the plasma membrane. Additionally, multiscale models of entire TLR receptors have been built to determine the link between ligand recognition, assembly and cross-membrane/downstream signaling. In particular, we have focused on TLR4, which recognizes lipopolysaccharide (LPS) from the outer membranes of Gram-negative bacteria, for which a variety of structural/biophysical and MD data are available. Modelling of mutant constructs containing variable linkers revealed the structural basis for experimentally demonstrated tight coupling between extra- and intra-cellular domains and the TM region, based on receptor stability and dimerization efficiency. These data improve our understanding of the assembly and signalling mechanisms in TLRs, and may facilitate design of ligands with specific immunomodulatory properties, paving the way for new therapeutic treatments of inflammatory diseases.

Multi‐stage tandem mass spectrometric analysis of novel β‐cyclodextrin‐substituted and novel bis‐pyridinium gemini surfactants designed as nanomedical drug delivery agents

This study aimed at evaluating the collision-induced dissociation tandem mass spectrometric (CID-MS/MS) fragmentation patterns of novel β-cyclodextrin-substituted- and bis-pyridinium gemini surfactants currently being explored as nanomaterial drug delivery agents. In the β-cyclodextrin-substituted gemini surfactants, a β-cyclodextrin ring is grafted onto an N,N-bis(dimethylalkyl)-α,ω-aminoalkane-diammonium moiety using variable succinyl linkers. In contrast, the bis-pyridinium gemini surfactants are based on a 1,1'-(1,1'-(ethane-1,2-diylbis(sulfanediyl))bis(alkane-2,1-diyl))dipyridinium template, defined by two symmetrical N-alkylpyridinium parts connected through a fixed ethane dithiol spacer. Detection of the precursor ion [M](2+) species of the synthesized compounds and the determination of mass accuracies were conducted using a QqTOF-MS instrument. A multi-stage tandem MS analysis of the detected [M](2+) species was conducted using the QqQ-LIT-MS instrument. Both instruments were equipped with an electrospray ionization (ESI) source. Abundant precursor ion [M](2+) species were detected for all compounds at sub-1 ppm mass accuracies. The β-cyclodextrin-substituted compounds, fragmented via two main pathways: Pathway 1: the loss of one head-tail region produces a [M-(N(Me)2-R)](2+) ion, from which sugar moieties (Glc) are sequentially cleaved; Pathway 2: both head-tail regions are lost to give [M-2(N(Me)2-R)](+), followed by consecutive loss of Glc units. Alternatively, the cleavage of the Glc units could also have occurred simultaneously. Nevertheless, the fragmentation evolved around the quaternary ammonium cations, with characteristic cleavage of Glc moieties. For the bis-pyridinium gemini compounds, they either lost neutral pyridine(s) to give doubly charged ions (Pathway A) or formed complementary pyridinium alongside other singly charged ions (Pathway B). Similar to β-cyclodextrin-substituted compounds, the fragmentation was centered on the pyridinium functional groups. The MS(n) analyses of these novel gemini surfactants, reported here for the first time, revealed diagnostic ions for each compound, with a universal fragmentation pattern for each compound series. The diagnostic ions will be employed within liquid chromatography (LC)/MS/MS methods for screening, identification, and quantification of these compounds within biological samples.

Coconfinement of Fluorescent Proteins: Spatially Enforced Communication of GFP and mCherry Encapsulated within the P22 Capsid

The precise architectures of viruses and virus-like particles are proving to be highly advantageous in synthetic materials applications. Not only can these nanocontainers be harnessed as active materials, but they can be exploited for examining the effects of in vivo "cell-like" crowding and confinement on the properties of the encapsulated cargo. Here we report the first example of intermolecular communication between two proteins coencapsulated within the capsid architecture of the bacteriophage P22. Using a genetically engineered three-protein fusion between the P22 scaffold protein, and the FRET pair, GFP, and a red fluorescent protein (mCherry), we were able to direct the encapsulation of the genetic fusion when coexpressed with P22 coat protein. These self-assembled P22 capsids are densely packaged, occupying more than 24% of the available volume, and the molecular design assures a 1:1 ratio of the interacting proteins. To probe the effect of crowding and confinement on the FRET communication in this nanoenvironment, we spaced the donor-acceptor pair with variable length flexible linkers and examined the effect on FRET inside the capsid compared to the same tethered FRET pairs free in solution. The P22 system is unique in that the capsid morphology can be altered, without losing the encapsulated cargo, resulting in a doubling of the capsid volume. Thus, we have additionally examined the encapsulated fusions at two different internal concentrations. Our results indicate that FRET is sensitive to the expansion of the capsid and encapsulation enforces significant intermolecular communication, increasing FRET by 5-fold. This P22 coencapsulation system is a promising platform for studying crowding, enforced proximity, and confinement effects on communication between active proteins.

Sequence evolution of the intrinsically disordered and globular domains of a model viral oncoprotein.

In the present work, we have used the papillomavirus E7 oncoprotein to pursue structure-function and evolutionary studies that take into account intrinsic disorder and the conformational diversity of globular domains. The intrinsically disordered (E7N) and globular (E7C) domains of E7 show similar degrees of conservation and co-evolution. We found that E7N can be described in terms of conserved and coevolving linear motifs separated by variable linkers, while sequence evolution of E7C is compatible with the known homodimeric structure yet suggests other activities for the domain. Within E7N, inter-residue relationships such as residue co-evolution and restricted intermotif distances map functional coupling and co-occurrence of linear motifs that evolve in a coordinate manner. Within E7C, additional cysteine residues proximal to the zinc-binding site may allow redox regulation of E7 function. Moreover, we describe a conserved binding site for disordered domains on the surface of E7C and suggest a putative target linear motif. Both homodimerization and peptide binding activities of E7C are also present in the distantly related host PHD domains, showing that these two proteins share not only structural homology but also functional similarities, and strengthening the view that they evolved from a common ancestor. Finally, we integrate the multiple activities and conformations of E7 into a hierarchy of structure-function relationships.