Mechanism‐Dependent Modulation of Ultrafast Interfacial Water Dynamics in Intrinsically Disordered Protein Complexes

The recognition of intrinsically disordered proteins (IDPs) is highly dependent on dynamics owing to the lack of structure. Here we studied the interplay between dynamics and molecular recognition in IDPs with a combination of time‐resolving tools on timescales ranging from femtoseconds to nanoseconds. We interrogated conformational dynamics and surface water dynamics and its attenuation upon partner binding using two IDPs, IBB and Nup153FG, both of central relevance to the nucleocytoplasmic transport machinery. These proteins bind the same nuclear transport receptor (Importinβ) with drastically different binding mechanisms, coupled folding–binding and fuzzy complex formation, respectively. Solvent fluctuations in the dynamic interface of the Nup153FG‐Importinβ fuzzy complex were largely unperturbed and slightly accelerated relative to the unbound state. In the IBB‐Importinβ complex, on the other hand, substantial relative slowdown of water dynamics was seen in a more rigid interface. These results show a correlation between interfacial water dynamics and the plasticity of IDP complexes, implicating functional relevance for such differential modulation in cellular processes, including nuclear transport.     

Increasing the Chemical‐Shift Dispersion of Unstructured Proteins with a Covalent Lanthanide Shift Reagent

The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical‐shift assignments, and data analysis that relies on well‐separated resonances. We present a covalent paramagnetic lanthanide‐binding tag (LBT) for increasing the chemical‐shift dispersion and facilitating the chemical‐shift assignment of challenging, repeat‐containing IDPs. Linkage of the DOTA‐based LBT to a cysteine residue induces pseudo‐contact shifts (PCS) for resonances more than 20 residues from the spin‐labeling site. This leads to increased chemical‐shift dispersion and decreased signal overlap, thereby greatly facilitating chemical‐shift assignment. This approach is applicable to IDPs of varying sizes and complexity, and is particularly helpful for repeat‐containing IDPs and low‐complexity regions. This results in improved efficiency for IDP analysis and binding studies.     

The Dynamic Multisite Interactions between Two Intrinsically Disordered Proteins

Protein interactions involving intrinsically disordered proteins (IDPs) comprise a variety of binding modes, from the well‐characterized folding upon binding to dynamic fuzzy complexes. To date, most studies concern the binding of an IDP to a structured protein, while the interaction between two IDPs is poorly understood. In this study, NMR, smFRET, and molecular dynamics (MD) simulation are combined to characterize the interaction between two IDPs, the C‐terminal domain (CTD) of protein 4.1G and the nuclear mitotic apparatus (NuMA) protein. It is revealed that CTD and NuMA form a fuzzy complex with remaining structural disorder. Multiple binding sites on both proteins were identified by molecular dynamics and mutagenesis studies. This study provides an atomic scenario in which two IDPs bearing multiple binding sites interact with each other in dynamic equilibrium. The combined approach employed here could be widely applicable for investigating IDPs and their dynamic interactions.     

A free-energy landscape for coupled folding and binding of an intrinsically disordered protein in explicit solvent from detailed all-atom computations

The N-terminal repressor domain of neural restrictive silencer factor (NRSF) is an intrinsically disordered protein (IDP) that binds to the paired amphipathic helix (PAH) domain of mSin3. An NMR experiment revealed that the minimal binding unit of NRSF is a 15-residue segment that adopts a helical structure upon binding to a cleft of mSin3. We computed a free-energy landscape of this system by an enhanced conformational sampling method, all-atom multicanonical molecular dynamics. The simulation started from a configuration where the NRSF segment was fully disordered and distant from mSin3 in explicit solvent. In the absence of mSin3, the disordered NRSF segment thermally fluctuated between hairpins, helices, and bent structures. In the presence of mSin3, the segment bound to mSin3 by adopting the structures involved in the isolated state, and non-native and native complexes were formed. The free-energy landscape comprised three superclusters, and free-energy barriers separated the superclusters. The native complex was located at the center of the lowest free-energy cluster. When NRSF landed in the largest supercluster, the generated non-native complex moved on the landscape to fold into the native complex, by increasing the interfacial hydrophobic contacts and the helix content. When NRSF landed in other superclusters, the non-native complex overcame the free-energy barriers between the various segment orientations in the binding cleft of mSin3. Both population-shift and induced-fit (or induced-folding) mechanisms work cooperatively in the coupled folding and binding. The diverse structural adaptability of NRSF may be related to the hub properties of the IDP.     

Two‐Dimensional Crowding Uncovers a Hidden Conformation of α‐Synuclein

The intrinsically disordered protein (IDP), α‐synuclein (αS), is well‐known for phospholipid membrane binding‐coupled folding into tunable helical conformers. Here, using single‐molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction–folding landscape of αS can be tuned by two‐dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS–membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher‐affinity module remains membrane‐bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane‐binding IDPs with multiple binding modes.     

Design and Synthesis of Nucleoproline Amino Acids for the Straightforward Preparation of Chiral and Conformationally Constrained Nucleopeptides

A straightforward synthesis of orthogonally protected nucleoproline (Nup) amino acids and their coupling to oligomers are described. A key step is the attachment of alkynylated nucleobases to Fmoc‐protected 4‐azidoproline (Fmoc‐Azp‐OH) by a Cu‐catalyzed 1,3‐dipolar cycloaddition (‘click reaction’). The developed protocol allows preparation of the nucleoprolines in scales of >30 g. Solid‐phase peptide synthesis proved to be straightforward with these Nup amino acids. The resulting oligonucleoproline peptides adopt defined helices, are very well H₂O soluble, and show comparable cell‐penetrating properties as recently reported α‐nucleoalanine peptides.     

Compensatory Adaptations of Structural Dynamics in an Intrinsically Disordered Protein Complex

Intrinsically disordered proteins (IDPs) play crucial roles in protein interaction networks and in this context frequently constitute important hubs and interfaces. Here we show by a combination of NMR and EPR spectroscopy that the binding of the cytokine osteopontin (OPN) to its natural ligand, heparin, is accompanied by thermodynamically compensating structural adaptations. The core segment of OPN expands upon binding. This “unfolding‐upon‐binding” is governed primarily through electrostatic interactions between heparin and charged patches along the protein backbone and compensates for entropic penalties due to heparin–OPN binding. It is shown how structural unfolding compensates for entropic losses through ligand binding in IDPs and elucidates the interplay between structure and thermodynamics of rapid substrate‐binding and ‐release events in IDP interaction networks.     

Computational strategy for intrinsically disordered protein ligand design leads to the discovery of p53 transactivation domain I binding compounds that activate the p53 pathway

Intrinsically disordered proteins or intrinsically disordered regions (IDPs) have gained much attention in recent years due to their vital roles in biology and prevalence in various human diseases. Although IDPs are perceived as attractive therapeutic targets, rational drug design targeting IDPs remains challenging because of their conformational heterogeneity. Here, we propose a hierarchical computational strategy for IDP drug virtual screening (IDPDVS) and applied it in the discovery of p53 transactivation domain I (TAD1) binding compounds. IDPDVS starts from conformation sampling of the IDP target, then it combines stepwise conformational clustering with druggability evaluation to identify potential ligand binding pockets, followed by multiple docking screening runs and selection of compounds that can bind multi-conformations. p53 is an important tumor suppressor and restoration of its function provides an opportunity to inhibit cancer cell growth. TAD1 locates at the N-terminus of p53 and plays key roles in regulating p53 function. No compounds that directly bind to TAD1 have been reported due to its highly disordered structure. We successfully used IDPDVS to identify two compounds that bind p53 TAD1 and restore wild-type p53 function in cancer cells. Our study demonstrates that IDPDVS is an efficient strategy for IDP drug discovery and p53 TAD1 can be directly targeted by small molecules.

A hierarchical computational strategy for IDP drug virtual screening (IDPDVS) was proposed and successfully applied to identify compounds that bind p53 TAD1 and restore wild-type p53 function in cancer cells.     

Time Window Expansion for HDX Analysis of an Intrinsically Disordered Protein

Application of typical HDX methods to examine intrinsically disordered proteins (IDP), proteins that are natively unstructured and highly dynamic at physiological pH, is limited because of the rapid exchange of unprotected amide hydrogens with solvent. The exchange rates of these fast exchanging amides are usually faster than the shortest time scale (10 s) employed in typical automated HDX-MS experiments. Considering the functional importance of IDPs and their association with many diseases, it is valuable to develop methods that allow the study of solution dynamics of these proteins as well as the ability to probe the interaction of IDPs with their wide range of binding partners. Here, we report the application of time window expansion to the millisecond range by altering the on-exchange pH of the HDX experiment to study a well-characterized IDP; the activation domain of the nuclear receptor coactivator, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α). This method enabled mapping the regions of PGC-1α that are stabilized upon binding the ligand binding domain (LBD) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). We further demonstrate the method’s applicability to other binding partners of the IDP PGC-1α and pave the way for characterizing many other biologically important ID proteins.

Tuning Riboswitch‐Mediated Gene Regulation by Rational Control of Aptamer Ligand Binding Properties

Riboswitch‐mediated control of gene expression depends on ligand binding properties (kinetics and affinity) of its aptamer domain. A detailed analysis of interior regions of the aptamer, which affect the ligand binding properties, is important for both understanding natural riboswitch functions and for enabling rational design of tuneable artificial riboswitches. Kinetic analyses of binding reaction between flavin mononucleotide (FMN) and several natural and mutant aptamer domains of FMN‐specific riboswitches were performed. The strong dependence of the dissociation rate (52.6‐fold) and affinity (100‐fold) on the identities of base pairs in the aptamer stem suggested that the stem region, which is conserved in length but variable in base‐pair composition and context, is the tuning region of the FMN‐specific aptamer. Synthetic riboswitches were constructed based on the same aptamer domain by rationally modifying the tuning regions. The observed 9.31‐fold difference in the half‐maximal effective concentration (EC₅₀) corresponded to a 11.6‐fold difference in the dissociation constant (K_D) of the aptamer domains and suggested that the gene expression can be controlled by rationally adjusting the tuning regions.     

Fluorescence Dynamics of Monocyclodextrin– and Bis(thiol‐cyclodextrin)–Coumarin C153 Complexes

The solvation and confinement of coumarin C153 within supramolecular host/guest complexes based on β‐cyclodextrin (β‐CD) and 6‐deoxy‐6‐thio‐β‐cyclodextrin (β‐CD‐SH) in water are studied by fluorescence spectroscopy. For β‐CD/C153, the 1:1 complex is proposed, and for β‐CD‐SH/C153 both the 1:1 and 2:1 complexes are believed to be formed. The 2:1 β‐CD‐SH/C153 complex has an association constant of 4.2×10⁵ M ^?1 and a C153 population of 82 %, which are interestingly high values, indicating that the proposed β‐CD‐SH dimers structure are connected by covalent disulfide bonds; this is supported by mass spectrometry. Solvation related to fast hydrogen‐bond rearrangement as a part of fluorescence relaxation is determined by the ultrafast components of time‐resolved spectroscopy to be 3 and 7 ps for the 1:1 β‐CD/C153 and 2:1 β‐CD‐SH/C153 complexes, respectively.     

A novel ionic‐conduction mechanism based on polyurethane electrolyte

A series of poly(ethylene glycol)–polyurethane (PEG–PU)/sodium perchlorate (NaClO₄) solid electrolytes were prepared, and their properties were characterized with Fourier transform infrared spectroscopy, differential scanning calorimetry, complex impedance analysis, and atomic force microscopy. Results showed that the oxygen atoms of carbonyl and ether oxygen groups had different activities on cations. Both carbonyl and ether oxygen groups participated in the ionic‐transport process in PU‐based electrolytes. There existed a coordination competition between sodium cations and different oxygen atoms in soft and hard segments of PU. For the PEG–PU/NaClO₄ system investigated, amorphous regions and interfacial regions between the amorphous and microcrystalline phases were responsible for ionic conduction. A new ionic‐transport mechanism, based on the existence of conduction pathways not only in amorphous regions but also in interfacial regions of microphase‐separated PU‐based electrolytes, is sketched. Moreover, at a particular concentration of doped salt (EO/NaClO₄ 12), the PEG–PU/NaClO₄ complex revealed a phase‐transition point in the morphology and exhibited minimum apparent activation energy and maximum ionic conductivity. © 2001 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 39: 1246–1254, 2001     

Synthesis,Crystal Structure,DNA‐binding Properties and Antioxidant Activity of a Copper(II) Complex with Naphthalimide Schiff Base

The Schiff N‐allylamine‐4‐(ethylenediamine‐5‐methylsalicylidene)‐1,8‐naphthalimide (H₂L) and its copper(II) complex, [Cu(HL)₂] · 0.5DMF, were synthesized and characterized. The crystal structure of the Cu^II complex reveals a slightly distorted square‐planar arrangement provided by two N and O donors from two deprotonated ligands. In addition, the DNA‐binding properties of the ligand and Cu^II complex were investigated by fluorescence spectra, electronic absorption, and viscosity measurements. The experimental studies of the DNA‐binding properties indicated that the ligand and Cu^II complex reacted with DNA via intercalation binding mode, and binding affinity for DNA takes the order: ligand > Cu^II complex. The antioxidant assay in vitro suggested that both exhibited potential intensely antioxidant properties, and the ligand is more effective than its Cu^II complex.     

DFT/TDDFT Investigation of the Modulation of Photochromic Properties in an Organoboron‐Based Diarylethene by Fluoride Ions

The diarylethene derivative 1,2‐bis‐(5′‐dimesitylboryl‐2′‐methylthieny‐3′‐yl)‐cyclopentene ( 1 ) containing dimesitylboryl groups is an interesting photochromic material. The dimesitylboryl groups can bind to F^?, which tunes the optical and electronic properties of the diarylethene compound. Hence, the diarylethene derivative 1 containing dimesitylboryl groups is sensitive to both light and F^?, and its photochromic properties can be tuned by a fluoride ion. Herein, we studied the substituent effect of dimesitylboron groups on the optical properties of both the closed‐ring and open‐ring isomers of the diarylethene molecule by DFT/TDDFT calculations and found that these methods are reliable for the determination of the lowest singlet excitation energies of diarylethene compounds. The introduction of dimesitylboron groups to the diarylethene compound can elongate its conjugation length and change the excited‐state properties from π→π* transition to a charge‐transfer state. This explains the modulation of photochromic properties through the introduction of dimesitylboron groups. Furthermore, the photochromic properties can be tuned through the binding of F^? to a boron center and the excited state of the diarylethene compound is changed from a charge‐transfer state to a π→π* transition. Hence, a subtle control of the photochromic spectroscopic properties was realized. In addition, the changes of electronic characteristics by the isomerization reaction of diarylethene compounds were also investigated with theoretical calculations. For the model compound 2 without dimesitylboryl groups, the closed‐ring isomer has better hole‐ and electron‐injection abilities, as well as higher charge‐transport rates, than the open‐ring isomer. The introduction of dimesitylboron groups to diarylethene can dramatically improve the charge‐injection and ‐transport abilities. The closed isomer of compound 1 ( 1 C ) has the best hole‐ and electron‐injection abilities, whereas the charge‐transport rates of the open isomer of compound 1 ( 1 O ) are higher than those of 1 C . Importantly, 1 O is an electron‐accepting and ‐transport material. These results show that the diarylethene compound containing dimesitylboryl groups has promising potential to be applied in optoelectronic devices and thus is worth to be further investigated.     

Binding of Organic Dyes with Human Serum Albumin: A Single‐Molecule Study

Kinetics of binding of dyes at different sites of human serum albumin (HSA) has been studied by single‐molecule spectroscopy. The protein was immobilized on a glass surface. To probe different binding sites (hydrophobic and hydrophilic) two dyes, coumarin 153 ( C153 , neutral) and rhodamine 6G ( R6G , cationic) were chosen. For both the dyes, a major (ca. 96‐98 %) and minor (ca. 3 %) binding site were detected. Rate constants of association and dissociation were simultaneously determined from directly measuring fluctuations in fluorescence intensity (τ_off and τ_on) and from this the equilibrium (binding) constants were calculated. Fluorescence lifetimes at individual sites were obtained from burst‐integrated lifetime analysis. Distributions of lifetime histograms for both the probes ( C153 and R6G ) exhibit two maxima, which indicates the presence of two binding domains in the protein. Unfolding of the protein has been studied by adding guanidinium hydrochloride (GdnHCl) to the solution. It is observed that addition of GdnHCl affects the dissociation and association kinetics and hence, binding equilibrium of the association of C153 . However, the effect of binding of R6G is not affected much. It is proposed that GdnHCl affects the hydrophobic binding sites more than the hydrophilic site.     

Contrasting Response of Two Dipolar Fluorescence Probes in a Leucine‐Based Organogel and Its Implications

The microenvironments of a leucine‐based organogel are probed by monitoring the fluorescence behavior of coumarin 153 (C153) and 4‐aminophthalimide (AP). The steady‐state data reveals distinctly different locations of the two molecules in the gel. Whereas AP resides close to the hydroxyl moieties of the gelator and engages in hydrogen‐bonding interactions, C153 is found in bulk‐toluene‐like regions. In contrast to C153, AP exhibits excitation‐wavelength‐dependent emission, indicating that the environments of the hydrogen‐bonded AP molecules are not all identical. A two‐component fluorescence decay of AP in gel, unlike C153, supports this model. A time‐resolved fluorescence anisotropy study of the rotational motion of the molecules also reveals the strong association of only AP with the gelator. That AP influences the critical gelation concentration implies its direct involvement in the gel‐formation process. The results highlight the importance of guest–gelator interactions in gels containing guest molecules.     

Conformational Control of Organocatalyst in Strongly Brønsted-Acidic Metal-Organic Frameworks for Enantioselective Catalysis

Chiral imidodiphosphates (IDPs) have emerged as strong Brønsted acid catalysts for many enantioselective processes. However, the dynamic transformation between O,O-syn and O,O-anti conformers typically results in low enantioselectivity. Here we demonstrate that topologies of metal-organic frameworks (MOFs) can be exploited to control IDP conformations and local chiral microenvironments for enantioselective catalysis. Two porous Dy-MOFs with different topologies are obtained from an enantiopure 1,1′-biphenol IDP-based tetracarboxylate ligand. While the ligand adopts a 4- or 3-connected (c) binding mode, all IDPs are rigidified to get only a single O,O-syn conformation and display greatly enhanced Brønsted acidity relative to the free IDP. The MOF with the 4-c IDP that has a relatively less compact shape than the 3-c IDP can be an efficient and recyclable heterogeneous Brønsted acid catalysing the challenging asymmetric O,O-acetalization reaction with up to 96 % enantiomeric excess.     

Studies on the Interaction of Dinitratobis（phen） Cadmium Complex with DNA

DNA-binding properties of the dinitratobis（phen） cadmium complex [Cd（phen）2（NO3）2] （where phen = 1,10-phenanthroline） have been investigated with absorption titration, fluorescence spectroscopy, viscosity measurement, molecular modeling and density functional theory （DFT） calculations. The results indictate DNA-binding mode of the complex to be weak groove binding rather than partial intercalative interaction expected of the extended planar aromatic phen ring. In addition, the DNA cleavage study was carried out by gel electrophoresis experiment. The results showed that the complex both hardly cleaves pBR322 DNA in the absence and present ascorbate. So it is suggested that the formation of cadmium complex can decrease cadmium toxicity to some extents.     

Reversible Oxygen Binding to the Polymeric Cobalt Tetraazaporphyrin Complex and Oxygen‐Facilitated Transport through Its Membrane

Summary: A membrane of a cobalt tetraazaporphyrin polymer complex was prepared with a nanometer thickness and used as an oxygen‐facilitated transport membrane. Rapid and reversible oxygen binding to the cobalt tetraazaporphyrin complex with a polymeric imidazole ligand was observed at low temperature. Oxygen transport through the membrane was facilitated and a high (oxygen/nitrogen) permselectivity of 28 was obtained.

Oxygen‐facilitated transport through a cobalt tetraazaporphyrin complex‐polymer membrane of nanometer thickness.