Chemical Perturbation of Oncogenic Protein Folding: from the Prediction of Locally Unstable Structures to the Design of Disruptors of Hsp90–Client Interactions

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein-90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co-chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c-Abl, c-Src, Cdk4, B-Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90-system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein–protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation.     

Conjugated‐Protein Mimics with Molecularly Imprinted Reconstructible and Transformable Regions that are Assembled Using Space‐Filling Prosthetic Groups

Conjugated‐protein mimics were obtained using a new molecular imprinting strategy combined with post‐imprinting modifications. An antibiotic was employed as a model template molecule, and a polymerizable template molecule was designed, which was composed of the antibiotic and two different prosthetic groups attached through a disulfide bond and Schiff base formation. After co‐polymerization with a cross‐linker, the template molecule was removed together with the prosthetic groups, yielding the apo‐type scaffold. Through conjugation of the two different prosthetic groups at pre‐determined positions within the apo‐type scaffold, the apo cavity was transformed into a functionalized holo cavity, which enables the on/off switching of the molecular recognition ability, signal transduction activity for binding events, and photoresponsive activity.     

Designing Molecular Spanners to Throw in the Protein Networks

Proteins govern most aspects of cellular life and, through specific interfaces, are typically involved in intricate protein–protein interaction (PPI) networks and signaling pathways. Subtle up- or downregulation of key protein functions and PPIs results in disease; still, the preferred option to contrast the role of a protein in disease and healthy conditions alike remains its outright shutdown through orthosteric ligands that block its active site. Here, we explore subtler alternatives to modulate proteins and PPIs. Driven by a view of proteins as dynamic entities, we discuss ways to identify allosteric binding sites, which, when targeted by tailored ligands, can induce significant changes in the active site of a protein, and lead to agonistic or antagonistic effects. We also summarize the selective regulation of specific PPIs—either direct or allosteric—and show that effects can be stabilizing as well as destabilizing, depending on how the conformational equilibrium of a protein is shifted.     

A New Colorimetric Platform for Protein Detection Based on Recognition‐Induced Cascade of Polymeric Nanoparticles Disassembly

Despite great progress, it is still of high interest to explore new homogeneous assays for simple, visual, and selective protein detection. Herein, one new colorimetric sensor has been developed for visual detection of protein by using polymeric micelles as a sensing scaffold and the molecular recognition between protein and the ligand on the surface of the polymeric micelles as the driving force to trigger the readout of the detection signal. The polymeric micelles formed via the self‐assembly of the amphiphilic block polymer biotin‐labeled poly(ethylene glycol)‐block‐poly(3‐acryl aminophenylboronic acid) are endowed with colorful feature by incorporation of alizarin red S (ARS) into the hydrophobic core. Based on the response to streptavidin recognition, these micelles are further disintegrated through the competitive binding of α‐cyclodextrin with boronic acid for disassociation of ARS, which achieves orange–yellow to pink–purple transition in 2 h. This work will open the way to develop one new mix‐and‐measure, visual, and homogeneous assay.     

Surface glycosylation of polymeric membranes

Surface glycosylation of polymeric membranes has been inspired by the structure of natural biomem-branes. It refers to that glycosyl groups are introduced onto the membrane surface by various strate-gies, which combine the separation function of the membrane with the biological function of the sac-charides in one system. In this review, progress in the surface glycosylation of polymeric membranes is highlighted in two aspects, i.e. the glycosylation methods and the potential applications of the sur-face-glycosylated membranes.     

蛋白表面分子印迹技术

蛋白印迹材料在生物分离、生物传感和医用生物材料等领域具有很强的应用价值并受到广泛关注。尽管小分子印迹技术已经成功应用于很多领域,但蛋白分子印迹仍然是挑战性研究课题。本文总结了蛋白表面印迹技术领域的研究进展,根据不同的蛋白表面印迹材料,详细叙述了蛋白表面分子印迹薄膜、核壳微球、纳米线、凝胶微粒、单层膜等印迹材料的制备过程、印迹方法和选择识别性能,讨论了蛋白表面分子印迹方法的优缺点,阐明了蛋白分子印迹未来发展的方向。     

Affinity Polymers Tailored for the Protein A Binding Site of Immunoglobulin G Proteins

Rational design in combination with a screening process was used to develop affinity polymers for a specific binding site on the surface of immunoglobulin G (IgG) proteins. The concept starts with the identification of critical amino acid residues on the protein interface and their topological arrangement. Appropriate binding monomers were subsequently synthesized. Together with a sugar monomer (2–5 equiv) for water solubility and a dansyl monomer (0.5 equiv) as a fluorescent label, they were subjected in aqueous solution to linear radical copolymerization in various compositions (e.g., azobisisobutyronitrile (AIBN), homogeneous water/DMF mixtures). After ultrafiltration and lyophilization, colorless dry water‐soluble powders were obtained. NMR spectroscopic and gel permeation chromatography (GPC) characterization indicated molecular weights between 30 and 500 kD and confirmed retention of monomer composition as well as the absence of monomers. In a competitive enzyme‐linked immunosorbent assay (ELISA) screen of the polymer libraries (20–50 members), few copolymers qualified as strong and selective binders for the protein A binding site on the Fc fragment of the antibody. Their monomer composition precisely reflected the critical amino acids found at the interface. The simple combination of a charged and a nonpolar binding monomer sufficed for selective submicromolar IgG recognition by the synthetic polymer. Affinities were confirmed by fluorescence titrations; they increased with decreasing salt load but remained largely unaltered at lowered pH. Other proteins, including those of similar size and isoelectric point (pI), were bound 10–1000 times less tightly. This example indicates that interaction domains in other proteins may also be targeted by synthetic polymers if their comonomer composition reflects the nature and arrangement of amino acid residues on the protein surface.     

基于靶向多肽探针的蛋白质分析检测新方法

高选择性、高灵敏度的蛋白质分析检测方法是复杂生命体系研究的有力工具,能为疾病的发现、治疗以及分子机制研究提供新的、有价值的思路和手段.该文围绕蛋白质与多肽的分子识别,针对疾病相关多肽、蛋白质的分离、分析与检测新方法,综述了近几年取得的研究进展,可为复杂生命体系中生理活性分子的结构与功能研究提供借鉴.     

Proteo-dendrimers designed for complementary recognition of cytochrome c: dendrimer architecture toward nanoscale protein complexation

"Proteo-dendrimers" in which polyanionic hepta(glutamic acids), fluorescent zinc porphyrinate cores, hydrophilic polyether surfaces, and nonpeptide hydrophobic dendrons are combined, were developed as a new series of synthetic receptors for protein recognition. They have polyanionic "patch" structures on their surfaces and undergo complementary electrostatic interactions with a positively charged cytochrome c patch, as observed in biological protein-protein recognition systems. Stability constants of the resulting supramolecular complexes were determined in phosphate buffer (pH 7) by monitoring the fluorescence quenching of the zinc porphyrinates. These proteo-dendrimer receptors exhibited higher affinities with cytochrome c proteins in aqueous solutions than with biological cytochrome b5. Furthermore, they effectively blocked complexation of biological cytochrome b5 with cytochrome c, indicating that the proteo-dendrimers and cytochrome b5 similarly occupy the polycationic patch of cytochrome c.     

Transglutaminase surface recognition by peptidocalix[4]arene diversomers

A series of N-linked tetrakis(tetrapeptido)calix[4]arene diversomers, 3A-P, has been synthesized by coupling of a cone calix[4]arene tetracarboxylic acid chloride with tetrapeptides 1A-P obtained in a parallel fashion. The inhibition activity of 3A-P towards tissue and microbial transglutaminase was evaluated by in vitro assays with a labeled substrate. Kinetic analysis using one of the most active derivatives (3A) showed a noncompetitive inhibition with respect to the amino acceptor substrate and an uncompetitive inhibition with respect to amino donor substrate. Experimental results are in accordance with an inhibition due to a protein specific surface recognition on a region noncomprising the enzyme active site.     

Protein sequence threading,the alignment problem,and a two-step strategy

Conventionally, protein structure prediction via “threading” relies on some nonoptimal method to align a protein sequence to each member of a library of known structures. We show how a score function (force field) can be modified so as to allow the direct application of a dynamic programming algorithm to the problem. This involves an approximation whose damage can be minimized by an optimization process during score function parameter determination. The method is compared to sequence to structure alignments using a more conventional pair-wise score function and the frozen approximation. The new method produces results comparable to the frozen approximation, but is faster and has fewer adjustable parameters. It is also free of memory of the template's original amino acid sequence, and does not suffer from a problem of nonconvergence, which can be shown to occur with the frozen approximation. Alignments generated by the simplified score function can then be ranked using a second score function with the approximations removed. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 1455–1467, 1999     

Common Patterns in Chaperone Interactions with a Native Client Protein

Many molecular chaperones are promiscuous and interact with a wide range of unfolded, quasi‐native, and native client proteins. The mechanisms by which chaperones interact with the highly diverse structures of native clients thus remain puzzling. In this work, we investigate at the atomic level how three ATP‐independent chaperones interact with a β‐sheet‐rich protein, the Fyn SH3 domain. The results reveal that the chaperone Spy recognizes the locally frustrated surface of the client Fyn SH3 and that the interaction is transient and highly dynamic, leaving the chaperone‐interacting surface on Fyn SH3 solvent accessible. The two alternative molecular chaperones SurA and Skp recognize the same locally frustrated surface of the Fyn SH3 domain. These results indicate dynamic recognition of frustrated segments as a common mechanism underlying the chaperone–native client interaction, which also provides a basis for chaperone promiscuousness.     

Recognition of proline-rich motifs by protein-protein-interaction domains

Protein-protein interactions are essential in every aspect of cellular activity. Multiprotein complexes form and dissociate constantly in a specifically tuned manner, often by conserved mechanisms. Protein domains that bind proline-rich motifs (PRMs) are frequently involved in signaling events. The unique properties of proline provide a mechanism for highly discriminatory recognition without requiring high affinities. We present herein a detailed, quantitative assessment of the structural features that define the interfaces between PRM-binding domains and their target PRMs, and investigate the specificity of PRM recognition. Together with the analysis of peptide-library screens, this approach has allowed the identification of several highly conserved key interactions found in all complexes of PRM-binding domains. The inhibition of protein-protein interactions by using small-molecule agents is very challenging. Therefore, it is important to first pinpoint the critical interactions that must be considered in the design of inhibitors of PRM-binding domains.     

稀土离子对钙调蛋白与单克隆抗体分子识别的影响

分别采用酶联免疫吸附(ELISA)法和荧光标记技术比较了Ca²⁺,La³⁺,Eu³⁺和Yb³⁺离子对钙调蛋白与单克隆抗体2C3之间分子识别的影响.结果表明,金属离子与钙调蛋白作用后会诱导其发生不同的构象变化,并进一步影响到钙调蛋白与单克隆抗体2C3分子之间的结合强度.当钙调蛋白分别与La³⁺,Eu³⁺,Yb³⁺作用后,它与单抗2C3分子之间的解离常数为(26.8±2.5),(21.8±3.4)和(64.8±5.1)nmol/L,而结合Ca²⁺前后的钙调蛋白与单抗分子的解离常数分别为(177.2±2.8)和(157±4.2)nmol/L.这一结果表明,稀土离子诱导钙调蛋白发生的构象变化明显不同于钙离子的作用,这种差异可能是稀土与钙离子对钙调蛋白调控作用表现出差别的原因.     

Two-dimensional electrophoresis analysis of proteomics based on image feature and mathematical morphology

Protein analysis techniques, including 2-D electro- phoresis, image analysis, biological mass spectrometry, database search, etc., are fundamental technologies of proteomics, a front area in biochemistry and life sci- ences[1―3]. Among them image analysi…