模型多肽Trp-Cage折叠形成机制的研究进展

蛋白质折叠是目前结构生物学领域的核心问题之一, 理解蛋白质结构折叠机制及其与生物功能之间的相互关系一直是生命科学家非常重要的研究内容, 并且该研究受到越来越多不同学科领域研究工作者的高度重视. 蛋白质大多数在数十毫秒、微秒或几秒内完成自我折叠过程, 但其折叠过程中所发生的分子结构精细转变却在纳秒甚至更短时间尺度内完成. 由于其折叠时间分辨率的限制, 目前无论是从常规实验还是理论计算角度对其研究都存在一定的难度. 本文首先概述了蛋白质折叠研究在实验和理论模拟方面存在的一些问题,然后以结构典型且可快速折叠的人工设计多肽Trp-cage为例,主要对其折叠过渡温度、折叠形成模型及其肽链上关键氨基酸残基在折叠过程中的作用三个方面进行了详细讨论, 综述了模型多肽Trp-cage的折叠动力学行为分别在实验和理论模拟方面的研究进展. 最后就如何有效化解蛋白质残基间相互作用网络进而降低其折叠机制的复杂性提出了一些新的建议, 不仅有助于阐明该迷你蛋白Trp-cage快速折叠、稳定形成的驱动力成因, 而且也能为蛋白质折叠机制研究和多肽设计提供有益参考.     

Forward chemical genetics: library scaffold design

With the unraveling of the entire human genome, it has become imperative to understand the function of the gene products, proteins. Within the past several years, chemical genetics has gained recognition as a powerful approach to study protein function by using small molecules as gene knock-out or knock-in mimics. Forward chemical genetics is a three-step process; the design and synthesis of a small molecule library represents the first step followed secondly by the search for novel phenotypes and then by isolation and identification of target protein(s). This review will focus on the first step, the design of the scaffold for small molecule libraries. It will also examine the connection between the choice of a scaffold and the propensity of that library to demonstrate enhanced biological activity when tested in certain cellular systems.     

Identification of small molecule binding sites within proteins using phage display technology

Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.     

Mapping protein pockets through their potential small-molecule binding volumes: QSCD applied to biological protein structures

Previously we demonstrated a method, Quantized Surface Complementarity Diversity (QSCD), of defining molecular diversity by measuring shape and functional complementarity of molecules to a basis set of theoretical target surfaces [Wintner E.A. and Moallemi C.C., J. Med. Chem., 43 (2000) 1993]. In this paper we demonstrate a method of mapping actual protein pockets to the same basis set of theoretical target surfaces, thereby allowing categorization of protein pockets by their properties of shape and functionality. The key step in the mapping is a `dissection' algorithm that breaks any protein pocket into a set of potential small molecule binding volumes. It is these binding volumes that are mapped to the basis set of theoretical target surfaces, thus measuring a protein pocket not as a single surface but as a collection of molecular recognition environments.     

Spatial control of protein binding on lipid bimembrane using photoeliminative linker

Protein adsorption and dissociation on cell membrane surfaces is a topic of important study to reveal biological processes including signal transduction and protein trafficking. We demonstrated here the establishment of a mimic model system for the spatial control of protein adsorption/elimination on a lipid bimembrane using a photochemical technique. The novel photoeliminative linker that we synthesized here consists of three distinct components: a substrate (biotin), a photoeliminative group (4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid), and a lipid bimembrane-adsorbent group (farnesyl). The photoeliminative linker was inserted on the entire surface of the lipid bimembrane and two-dimensionally eliminated by spatial UV irradiation onto the membrane to create a biotin pattern. A target protein, streptavidin was selectively immobilized on the patterned biotin, although it was almost not attached on the nonirradiated region. The streptavidin array was selectively dissociated by UV irradiation onto the entire membrane.     

Enhanced selectivity for inhibition of analog-sensitive protein kinases through scaffold optimization

The ability to inhibit any protein kinase of interest with a small molecule is enabled by a combination of genetics and chemistry. Genetics is used to modify the active site of a single kinase to render it distinct from all naturally occurring kinases. Next, organic synthesis is used to develop a small molecule, which does not bind to wild-type kinases but is a potent inhibitor of the engineered kinase. This approach, termed chemical genetics, has been used to generate highly potent mutant kinase-specific inhibitors based on a pyrazolopyrimidine scaffold. Here, we asked if the selectivity of the resulting pyrazolopyrimidines could be improved, as they inhibit several wild-type kinases with low-micromolar IC₅₀ values. Our approach to improve the selectivity of allele-specific inhibitors was to explore a second kinase inhibitor scaffold. A series of 6,9-disubstituted purines was designed, synthesized, and evaluated for inhibitory activity against several kinases in vitro and in vivo. Several purines proved to be potent inhibitors against the analog-sensitive kinases and exhibited greater selectivity than the existing pyrazolopyrimidines.     

Synthesis of a phosphoserine mimetic prodrug with potent 14-3-3 protein inhibitory activity

Many protein-protein interactions in cells are mediated by functional domains that recognize and bind to motifs containing phosphorylated serine and threonine residues. To create small molecules that inhibit such interactions, we developed methodology for the synthesis of a prodrug that generates a phosphoserine peptidomimetic in cells. For this study, we synthesized a small molecule inhibitor of 14-3-3 proteins that incorporates a nonhydrolyzable difluoromethylenephosphoserine prodrug moiety. The prodrug is cytotoxic at low micromolar concentrations when applied to cancer cells and induces caspase activation resulting in apoptosis. The prodrug reverses the 14-3-3-mediated inhibition of FOXO3a resulting from its phosphorylation by Akt1 in a concentration-dependent manner that correlates well with its ability to inhibit cell growth. This methodology can be applied to target a variety of proteins containing phosphoserine and other phosphoamino acid binding domains.     

XPS and AFM characterization of the enzyme glucose oxidase immobilized on SiO(2) surfaces

A process to immobilize the enzyme glucose oxidase on SiO2 surfaces for the realization of integrated microbiosensors was developed. The sample characterization was performed by monitoring, step by step, oxide activation, silanization, linker molecule (glutaraldehyde) deposition, and enzyme immobilization by means of XPS, AFM, and contact angle measurements. The control of the environment during the procedure, to prevent silane polymerization, and the use of oxide activation to obtain a uniform enzyme layer are issues of crucial importance. The correct protocol application gives a uniform layer of the linker molecule and the maximum sample surface coverage. This result is fundamental for maximizing the enzyme bonding sites on the sample surface and achieving the maximum surface coverage. Thin SiO2 layers thermally grown on a Si substrate were used. The XPS Si 2p signal of the substrate was monitored during immobilization. Such a signal is not completely shielded by the thin oxide layer and it is fully suppressed after the completion of the whole protocol. A power spectral density analysis on the AFM measurements showed the crucial role of both the oxide activation and the intermediate steps (silanization and linker molecule deposition) to obtain uniform immobilized enzyme coverage. Finally, enzymatic activity measurements confirmed the suitability of the optimized protocol.     

Miniature homeodomains: high specificity without an N-terminal arm

Recently, we described a strategy for the design of miniature proteins that bind DNA and protein surfaces with high affinity and selectivity. This strategy involves identifying the functional epitope required for macromolecular recognition by a natural protein and presenting it on a small, stable protein scaffold. In previous work, high-affinity DNA recognition was achieved only when the miniature protein contained the complete functional epitope. Here we report a miniature homeodomain that recognizes its 6-bp target site in the nanomolar concentration range at 25 degrees C, despite the absence of DNA contact residues located along the homeodomain N-terminal arm. We conclude that miniature proteins can achieve high affinity and selectivity for DNA by design even when the functional epitope is incomplete by using pre-organization to effectively compensate for lost protein-DNA contacts. In this case it has been possible to miniaturize both the recognition surface and the structural framework of a globular protein fold.     

A new strategy for preparing molecular imaging and therapy agents using fluorine-rich (fluorous) soluble supports

A convenient new strategy for producing radiolabeled compounds in high effective specific activity was developed using soluble fluorous supports. The reported methodology involves a fluorous linker group that is released from the substrate of interest upon reaction with radioiodine. The desired product can then be selectively separated from unreacted starting material and reaction byproducts using a simple fluorous solid-phase extraction procedure. The utility of this approach was demonstrated by labeling a series of benzoic acid derivatives which are commonly used to prepare molecular imaging agents. All compounds were produced in high radiochemical yields, purities, and effective specific activities. The strategy was further elaborated in that it was used to prepare a small collection of radiolabeled benzamides as a way of demonstrating the potential utility of this method for creating libraries of molecular imaging agents.     

Second-generation DNA-encoded multiple display on a constant macrocyclic scaffold enabled by an orthogonal protecting group strategy

DNA-encoded chemical library(DEL) represents an emerging drug discovery technology to construct compound libraries with abundant chemical combinations. While drug-like small molecule DELs facilitate the discovery of binders against targets with defined pockets, macrocyclic DELs harboring extended scaffolds enable targeting of the protein–protein interaction(PPI) interface. We previously demonstrated the design of the first-generation DNA-encoded multiple display based on a constant macrocyclic s...     

Do benzodiazepines mimic reverse-turn structures?

The role of benzodiazepine derivatives (BZD) as a privileged scaffold that mimics beta-turn structures (Ripka et al. (1993) Tetrahedron 49:3593-3608) in peptide/protein recognition was reexamined in detail. Stable BZD ring conformers were determined with MM3, and experimental reverse-turn structures were extracted from the basis set of protein crystal structures previously defined by Ripka et al. Ideal beta-turns were also modeled and similarly compared with BZD conformers. Huge numbers of conformers were generated by systematically scanning the torsional degrees of freedom for BZDs, as well as those of ideal beta-turns for comparison. Using these structures, conformers of BZDs were fit to experimental structures as suggested by Ripka et al., or modeled classical beta-turn conformers, and the root-mean-square deviation (RMSD) values were calculated for each pairwise comparison. Pairs of conformers with the smallest RMSD values for overlap of the four alpha-beta side-chain orientations were selected. All overlaps of BZD conformers with experimental beta-turns yielded one or more comparisons where the least RMSD was significantly small, 0.48-0.86 angstroms, as previously suggested. Utilizing a different methodology, the overall conclusion that benzodiazepines could serve as reverse-turn mimetics of Ripka et al. is justified. The least RMSD values for the overlap of BZDs and modeled classical beta-turns were also less than 1 angstrom. When comparing BZDs with experimental or classical beta-turns, the set of experimental beta-turns selected by Ripka et al. fit the BZD scaffolds better than modeled classical beta-turns; however, all the experimental beta-turns did not fit a particular BZD scaffold better. A single BZD ring conformation, and/or chiral orientation, can mimic some, but not all, of the experimental beta-turn structures. BZD has two central ring conformations and one chiral center that explains why the four variations of the BZD scaffold can mimic all types of beta-turn structure examined. It was found, moreover, that the BZD scaffold also mimics each of the nine clusters of experimental orientations of side chains of reverse turns in the Protein Data Bank, when the new classification scheme for the four side-chain directions (the relative orientations of alpha-beta vectors of residues i through i+3) was considered (Tran et al. (2005) J Comput-Aided Mol Des 19:551-566).     

A modular system for the synthesis of multiplexed magnetic resonance probes

We have developed a modular architecture for preparing high-relaxivity multiplexed probes utilizing click chemistry. Our system incorporates azide bearing Gd(III) chelates and a trialkyne scaffold with a functional group for subsequent modification. In optimizing the relaxivity of this new complex, we undertook a study of the linker length between a chelate and the scaffold to determine its effect on relaxivity. The results show a strong dependence on flexibility between the individual chelates and the scaffold with decreasing linker length leading to significant increases in relaxivity. Nuclear magnetic resonance dispersion (NMRD) spectra were obtained to confirm a 10-fold increase in the rotational correlation time from 0.049 to 0.60 ns at 310 K. We have additionally obtained a crystal structure demonstrating that modification with an azide does not impact the coordination of the lanthanide. The resulting multinuclear center has a 500% increase in per Gd (or ionic) relaxivity at 1.41 T versus small molecule contrast agents and a 170% increase in relaxivity at 9.4 T.     

Novel guanidine-containing molecular transporters based on lactose scaffolds: lipophilicity effect on the intracellular organellar selectivity

We have synthesized two lactose-based molecular transporters, each containing seven guanidine residues attached to the lactose scaffold through omega-aminocarboxylate linker chains of two different lengths, and have examined their cellular uptakes and intracellular and organellar localizations in HeLa cells, as well as their tissue distributions in mice. Both molecular transporters showed higher cellular uptake efficiencies than Arg8, and wide tissue distributions including the brain. Mitochondrial localization is of special interest because of its potential relevance to "mitochondrial diseases". Interestingly, it has been found that the intracellular localization sites of the G7 molecular transporters-namely either mitochondria or lysosomes and endocytic vesicles-are largely determined by the linker chain lengths, or their associated lipophilicities.     

Assembly of triple-stranded beta-sheet peptides at interfaces

A 30-residue peptide, BS30, which incorporates two proline residues to induce reverse turns, was designed to form a triple-stranded beta-sheet monolayer at the air-water interface. To discern the structural role of proline, a second peptide, BS30G, identical to BS30 but with glycine residues replacing proline, was prepared and examined in parallel fashion. Surface pressure-molecular area isotherms indicated a limiting area per molecule (ca. 460 A(2)) for BS30 that corresponds well to that estimated from the known dimensions of crystalline beta-sheet monolayers (492 A(2)). Comparable measurements on BS30G yielded a smaller molecular area (380 A(2)). Grazing incidence X-ray diffraction measurements performed on the BS30 monolayer at nominal area per molecule of 500 A(2), exhibited two Bragg peaks corresponding to 4.79 and 34.9 A spacings, consistent with formation of triple-stranded beta-sheet structures that assemble into two-dimensional crystallites at the air-water interface. Visualized by Brewster angle microscopy, BS30 monolayers displayed uniform, solidlike domains, whereas BS30G appeared to be disordered.     

Molecular dynamics studies show solvation structure of type III antifreeze protein is disrupted at low pH

Antifreeze proteins are a class of biological molecules of interest in many research and industrial applications due to their highly specialized function, but there is little information of their stability and properties under varied pH derived from computational studies. To gain novel insights in this area, we conducted molecular dynamics (MD) simulations with the antifreeze protein 1KDF at varied temperatures and pH. Water solvation and H-bond formation around specific residues – ASN14, THR18 and GLN44 – involved in its antifreeze activity were extensively studied. We found that at pH1 there was a disruption in water solvation around the basal and the ice binding surfaces of the molecule. This was induced by a small change in the secondary structure propensities of some titrable residues, particularly GLU35. This change explains the experimentally observed reduction in antifreeze activity previously reported for this protein at pH1. We also found that THR18 showed extremely low H-bond formation, and that the three antifreeze residues all had very low average H-bond lifetimes. Our results confirm long-standing assumptions that these small, compact molecules can maintain their antifreeze activity in a wide range of pH, while demonstrating the mechanism that may reduce antifreeze activity at low pH. This aspect is useful when considering industrial and commercial use of antifreeze proteins subject to extreme pH environments, in particular in food industrial applications.     

Voltammetric Determination of Surface‐Confined Biomolecules with N‐(2‐Ethyl‐ferrocene)maleimide

A thiol‐specific electroactive cross‐linker, N‐(2‐ethyl‐ferrocene)maleimide (Fc‐Mi), has been used to tag surface‐confined peptides containing cysteine residues or oligodeoxynucleotides (ODNs) whose 3′ ends have been modified with thiol groups. The peptides studied herein include both the oxidized and reduced forms of glutathione and a hexapeptide. Cyclic voltammograms (CVs) of the Fc‐Mi groups attached to the surfaces were used to quantify the total number of cysteine residues that are tagged and/or can undergo facile electron transfer reactions with the underlying electrodes. A quartz crystal microbalance was used in conjunction with CV to estimate the total number of cysteine groups labeled by Fc‐Mi per peptide molecule. By comparing to mass spectrometric studies, it is confirmed that not all of the Fc‐Mi linked to the cysteine groups can participate in the electron transfer reactions. The methodology is further extended to the determination of ODN samples in a sandwich assay wherein the thiol linker on the 3′ end can be tagged with Fc‐Mi. The analytical performance was evaluated through determinations of a complementary ODN target and targets with varying numbers of mismatching bases. ODN samples as low as 10 fmol can be detected. Such a low detection level is remarkable considering that no signal amplification scheme is involved in the current method. The approach is shown to be sequence‐ and/or structure‐specific and does not require sophisticated instrumentation and complex experimental procedure.     

Synthesis of functionalized furopyrazines as restricted dipeptidomimetics

Herein, an efficient synthetic approach to a furopyrazine scaffold with four points of diversity, starting from 2(1H)-pyrazinones, with dipeptomimetic properties, is presented. R-groups corresponding to amino acid side chains were introduced during the 2(1H)-pyrazinone and subsequent furopyrazine formation. The furopyrazine scaffold was further functionalized with an amino- and a carboxy-terminus resulting in a conformationally restricted dipeptidomimetic scaffold. The carboxy-terminus was introduced via a chemoselective vinylation of the 7-position followed by oxidative cleavage, while the amino-terminus was obtained via Buchwald–Hartwig amidation of the 2-position of the scaffold. The versatility of the synthetic method was demonstrated by the synthesis of a small library of diversely substituted furopyrazines having various amino acid side chains on the four points of diversity. Evaluation with an X-ray structure of the scaffold and computational analysis supports the exploitation of the furopyrazine scaffold as a restricted dipeptide mimic, which can mimic the two central residues of a β-turn.     

Macrocyclic beta-sheet peptides that mimic protein quaternary structure through intermolecular beta-sheet interactions

This paper reports the design, synthesis, and characterization of a family of cyclic peptides that mimic protein quaternary structure through beta-sheet interactions. These peptides are 54-membered-ring macrocycles comprising an extended heptapeptide beta-strand, two Hao beta-strand mimics [JACS 2000, 122, 7654] joined by one additional alpha-amino acid, and two delta-linked ornithine beta-turn mimics [JACS 2003, 125, 876]. Peptide 3a, as the representative of these cyclic peptides, contains a heptapeptide sequence (TSFTYTS) adapted from the dimerization interface of protein NuG2 [PDB ID: 1mio]. 1H NMR studies of aqueous solutions of peptide 3a show a partially folded monomer in slow exchange with a strongly folded oligomer. NOE studies clearly show that the peptide self-associates through edge-to-edge beta-sheet dimerization. Pulsed-field gradient (PFG) NMR diffusion coefficient measurements and analytical ultracentrifugation (AUC) studies establish that the oligomer is a tetramer. Collectively, these experiments suggest a model in which cyclic peptide 3a oligomerizes to form a dimer of beta-sheet dimers. In this tetrameric beta-sheet sandwich, the macrocyclic peptide 3a is folded to form a beta-sheet, the beta-sheet is dimerized through edge-to-edge interactions, and this dimer is further dimerized through hydrophobic face-to-face interactions involving the Phe and Tyr groups. Further studies of peptides 3b-3n, which are homologues of peptide 3a with 1-6 variations in the heptapeptide sequence, elucidate the importance of the heptapeptide sequence in the folding and oligomerization of this family of cyclic peptides. Studies of peptides 3b-3g show that aromatic residues across from Hao improve folding of the peptide, while studies of peptides 3h-3n indicate that hydrophobic residues at positions R3 and R5 of the heptapeptide sequence are important in oligomerization.