共查询到20条相似文献,搜索用时 15 毫秒
1.
Qing Li Zackary N. Scholl Prof. Piotr E. Marszalek 《Angewandte Chemie (International ed. in English)》2014,53(49):13429-13433
The folding behaviors and mechanisms of large multidomain proteins have remained largely uncharacterized, primarily because of the lack of appropriate research methods. To address these limitations, novel mechanical folding probes have been developed that are based on antiparallel coiled‐coil polypeptides. Such probes can be conveniently inserted at the DNA level, at different positions within the protein of interest where they minimally disturb the host protein structure. During single‐molecule force spectroscopy measurements, the forced unfolding of the probe captures the progress of the unfolding front through the host protein structure. This novel approach allows unfolding pathways of large proteins to be directly identified. As an example, this probe was used in a large multidomain protein with ten identical ankyrin repeats, and the unfolding pathway, its direction, and the order of sequential unfolding were unequivocally and precisely determined. This development facilitates the examination of the folding pathways of large proteins, which are predominant in the proteasomes of all organisms, but have thus far eluded study because of the technical limitations encountered when using traditional techniques. 相似文献
2.
Design of Ligand Binding to an Engineered Protein Cavity Using Virtual Screening and Thermal Up-shift Evaluation 总被引:1,自引:0,他引:1
Machicado C López-Llano J Cuesta-López S Bueno M Sancho J 《Journal of computer-aided molecular design》2005,19(6):421-443
Summary Proteins could be used to carry and deliver small compounds. As a tool for designing ligand binding sites in protein cores,
a three-step virtual screening method is presented that has been optimised using existing data on T4 lysozyme complexes and
tested in a newly engineered cavity in flavodoxin. The method can pinpoint, in large databases, ligands of specific protein
cavities. In the first step, physico-chemical filters are used to screen the library and discard a majority of compounds.
In the second step, a flexible, fast docking procedure is used to score and select a smaller number of compounds as potential
binders. In the third step, a finer method is used to dock promising molecules of the hit list into the protein cavity, and
an optimised free energy function allows discarding the few false positives by calculating the affinity of the modelled complexes.
To demonstrate the portability of the method, several cavities have been designed and engineered in the flavodoxin from Anabaena PCC 7119, and the W66F/L44A double mutant has been selected as a suitable host protein. The NCI database has then been screened
for potential binders, and the binding to the engineered cavity of five promising compounds and three tentative non-binders
has been experimentally tested by thermal up-shift assays and spectroscopic titrations. The five tentative binders (some apolar
and some polar), unlike the three tentative non-binders, are shown to bind to the host mutant and, importantly, not to bind
to the wild type protein. The three-step virtual screening method developed can thus be used to identify ligands of buried
protein cavities. We anticipate that the method could also be used, in a reverse manner, to identify natural or engineerable
protein cavities for the hosting of ligands of interest. 相似文献
3.
Yajie Liu Zelin Duan Jing Fang Fan Zhang Junyu Xiao Wen‐Bin Zhang 《Angewandte Chemie (International ed. in English)》2020,59(37):16122-16127
Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post‐translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS‐PAGE, LC‐MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X‐ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein‐topology engineering. 相似文献
4.
8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS–protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS–protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective. 相似文献
5.
Vytautas Iešmantavičius Dr. Jakob Dogan Dr. Per Jemth Prof. Kaare Teilum Dr. Magnus Kjaergaard 《Angewandte Chemie (International ed. in English)》2014,53(6):1548-1551
Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well‐ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence‐monitored stopped‐flow kinetic measurements we show that the secondary structure content in helix 1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins. 相似文献
6.
《Angewandte Chemie (International ed. in English)》2017,56(20):5598-5602
Owing to the difficulty in comprehensively characterizing nanocrystal (NC) surfaces, clear guidance for ligand design is lacking. In this work, a series of bidentate bis(pyridine) anthracene isomers (2,3‐PyAn, 3,3‐PyAn, 2,2‐PyAn) that differ in their binding geometries were designed to find the best complementary fit to the NC surface. The efficiency of triplet energy transfer (TET) from the CdSe NC donor to a diphenylanthracene (DPA) acceptor mediated by these isomers was used as a proxy for the efficacy of orbital overlap and therefore ligand binding. 2,3‐PyAn, with an intramolecular N–N distance of 8.2 Å, provided the best match to the surface of CdSe NCs. When serving as a transmitter for photon upconversion, 2,3‐PyAn yielded the highest upconversion quantum yield (QY) of 12.1±1.3 %, followed by 3,3‐PyAn and 2,2‐PyAn. The TET quantum efficiencies determined by ultrafast transient absorption measurements showed the same trend. 相似文献
7.
Dr. Jens Voskuhl Dr. Christian Wendeln M. Sc. Frank Versluis M. Sc. Eva‐Corinna Fritz M. Sc. Oliver Roling M. Sc. Harshal Zope Dr. Christian Schulz Dipl.‐Phys. Stefan Rinnen Prof. Dr. Heinrich F. Arlinghaus Prof. Dr. Bart Jan Ravoo Dr. Alexander Kros 《Angewandte Chemie (International ed. in English)》2012,51(50):12616-12620
8.
Yajie Liu Zelin Duan Jing Fang Fan Zhang Prof. Junyu Xiao Prof. Wen-Bin Zhang 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2020,132(37):16256-16261
Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post-translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS-PAGE, LC-MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X-ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein-topology engineering. 相似文献
9.
Dr. Caterina Bernacchioni Dr. Cecilia Pozzi Flavio Di Pisa Prof. Stefano Mangani Prof. Paola Turano 《Chemistry (Weinheim an der Bergstrasse, Germany)》2016,22(45):16213-16219
Ferritins are iron‐storage nanocage proteins that catalyze the oxidation of Fe2+ to Fe3+ at ferroxidase sites. By a combination of structural and spectroscopic techniques, Asp140, together with previously identified Glu57 and Glu136, is demonstrated to be an essential residue to promote the iron oxidation at the ferroxidase site. However, the presence of these three carboxylate moieties in close proximity to the catalytic centers is not essential to achieve binding of the Fe2+ substrate to the diferric ferroxidase sites with the same coordination geometries as in the wild‐type cages. 相似文献
10.
Tianshu Xiao Dr. Jing‐song Fan Hu Zhou Dr. Qingsong Lin Prof. Daiwen Yang 《Angewandte Chemie (International ed. in English)》2016,55(24):6869-6872
Fatty acid binding proteins are responsible for the transportation of fatty acids in biology. Despite intensive studies, the molecular mechanism of fatty acid entry to and exit from the protein cavity is still unclear. Here a cap‐closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the helical cap is locked to the β‐barrel by a disulfide linkage. Structure determination shows that this variant adopts a closed conformation, but still uptakes fatty acids. Stopped‐flow experiments indicate that a rate‐limiting step exists before the ligand association and this step corresponds to the conversion of the closed form to the open one. NMR relaxation dispersion and H‐D exchange data demonstrate the presence of two excited states: one is native‐like, but the other adopts a locally unfolded structure. Local unfolding of helix 2 generates an opening for ligands to enter the protein cavity, and thus controls the ligand association rate. 相似文献
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Dr. Servaas Michielssens Dr. Jan Henning Peters Dr. David Ban Supriya Pratihar Dr. Daniel Seeliger Monika Sharma Karin Giller Dr. Thomas Michael Sabo Dr. Stefan Becker Dr. Donghan Lee Prof. Dr. Christian Griesinger Prof. Dr. Bert L. de Groot 《Angewandte Chemie (International ed. in English)》2014,53(39):10367-10371
In a conformational selection scenario, manipulating the populations of binding‐competent states should be expected to affect protein binding. We demonstrate how in silico designed point mutations within the core of ubiquitin, remote from the binding interface, change the binding specificity by shifting the conformational equilibrium of the ground‐state ensemble between open and closed substates that have a similar population in the wild‐type protein. Binding affinities determined by NMR titration experiments agree with the predictions, thereby showing that, indeed, a shift in the conformational equilibrium enables us to alter ubiquitin’s binding specificity and hence its function. Thus, we present a novel route towards designing specific binding by a conformational shift through exploiting the fact that conformational selection depends on the concentration of binding‐competent substates. 相似文献
13.
众所周知,插入剂的DNA特性结合位点位于DNA碱基对之间,然而这种非共价相互作用对于含脱碱基(AP)位点的DNA来讲还没有引起足够的重视,虽然在生物细胞中总是存在着DNA脱碱基位点。本文以原黄素(proflavine,PF)为例研究了插入剂对DNA中AP位点的结合特性。实验结果表明,相对于插入位点而言,AP位点是PF的优先结合位点,AP位点的本征结合常数比插入结合常数高一个数量级以上。此外,PF的结合使含脱碱基位点DNA的热稳定性明显提高,表明PF在脱碱基位点的结合构像明显不同于插入结合时的分子定向。本文结果将有助于判断小分子的DNA结合方式所决定的药物的生物化学及生物物理效用。 相似文献
14.
Jun-Fang Du Dr. Wei Li Prof. Dr. Lianzhi Li Prof. Dr. Ge-Bo Wen Prof. Dr. Ying-Wu Lin Prof. Dr. Xiangshi Tan 《ChemistryOpen》2015,4(2):97-101
Heme coordination state determines the functional diversity of heme proteins. Using myoglobin as a model protein, we designed a distal hydrogen-bonding network by introducing both distal glutamic acid (Glu29) and histidine (His43) residues and regulated the heme into a bis-His coordination state with native ligands His64 and His93. This resembles the heme site in natural bis-His coordinated heme proteins such as cytoglobin and neuroglobin. A single mutation of L29E or F43H was found to form a distinct hydrogen-bonding network involving distal water molecules, instead of the bis-His heme coordination, which highlights the importance of the combination of multiple hydrogen-bonding interactions to regulate the heme coordination state. Kinetic studies further revealed that direct coordination of distal His64 to the heme iron negatively regulates fluoride binding and hydrogen peroxide activation by competing with the exogenous ligands. The new approach developed in this study can be generally applicable for fine-tuning the structure and function of heme proteins. 相似文献
15.
In this work we present an impedimetric detection system for DNA‐ligand interactions. The sensor system consists of thiol‐modified single‐stranded DNA chemisorbed to gold. Impedance measurements in the presence of the redox system ferri‐/ferrocyanide show an increase in charge transfer resistance (Rct) after hybridisation of a complementary target. Different amounts of capture strands, used for gold electrode modification, result in surface coverages between 3 and 15 pmol/cm2 ssDNA. The relative change in Rct upon hybridisation increases with increasing amount of capture probe on the electrode from 1.5‐ to 4.5‐fold. Impedimetric detection of binding events of a metal‐intercalator ([Ru(phen)3]2+) and a groove binder (spermine) to double‐stranded DNA is demonstrated. Binding of [Ru(phen)3]2+ and spermine exhibits a decrease in charge transfer resistance. Here, the ligand’s interaction leads to electrostatic shielding of the negatively charged DNA backbone. The impedance changes have been evaluated in dependence on the concentration of both DNA binders. Furthermore, the association of a single‐stranded binding protein (SSBP) is found to cause an increase in charge transfer resistance only when incubated with single‐stranded DNA. The specific binding of an anti‐dsDNA antibody to the dsDNA‐modified electrode surface decreases in contrast the interfacial impedance. 相似文献
16.
Dr. Fredj Ben Bdira Dr. Christopher A. Waudby Dr. Alexander N. Volkov Dr. Sybrin P. Schröder Dr. Eiso AB Prof. Dr. Jeroen D. C. Codée Prof. Dr. Hermen S. Overkleeft Prof. Dr. Johannes M. F. G. Aerts Dr. Hugo van Ingen Prof. Dr. Marcellus Ubbink 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2020,132(46):20689-20695
The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction. 相似文献
17.
Anion‐Controlled Switching of an X Ligand into a Z Ligand: Coordination Non‐innocence of a Stiboranyl Ligand 下载免费PDF全文
Iou‐Sheng Ke James S. Jones Prof. Dr. François P. Gabbaï 《Angewandte Chemie (International ed. in English)》2014,53(10):2633-2637
The tetravalent platinum stiboranyl complex [(o‐(Ph2P)C6H4)2(o‐C6Cl4O2)Sb]PtCl2Ph ( 2 ) has been synthesized by reaction of [(o‐(Ph2P)C6H4)2SbClPh]PtCl ( 1 ) with o‐chloranil. In the presence of fluoride anions, the stiboranyl moiety of 2 displays non‐innocent behavior and is readily converted into a fluorostiborane unit. This transformation, which is accompanied by elimination of a chloride ligand from the Pt center, results in the formation of [(o‐(Ph2P)C6H4)2(o‐C6Cl4O2)SbF]PtClPh ( 3 ). Structural, spectroscopic, and computational studies show that the conversion of 2 into 3 is accompanied by a cleavage of the covalent Pt? Sb bond present in 2 and formation of a longer and weaker Pt→Sb interaction in 3 . These results show that this new Pt–Sb platform supports the fluoride‐induced metamorphosis of a stiboranyl X ligand into a stiborane Z ligand. 相似文献
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Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy 下载免费PDF全文
Hadar Ivanir‐Dabora Evgeny Nimerovsky Prof. P. K. Madhu Dr. Amir Goldbourt 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(30):10778-10785
Magic‐angle spinning solid‐state NMR spectroscopy has been applied to study the dynamics of CBM3b–Cbh9A from Clostridium thermocellum (ctCBM3b), a cellulose binding module protein. This 146‐residue protein has a nine‐stranded β‐sandwich fold, in which 35 % of the residues are in the β‐sheet and the remainder are composed of loops and turns. Dynamically averaged 1H‐13C dipolar coupling order parameters were extracted in a site‐specific manner by using a pseudo‐three‐dimensional constant‐time recoupled separated‐local‐field experiment (dipolar‐chemical shift correlation experiment; DIPSHIFT). The backbone‐Cα and Cβ order parameters indicate that the majority of the protein, including turns, is rigid despite having a high content of loops; this suggests that restricted motions of the turns stabilize the loops and create a rigid structure. Water molecules, located in the crystalline interface between protein units, induce an increased dynamics of the interface residues thereby lubricating crystal water‐mediated contacts, whereas other crystal contacts remain rigid. 相似文献