Single-molecule photon-fueled DNA nanoscissors for DNA cleavage based on the regulation of substrate binding affinity by azobenzene

2013 ◽  
Vol 49 (77) ◽  
pp. 8716 ◽  
Author(s):  
Yuan Zou ◽  
Jie Chen ◽  
Zhi Zhu ◽  
Lianyu Lu ◽  
Yishun Huang ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Substrate Binding

scholarly journals Activation-induced Deaminase (AID)-directed Hypermutation in the Immunoglobulin Sμ Region

2002 ◽  
Vol 195 (4) ◽  
pp. 529-534 ◽  
Author(s):  
Hitoshi Nagaoka ◽  
Masamichi Muramatsu ◽  
Namiko Yamamura ◽  
Kazuo Kinoshita ◽  
Tasuku Honjo
Keyword(s):  
B Lymphocytes ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Somatic Hypermutation ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Variable Regions ◽  
Class Switch ◽  
Large Deletions ◽  
Activation Induced Deaminase

Somatic hypermutation (SHM) and class switch recombination (CSR) cause distinct genetic alterations at different regions of immunoglobulin genes in B lymphocytes: point mutations in variable regions and large deletions in S regions, respectively. Yet both depend on activation-induced deaminase (AID), the function of which in the two reactions has been an enigma. Here we report that B cell stimulation which induces CSR but not SHM, leads to AID-dependent accumulation of SHM-like point mutations in the switch μ region, uncoupled with CSR. These findings strongly suggest that AID itself or a single molecule generated by RNA editing function of AID may mediate a common step of SHM and CSR, which is likely to be involved in DNA cleavage.

scholarly journals Hierarchical coupling between ATP hydrolysis and Hsp90’s client binding site

2020 ◽  
Author(s):  
Steffen Wolf ◽  
Benedikt Sohmen ◽  
Björn Hellenkamp ◽  
Johann Thurn ◽  
Gerhard Stock ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Information Transfer ◽  
Atp Hydrolysis ◽  
Structural Information ◽  
Substrate Binding ◽  
Signal Propagation ◽  
Nonequilibrium Molecular Dynamics ◽  
Time Resolved ◽  
Substrate Binding Site

I.ABSTRACTSeveral indicators for a signal propagation from a binding site to a distant functional site have been found in the Hsp90 dimer. Here we determined a time-resolved pathway from ATP hydrolysis to changes in a distant folding substrate binding site. This was possible by combining single-molecule fluorescence-based methods with extensive atomistic nonequilibrium molecular dynamics simulations. We find that hydrolysis of one ATP effects a structural asymmetry in the full Hsp90 dimer that leads to the collapse of a central folding substrate binding site. Arg380 is the major mediator in transferring structural information from the nucleotide to the substrate binding site. This allosteric process occurs via hierarchical dynamics that involve timescales from picoto milliseconds and length scales from Ångstroms to several nanometers. We presume that similar hierarchical mechanisms are fundamental for information transfer through many other proteins.

scholarly journals Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase

2021 ◽  
Author(s):  
Chih-Wei Huang ◽  
Chi-Ching Hwang ◽  
Yung-Lung Chang ◽  
Jen-Tzu Liu ◽  
Sheng-Peng Wu ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Functional Role ◽  
Substrate Binding ◽  
Critical Role ◽  
Intermediate Formation ◽  
Hydroxyl Group ◽  
Double Mutation ◽  
Closed Conformation ◽  
Bond Network

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E’s Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine

1992 ◽  
Vol 12 (9) ◽  
pp. 3757-3765
Author(s):  
J W Chen ◽  
B R Evans ◽  
S H Yang ◽  
H Araki ◽  
Y Oshima ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Substrate Binding ◽  
Point Mutations ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Site Specific ◽  
Arginine Residues ◽  
Active Site Tyrosine ◽  
Half Site

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

scholarly journals Using Single Molecule Methods to Study Mechanisms of Site Specific DNA Cleavage

2019 ◽  
Vol 116 (3) ◽  
pp. 504a
Author(s):  
Allen C. Price ◽  
Stephen D. Parziale ◽  
Karissa Mehrtens ◽  
Anna D. Ware ◽  
Emily K. Matozel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Site Specific

Characterization and improvement of substrate-binding affinity of d-aspartate oxidase of the thermophilic fungus Thermomyces dupontii

2019 ◽  
Vol 103 (10) ◽  
pp. 4053-4064 ◽  
Author(s):  
Shouji Takahashi ◽  
Kohei Osugi ◽  
Yuya Shimekake ◽  
Akira Shinbo ◽  
Katsumasa Abe ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Substrate Binding ◽  
Thermophilic Fungus ◽  
Thermomyces Dupontii

scholarly journals Hyaluronan–CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9

2002 ◽  
Vol 158 (6) ◽  
pp. 1133-1144 ◽  
Author(s):  
Paola Spessotto ◽  
Francesca Maria Rossi ◽  
Massimo Degan ◽  
Raffaele Di Francia ◽  
Roberto Perris ◽  
...  
Keyword(s):  
Cell Migration ◽  
Bone Resorption ◽  
Cell Motility ◽  
Binding Affinity ◽  
Antisense Oligonucleotides ◽  
Substrate Binding ◽  
Down Regulation ◽  
Multinucleated Cells ◽  
Cell Substrate

Osteoclast (OC) precursors migrate to putative sites of bone resorption to form functionally active, multinucleated cells. The preOC FLG 29.1 cells, known to be capable of irreversibly differentiating into multinucleated OC-like cells, displayed several features of primary OCs, including expression of specific integrins and the hyaluronan (HA) receptor CD44. OC-like FLG 29.1 cells adhered to and extensively migrated through membranes coated with fibronectin, vitronectin, and laminins, but, although strongly binding to HA, totally failed to move on this substrate. Moreover, soluble HA strongly inhibited OC-like FLG 29.1 cell migration on the permissive matrix substrates, and this behavior was dependent on its engagement with CD44, as it was fully restored by function-blocking anti-CD44 antibodies. HA did not modulate the cell–substrate binding affinity/avidity nor the expression levels of the corresponding integrins. MMP-9 was the major secreted metalloproteinase used by OC-like FLG 29.1 cells for migration, because this process was strongly inhibited by both TIMP-1 and GM6001, as well as by MMP-9–specific antisense oligonucleotides. After HA binding to CD44, a strong down-regulation of MMP-9 mRNA and protein was detected. These findings highlight a novel role of the HA–CD44 interaction in the context of OC-like cell motility, suggesting that it may act as a stop signal for bone-resorbing cells.

Single-Molecule Analysis of Nucleotide-Dependent Substrate Binding by the Protein Unfoldase ClpA

2007 ◽  
Vol 129 (41) ◽  
pp. 12378-12379 ◽  
Author(s):  
Mary E. Farbman ◽  
Anne Gershenson ◽  
Stuart Licht
Keyword(s):  
Single Molecule ◽  
Substrate Binding ◽  
Single Molecule Analysis
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