scholarly journals The dissociation of avidin–biotin complexes by guanidinium chloride

1972 ◽  
Vol 130 (3) ◽  
pp. 707-711 ◽  
Author(s):  
N. M. Green ◽  
E. J. Toms
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Guanidinium Chloride ◽  
A Site ◽  
Avidin Biotin Complex

Avidin molecules in which a fraction of the four binding sites were occupied by biotin did not dissociate completely in 6.4m-guanidinium chloride. Only unoccupied subunits dissociated. The remainder recombined to form the tetrameric avidin–biotin complex. The rate at which unoccupied subunits were unfolded and dissociated was only decreased by one-half in species in which three of the four binding sites were occupied by biotin. These results can be explained by assuming that unfolding of unoccupied subunits followed by dissociation from the tetramer is initiated by penetration of guanidinium ions into the binding site and disorganization of this region of the subunit. When a site is occupied by biotin this pathway is blocked and the subunit does not unfold. Each subunit behaves independently and is not markedly stabilized when neighbouring subunits are occupied.

Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1230-1236 ◽  
Author(s):  
Zhong Q. Li ◽  
Weiyi Liu ◽  
Kwang S. Park ◽  
Brue S. Sachais ◽  
Gowthani M. Arepally ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Binding Sites ◽  
Heparin Therapy ◽  
Common Complication ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
The Third ◽  
N Terminus ◽  
A Site

Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant. The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin.

scholarly journals The effect of N-bromosuccinimide on the sub-unit structure of avidin and its complexes with biotin

1968 ◽  
Vol 110 (1) ◽  
pp. 59-66 ◽  
Author(s):  
N. M. Green ◽  
M. E. Ross
Keyword(s):  
Binding Sites ◽  
Oxidation Products ◽  
Guanidinium Chloride ◽  
Unit Structure ◽  
Tryptophan Residues ◽  
Biotin Binding ◽  
Tetrameric Complex ◽  
Chloride Concentrations ◽  
Avidin Biotin Complex

1. Each molecule of biotin bound to avidin protected four tryptophan residues from oxidation by N-bromosuccinimide, regardless of the occupancy of neighbouring binding sites in the four-sub-unit avidin molecule. 2. The oxidation products from avidin molecules in which some of the sites were occupied were separated on columns of Sephadex G-100. In the absence of biotin, oxidized avidin broke down into sub-units, which partly aggregated. When some of the sites were occupied by biotin, the only detectable products were completely oxidized avidin (sub-units and large aggregates) and unoxidized avidin–biotin complex (tetramer). Since the biotin-containing sub-units were randomly distributed before oxidation took place, they must have dissociated from the molecules containing oxidized sub-units and then reassociated to form the tetrameric avidin–biotin complex. 3. This reassociation still occurred in 3·5m-guanidinium chloride, which prevents the reassociation of unoccupied sub-units. During their brief existence in this medium, the sub-units of avidin–biotin complex were protected from oxidation by N-bromo-succinimide to the same extent as was the tetrameric complex. 4. It is concluded that sub-units of avidin–biotin complex do not readily lose their biotin, even in 3·5m-guanidinium chloride, and that monomeric biotin–binding species are probably present in solutions of avidin sub-units at guanidinium chloride concentrations between 3·0m and 3·5m.

scholarly journals Calmodulin Binding to Connexin 35: Specializations to Function as an Electrical Synapse

2020 ◽  
Vol 21 (17) ◽  
pp. 6346
Author(s):  
Jaya Aseervatham ◽  
Xiaofan Li ◽  
Cheryl K. Mitchell ◽  
Ya-Ping Lin ◽  
Ruth Heidelberger ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Electrical Synapse ◽  
Cytoplasmic Calcium ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
Connexin 36 ◽  
Calcium Calmodulin Dependent ◽  
C Terminus ◽  
A Site

Calmodulin binding is a nearly universal property of gap junction proteins, imparting a calcium-dependent uncoupling behavior that can serve in an emergency to decouple a stressed cell from its neighbors. However, gap junctions that function as electrical synapses within networks of neurons routinely encounter large fluctuations in local cytoplasmic calcium concentration; frequent uncoupling would be impractical and counterproductive. We have studied the properties and functional consequences of calmodulin binding to the electrical synapse protein Connexin 35 (Cx35 or gjd2b), homologous to mammalian Connexin 36 (Cx36 or gjd2). We find that specializations in Cx35 calmodulin binding sites make it relatively impervious to moderately high levels of cytoplasmic calcium. Calmodulin binding to a site in the C-terminus causes uncoupling when calcium reaches low micromolar concentrations, a behavior prevented by mutations that eliminate calmodulin binding. However, milder stimuli promote calcium/calmodulin-dependent protein kinase II activity that potentiates coupling without interference from calmodulin binding. A second calmodulin binding site in the end of the Cx35 cytoplasmic loop, homologous to a calmodulin binding site present in many connexins, binds calmodulin with very low affinity and stoichiometry. Together, the calmodulin binding sites cause Cx35 to uncouple only at extreme levels of intracellular calcium.

scholarly journals Binding of Ca2+ to the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum: equilibrium studies

1994 ◽  
Vol 297 (3) ◽  
pp. 615-624 ◽  
Author(s):  
I M Henderson ◽  
Y M Khan ◽  
J M East ◽  
A G Lee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Constants ◽  
Tryptophan Fluorescence ◽  
Equilibrium Studies ◽  
High Affinity Binding Site ◽  
Effects Of Ph ◽  
A Site ◽  
Cytoplasmic Side

Equilibrium fluorescence methods have been used to establish a model for Ca2+ binding to the (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding. The basic scheme proposed is: E2 <--> E1 <--> E1Ca <--> El'Ca <--> E1′Ca2. The E1 conformation of the ATPase initially has one high-affinity binding site for Ca2+ exposed to the cytoplasmic side of the sarcoplasmic reticulum, but in the E2 conformation this site is unable to bind Ca2+; Ca2+ does not bind to luminal sites on E2. The second, outer, Ca(2+)-binding site on the ATPase is formed after binding of Ca2+ to the first, inner, site on E1 and the E1Ca <--> E1′Ca conformation change. The pH- and Mg(2+)-dependence of the E2 <--> E1 equilibrium has been established after changes in the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole. It is proposed that Mg2+ from the cytoplasmic side of the sarcoplasmic reticulum can bind to the first Ca(2+)-binding site on both E1 and E2. It is proposed that the change in tryptophan fluorescence intensity after binding of Ca2+ follows from the E1Ca <--> E1′Ca change. The pH- and Mg(2+)-dependence of this change defines H(+)- and Mg(2+)-binding constants at the two Ca(2+)-binding sites. It is proposed that the change in tryptophan fluorescence observed on binding Mg2+ follows from binding at the second Ca(2+)-binding site. Effects of pH and Mg2+ on the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are proposed to follow from binding to a site on the ATPase, the ‘gating’ site, which affects the affinity of the first Ca(2+)-binding site for Ca2+ and affects the rate of dissociation of Ca2+ from the ATPase.

Covalent-Fragment Screening of Brd4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites

2019 ◽  
Author(s):  
Michael Olp ◽  
Daniel Sprague ◽  
Stefan Kathman ◽  
Ziyang Xu ◽  
Alexandar Statsyuk ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Site ◽  
Binding Sites ◽  
Computational Prediction ◽  
Chemical Probes ◽  
Computational Docking ◽  
Fragment Screening ◽  
Covalent Inhibitors ◽  
Bet Protein ◽  
Proof Of Principle

<p>Brd4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly-conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to non-homologous cysteine residues within the <i>C</i>-terminal Brd4 bromodomain (Brd4-BD2), we performed a mid-throughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify Brd4. Subsequent mass spectrometry, NMR and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to Brd4 among all human bromodomains. This site is orthogonal to the Brd4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays. Finally, we tethered covalent fragments to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace Brd4 from chromatin.</p>

Effects of Multiple Binding Sites on Studies of Hydrogen Bonding between Nitroxide Radicals and Solvent Molecules

1993 ◽  
Vol 58 (1) ◽  
pp. 47-52 ◽  
Author(s):  
Imad Al-Bala'a ◽  
Richard D. Bates
Keyword(s):  
Hydrogen Bonding ◽  
Magnetic Resonance ◽  
Binding Site ◽  
Binding Sites ◽  
Hyperfine Coupling Constant ◽  
Hydrogen Donor ◽  
Complex Formation Constants ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Solvent Molecules

The role of more than one binding site on a nitroxide free radical in magnetic resonance determinations of the properties of the complex formed with a hydrogen donor is examined. The expression that relates observed hyperfine couplings in EPR spectra to complex formation constants and concentrations of each species in solution becomes much more complex when multiple binding sites are present, but reduces to a simpler form when binding at the two sites occurs independently and the binding at the non-nitroxide site does not produce significant differences in the hyperfine coupling constant in the complexed radical. Effects on studies of hydrogen bonding between multiple binding site nitroxides and hydrogen donor solvent molecules by other magnetic resonance methods are potentially more extreme.

Tuning of Electronic Properties in Conducting Polymers

2001 ◽  
Vol 66 (8) ◽  
pp. 1208-1218 ◽  
Author(s):  
Guofeng Li ◽  
Mira Josowicz ◽  
Jiří Janata
Keyword(s):  
Hydrogen Bonding ◽  
Binding Site ◽  
Binding Sites ◽  
Polymer Chains ◽  
Electronic Transitions ◽  
Weakly Bound ◽  
Ordered Structures ◽  
Doped Polymer ◽  
Positively Charged ◽  
Repeated Cycling

Structural and electronic transitions in poly(thiophenyleneiminophenylene), usually referred to as poly(phenylenesulfidephenyleneamine) (PPSA) upon electrochemical doping with LiClO4 have been investigated. The unusual electrochemical behavior of PPSA indicates that the dopant anions are bound in two energetically different sites. In the so-called "binding site", the ClO4- anion is Coulombically attracted to the positively charged S or N sites on one chain and simultaneously hydrogen-bonded with the N-H group on a neighboring polymer chain. This strong interaction causes a re-organization of the polymer chains, resulting in the formation of a networked structure linked together by these ClO4- Coulombic/hydrogen bonding "bridges". However, in the "non-binding site", the ClO4- anion is very weakly bound, involves only the electrostatic interaction and can be reversibly exchanged when the doped polymer is reduced. In the repeated cycling, the continuous and alternating influx and expulsion of ClO4- ions serves as a self-organizing process for such networked structures, giving rise to a diminishing number of available "non-binding" sites. The occurrence of these ordered structures has a major impact on the electrochemical activity and the morphology of the doped polymer. Also due to stabilization of the dopant ions, the doped polymer can be kept in a stable and desirable oxidation state, thus both work function and conductivity of the polymer can be electrochemically controlled.

scholarly journals Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression.

1993 ◽  
Vol 13 (9) ◽  
pp. 5805-5813 ◽  
Author(s):  
M M Wang ◽  
R Y Tsai ◽  
K A Schrader ◽  
R R Reed
Keyword(s):  
Signal Transduction ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Olfactory Neuron ◽  
Promoter Regions ◽  
Transcriptional Initiation ◽  
Genes Encoding

Genes which mediate odorant signal transduction are expressed at high levels in neurons of the olfactory epithelium. The molecular mechanism governing the restricted expression of these genes likely involves tissue-specific DNA binding proteins which coordinately activate transcription through sequence-specific interactions with olfactory promoter regions. We have identified binding sites for the olfactory neuron-specific transcription factor, Olf-1, in the sequences surrounding the transcriptional initiation site of five olfactory neuron-specific genes. The Olf-1 binding sites described define the consensus sequence YTCCCYRGGGAR. In addition, we have identified a second binding site, the U site, in the olfactory cyclic nucleotide gated channel and type III cyclase promoters, which binds factors present in all tissue examined. These experiments support a model in which expression of Olf-1 in the sensory neurons coordinately activates a set of olfactory neuron-specific genes. Furthermore, expression of a subset of these genes may be modulated by additional binding factors.

scholarly journals Study of Endogen Substrates, Drug Substrates and Inhibitors Binding Conformations on MRP4 and Its Variants by Molecular Docking and Molecular Dynamics

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1051
Author(s):  
Edgardo Becerra ◽  
Giovanny Aguilera-Durán ◽  
Laura Berumen ◽  
Antonio Romo-Mancillas ◽  
Guadalupe García-Alcocer
Keyword(s):  
Molecular Dynamics ◽  
Molecular Docking ◽  
Binding Energy ◽  
Binding Site ◽  
Binding Sites ◽  
Adenosine Monophosphate ◽  
Competitive Inhibitor ◽  
Umbrella Sampling ◽  
Coarse Grained ◽  
Nucleotide Polymorphisms

Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.

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