scholarly journals A Spectroscopic Study on PtCl4(2−) Binding to Rabbit Skeletal Muscle G-Actin

1995 ◽  
Vol 2 (3) ◽  
pp. 127-136 ◽  
Author(s):  
Juan Zou ◽  
Hong-Ye Sun ◽  
Kui Wang
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Binding Constant ◽  
Intrinsic Fluorescence ◽  
Molar Ratio ◽  
Second Step ◽  
High Affinity ◽  
Α Helix ◽  
Discontinuous Mode ◽  
Sulfur Containing

It was found that the binding of PtCl42− to G-actin and the consequent conformational changes are different with those for hard acids. It is a two-step process depending on molar ratio PtCl42−/actin (R). In the first step, R less than 25, the PtCl42− ions are bound to sulfur-containing groups preferentially. These high-affinity sites determined by Scatchard approach are characterized by n1 = 30 with average binding constant K1=1.0×107M-1. The conformational changes are significant as characterized by N-(1-pyrenyl) maleimide(NPM) labeled fluorescence, intrinsic fluorescence and CD spectra. EPR spectroscopy of maleimide spin labeled(MSL) actin demonstrated that even PtCl42−binding is limited to a very small fraction of high-affinity sites(R<1), it can bring about a pronounced change of conformation. In the range of R=25-40, high-affinity sites accessible are saturated. In the second step(R>40) , deep-buried binding sites turn out to be accessible as a result of the accumulated conformational changes. These new binding sites are estimated to be n2=26 with average binding constant K2=2.1×106M-1. Although in this step the quenching of intrinsic fluorescence goes on and the NPM-labled thiols moves to more hydrophilic environment, no change in α-helix content was found. These results suggested that with increasing in PtCl42− binding, the G-actin turns to an open and loose structure in a discontinuous mode.

Activity and conformational changes of horseradish peroxidase in trifluoroethanol

2002 ◽  
Vol 80 (2) ◽  
pp. 205-213 ◽  
Author(s):  
Hong-Wei Zhou ◽  
Yan Xu ◽  
Hai-Meng Zhou
Keyword(s):  
Enzyme Activity ◽  
Horseradish Peroxidase ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Intrinsic Fluorescence ◽  
Two Phase ◽  
Main Effect ◽  
Α Helix ◽  
Kinetics Of

The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5–25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25–40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The α-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.Key words: horseradish peroxidase, trifluoroethanol, unfolding, Soret.

scholarly journals The accumulation and compartmentalization of isometamidium chloride in Trypanosoma congolense, monitored by its intrinsic fluorescence

1995 ◽  
Vol 312 (1) ◽  
pp. 319-327 ◽  
Author(s):  
J M Wilkes ◽  
A S Peregrine ◽  
D Zilberstein
Keyword(s):  
Binding Sites ◽  
Thermal Denaturation ◽  
Specific Binding ◽  
Trypanosoma Congolense ◽  
Emission Peak ◽  
Intrinsic Fluorescence ◽  
Drug Uptake ◽  
Membrane Transporter ◽  
High Affinity ◽  
Dnase 1

Interaction of the trypanocide isometamidium chloride with components of Trypanosoma congolense results in characteristic shifts in the intrinsic fluorescence of the drug. The specificity of this interaction was investigated by analysing the effects of various physicochemical manipulations on its fluorescence properties. The characteristic shifts involved a preferential increase in the intensity of one emission peak over the other, resulting in a systematic increase in the ratio of fluorescence intensities. These effects were apparently due to constraints on fluorophore free rotation in the solution (that is, viscosity). Purified DNA produced similar effects in a saturable manner displaying high affinity for the drug, indicating that the constraint involves binding of the drug to high-affinity binding sites within the DNA. Such binding sites were demonstrated in lysates derived from trypanosomal cells. The binding sites were associated with macromolecular species (M(r) > 12000), and were partly disrupted by thermal denaturation and proteolysis. Treatment with DNase 1 produced high levels of disruption of the binding sites (> 85%), indicating an involvement of DNA in the binding. BSA demonstrated weak non-specific binding of the drug. Entry of drug into live trypanosomal cells (monitored by 14C-labelled drug uptake) was paralleled by fluorescence shifts observed under comparable conditions of drug concentration and buffer conditions. Both systems (fluorescence shifts and accumulation of labelled drug) indicated the presence of a saturable membrane transporter with high affinity for the drug. We conclude that monitoring the fluorescence shifts of isometamidium constitutes a sensitive and highly specific probe for entry of the drug into trypanosomal cells, thereby enabling resolution of the transport events involved.

Fragment D Immunoreacivity: The Influence of Iodination and Calcium Environment

1979 ◽  
Author(s):  
B. Kudryk ◽  
M. Blombäck
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Specific Activity ◽  
High Specific Activity ◽  
Antigenic Determinants ◽  
Affinity Binding ◽  
Compact Structure ◽  
High Affinity Binding ◽  
High Affinity ◽  
Chloramine T

Human fragment D (Fg-Ds) has heen iodinated using both the Chloramine-T and lactoperoildaae methods. The specific activity was similar regardless of the method used. However, binding to a specific antibody was different for each preparation. The antigen labeled by the Chloramine-T method bound to a maximum of 40% the other labeled product bound up to 85%. A correlation between the decree of immunoreactivity and avidity for a fihrinmcnomer conjugate vas found also. Fibrinmonomer bound about twice the ajnount of lactoperoxidase iodinated Fg-Da ae it did the Chloramine-T product. The use of these conjugates in the purification of immunoreactive Fg-Ds of high specific activity will be discussed. High affinity binding sites for calcium have recently been demonstrated in fibrinogen. Tha presence of bound calcium is also believed to protect Fg-Ds f m further digestion by plasmin. This is probably due to the formation of a more compact structure. However, conformational changes for calcium bound fibrinogen or Fg-Ds have not been observed. We tested the immunoreactivity of the lactoperoxidase iodinated Fg-Ds in presence and absence of calcium. Differences were found and this data suggests that soma modification of antigenic determinants takes place as a consequence of calcium in the environment.

scholarly journals Structural and functional properties of heparin analogues obtained by chemical sulphation of Escherichia coli K5 capsular polysaccharide

1995 ◽  
Vol 309 (2) ◽  
pp. 465-472 ◽  
Author(s):  
N Razi ◽  
E Feyzi ◽  
I Björk ◽  
A Naggi ◽  
B Casu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Binding Sites ◽  
Capsular Polysaccharide ◽  
Factor Xa ◽  
Compositional Analysis ◽  
Basic Structure ◽  
Sulphate Content ◽  
High Affinity ◽  
Structural And Functional Properties

Capsular polysaccharide from Escherichia coli K5, with the basic structure (GlcA beta 1-4GlcNAc alpha 1-4)n, was chemically modified through N-deacetylation, N-sulphation and O-sulphation [Casu, Grazioli, Razi, Guerrini, Naggi, Torri, Oreste, Tursi, Zoppetti and Lindahl (1994) Carbohydr. Res. 263, 271-284]. Depending on the reaction conditions, the products showed different proportions of components with high affinity for antithrombin (AT). A high-affinity subfraction, M(r) approx. 36,000, was shown by near-UV CD, UV-absorption difference spectroscopy and fluorescence to cause conformational changes in the AT molecule very similar to those induced by high-affinity heparin. Fluorescence titrations demonstrated about two AT-binding sites per polysaccharide chain, each with a Kd of approx. 200 nM. The anti-(Factor Xa) activity was 170 units/mg, similar to that of the IIId international heparin standard and markedly higher than activities of previously described heparin analogues. Another preparation, M(r) approx. 13,000, of higher overall O-sulphate content, exhibited a single binding site per chain, with Kd approx. 1 microM, and an anti-(Factor Xa) activity of 70 units/mg. Compositional analysis of polysaccharide fractions revealed a correlation between the contents of -GlcA-GlcNSO3(3,6-di-OSO3)- disaccharide units and affinity for AT; the 3-O-sulphated GlcN unit has previously been identified as a marker component of the AT-binding pentasaccharide sequence in heparin. The abundance of the implicated disaccharide unit approximately equalled that of AT-binding sites in the 36,000-M(r) polysaccharide fraction, and approached one per high-affinity oligosaccharide (predominantly 10-12 monosaccharide units) isolated after partial depolymerization of AT-binding polysaccharide. These findings suggest that the modified bacterial polysaccharide interacts with AT and promotes its anticoagulant action in a manner similar to that of heparin.

scholarly journals ATP hydrolysis by KaiC promotes its KaiA binding in the cyanobacterial circadian clock system

2019 ◽  
Vol 2 (3) ◽  
pp. e201900368 ◽  
Author(s):  
Yasuhiro Yunoki ◽  
Kentaro Ishii ◽  
Maho Yagi-Utsumi ◽  
Reiko Murakami ◽  
Susumu Uchiyama ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Conformational Changes ◽  
Binding Sites ◽  
Atp Hydrolysis ◽  
Periodic Oscillation ◽  
Mass Spectrometric ◽  
High Affinity ◽  
Clock System ◽  
Determining Factor

The cyanobacterial clock is controlled via the interplay among KaiA, KaiB, and KaiC, which generate a periodic oscillation of KaiC phosphorylation in the presence of ATP. KaiC forms a homohexamer harboring 12 ATP-binding sites and exerts ATPase activities associated with its autophosphorylation and dephosphorylation. The KaiC nucleotide state is a determining factor of the KaiB–KaiC interaction; however, its relationship with the KaiA–KaiC interaction has not yet been elucidated. With the attempt to address this, our native mass spectrometric analyses indicated that ATP hydrolysis in the KaiC hexamer promotes its interaction with KaiA. Furthermore, our nuclear magnetic resonance spectral data revealed that ATP hydrolysis is coupled with conformational changes in the flexible C-terminal segments of KaiC, which carry KaiA-binding sites. From these data, we conclude that ATP hydrolysis in KaiC is coupled with the exposure of its C-terminal KaiA-binding sites, resulting in its high affinity for KaiA. These findings provide mechanistic insights into the ATP-mediated circadian periodicity.

What the structure of a calcium pump tells us about its mechanism

2001 ◽  
Vol 356 (3) ◽  
pp. 665-683 ◽  
Author(s):  
Anthony G. LEE ◽  
J. Malcolm EAST
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Calcium Pump ◽  
Transmembrane Region ◽  
Site Model ◽  
High Affinity ◽  
Cytoplasmic Surface ◽  
Α Helix ◽  
Access Pathway ◽  
Phosphorylation Domain

The report of the crystal structure of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum in its Ca2+-bound form [Toyoshima, Nakasako and Ogawa (2000) Nature (London) 405, 647–655] provides an opportunity to interpret much kinetic and mutagenic data on the ATPase in structural terms. There are no large channels leading from the cytoplasmic surface to the pair of high-affinity Ca2+ binding sites within the transmembrane region. One possible access pathway involves the charged residues in transmembrane α-helix M1, with a Ca2+ ion passing through the first site to reach the second site. The Ca2+-ATPase also contains a pair of binding sites for Ca2+ that are exposed to the lumen. In the four-site model for transport, phosphorylation of the ATPase leads to transfer of the two bound Ca2+ ions from the cytoplasmic to the lumenal pair of sites. In the alternating four-site model for transport, phosphorylation leads to release of the bound Ca2+ ions directly from the cytoplasmic pair of sites, linked to closure of the pair of lumenal binding sites. The lumenal pair of sites could involve a cluster of conserved acidic residues in the loop between M1 and M2. Since there is no obvious pathway from the high-affinity sites to the lumenal surface of the membrane, transport of Ca2+ ions must involve a significant change in the packing of the transmembrane α-helices. The link between the phosphorylation domain and the pair of high-affinity Ca2+ binding sites is probably provided by two small helices, P1 and P2, in the phosphorylation domain, which contact the loop between transmembrane α-helices M6 and M7.

Substrate recognition and translocation by polyspecific organic cation transporters

2011 ◽  
Vol 392 (1-2) ◽  
Author(s):  
Hermann Koepsell
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Organic Cation ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Organic Cation Transporters ◽  
Cationic Drugs ◽  
High Affinity ◽  
Cation Transporters ◽  
Substrate Binding Sites

Abstract Organic cation transporters (OCTs) of the SLC22 family play a pivotal role in distribution and excretion of cationic drugs. They mediate electrogenic translocation of cations in both directions. OCTs are polyspecific transporters. During substrate translocation they perform a series of conformational changes involving an outward-facing conformation, an occluded state and an inward-facing conformation. Mutagenesis of OCT1 in combination with homology modeling showed that identical amino acids form the innermost parts of the outward-open and inward-open binding clefts. In addition to low affinity substrate binding sites, OCT1 contains high affinity substrate binding sites that can mediate inhibition via non-transported compounds.

scholarly journals Reactions of Platinum II Complexes with Sulfur- and Nitrogen-Containing Biomolecules: Selective Intermolecular Migration of the Thioether-Bound Platinum II Complex to the N7 Nitrogen Atom of Guanosine-5’-Monophosphate

2018 ◽  
Vol 19 (4) ◽  
pp. 325-330
Author(s):  
Ivana Vasic ◽  
Marija D. Zivkovic
Keyword(s):  
Nmr Spectroscopy ◽  
Side Effects ◽  
Nitrogen Atom ◽  
Antitumor Activity ◽  
1H Nmr Spectroscopy ◽  
Room Temperature ◽  
Molar Ratio ◽  
Second Step ◽  
Guanine Base ◽  
Sulfur Containing

Abstract The interactions of platinum(II) complexes with nitrogen- and sulfur-containing biomolecules are responsible for aider antitumor activity due to attack on DNA or a variety of toxic side effects. Therefore, the monofunctional Pt(II) complexes, [Pt(Gly-Gly-N,N,O)I]+ (Gly-Gly is the dipeptide glycyl-glycine coordinated through the oxygen and two nitrogen atoms) and [Pt(Gly-L-Met-S,N,N)Cl] (Gly-L-Met is the dipeptide glycyl-L-methionine coordinated through the sulfur and two nitrogen atoms) have been used to study their interactions with S-methylglutathione (GS-Me) and guanosine- 5’-monophosphate (5’-GMP). All reactions have been studied by 1H NMR spectroscopy and at room temperature in 50 mM phosphate buffer at pD 7.4. The investigation of the competitive binding of 5’-GMP and GS-Me to the Pt(II) complexes (1:1:1 molar ratio) has shown that in the initial stages of the reaction the corresponding Pt(II) complex only reacts with GS-Me, and second step of the reaction is very slow intermolecular displacement of the S-bound thioether ligand with N7 atom of the guanine base of 5’-GMP. The obtained results have been analyzed in relation to the antitumor activity and toxicity of Pt(II) complexes.

RNase A – tRNA binding alters protein conformation

2007 ◽  
Vol 85 (3) ◽  
pp. 311-318 ◽  
Author(s):  
C.N. N’soukpoé-Kossi ◽  
C. Ragi ◽  
H.A. Tajmir-Riahi
Keyword(s):  
Binding Sites ◽  
Binding Constant ◽  
Structural Changes ◽  
Structural Information ◽  
Binding Mode ◽  
Rnase A ◽  
Random Coil ◽  
Pancreatic Ribonuclease A ◽  
Α Helix ◽  
Rna Interaction

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P-O5′ bonds in RNA on the 3′ side of pyrimidine to form cyclic 2′,5′-phosphates. Even though extensive structural information is available on RNase A complexes with mononucleotides and oligonucleotides, the interaction of RNase A with tRNA has not been fully investigated. We report the complexation of tRNA with RNase A in aqueous solution under physiological conditions, using a constant RNA concentration and various amounts of RNase A. Fourier transform infrared, UV-visible, and circular dichroism spectroscopic methods were used to determine the RNase binding mode, binding constant, sequence preference, and biopolymer secondary structural changes in the RNase–tRNA complexes. Spectroscopic results showed 2 major binding sites for RNase A on tRNA, with an overall binding constant of K = 4.0 × 105 (mol/L)–1. The 2 binding sites were located at the G-C base pairs and the backbone PO2 group. Protein–RNA interaction alters RNase secondary structure, with a major reduction in α helix and β sheets and an increase in the turn and random coil structures, while tRNA remains in the A conformation upon protein interaction. No tRNA digestion was observed upon RNase A complexation.

scholarly journals The Conformational Structural Change of Soy Glycinin via Lactic Acid Bacteria Fermentation Reduced Immunoglobulin E Reactivity

Foods ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2969
Author(s):  
Zhen Liu ◽  
Yaqiong Wang ◽  
Yifei Liu ◽  
Qiuqin Zhang ◽  
Wei Li ◽  
...  
Keyword(s):  
Particle Size ◽  
Conformational Changes ◽  
Fluorescence Intensity ◽  
Immunoglobulin E ◽  
Principal Component ◽  
Surface Hydrophobicity ◽  
Intrinsic Fluorescence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ige Reactivity ◽  
Α Helix

This study investigated the fermentation of isolated soy glycinin by using the Lactiplantibacillus plantarum B1-6 strain, its reduction effect on immunoglobulin E (IgE) reactivity, the relationship with protein aggregation/gelation state and conformational changes. Fermentation was performed under different glycinin concentrations (0.1%, 0.5%, 1% and 2%, w/v) and varied fermentation terminal pH levels (FT-pH) (pH 6.0, 4.5, 4.0 and 3.5). L. plantarum B1-6 showed potency in reducing immunoreactivity to 0.10–69.85%, as determined by a sandwich enzyme-linked immunosorbent assay. At a FT-pH of 6.0 and 4.5, extremely low IgE reactivity (0.1–22.32%) was observed. Fermentation resulted in a great increase (2.31–6.8-fold) in particle size and a loss of intensity in A3 and basic subunits. The conformation of glycinin was altered, as demonstrated by improved surface hydrophobicity (1.33–7.39-fold), decreased intrinsic fluorescence intensity and the α-helix structure. Among the four selected concentrations, glycinin at 1% (w/v, G-1) evolved the greatest particles during fermentation and demonstrated the lowest immunoreactivity. Principal component analysis confirmed that particle size, intrinsic fluorescence intensity, α-helix and ionic bond were closely related to immunoreactivity reduction.

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