scholarly journals ATP Ground- and Transition States of Bacterial Enhancer Binding AAA+ ATPases Support Complex Formation with Their Target Protein, σ54

Structure ◽  
2007 ◽  
Vol 15 (4) ◽  
pp. 429-440 ◽  
Author(s):  
Baoyu Chen ◽  
Michaeleen Doucleff ◽  
David E. Wemmer ◽  
Sacha De Carlo ◽  
Hector H. Huang ◽  
...  
Keyword(s):  
Complex Formation ◽  
Transition States ◽  
Target Protein ◽  
Aaa Atpases

scholarly journals Kinetic analysis of copper transfer from a chaperone to its target protein mediated by complex formation

2017 ◽  
Vol 53 (8) ◽  
pp. 1397-1400 ◽  
Author(s):  
Kristine L. Kay ◽  
Liang Zhou ◽  
Leonardo Tenori ◽  
Justin M. Bradley ◽  
Chloe Singleton ◽  
...  
Keyword(s):  
Complex Formation ◽  
Kinetic Analysis ◽  
Target Protein ◽  
P Rate ◽  
Copper Transfer

Rate of Cu(i) transfer between chaperone and target protein is enhanced dramatically by complex formation.

scholarly journals Rationalizing PROTAC-mediated ternary complex formation using Rosetta

2020 ◽  
Author(s):  
Nan Bai ◽  
Palani Kirubakaran ◽  
John Karanicolas
Keyword(s):  
Complex Formation ◽  
Ternary Complex ◽  
E3 Ligase ◽  
Computational Method ◽  
Target Protein ◽  
Binding Modes ◽  
Trial And Error ◽  
Protein Targets ◽  
E3 Ligases ◽  
Ternary Complex Formation

AbstractPROTACs are molecules that combine a target-binding warhead with an E3 ligase-recruiting moiety; by drawing the target protein into a ternary complex with the E3 ligase, PROTACs induce target protein degradation. While PROTACs hold exciting potential as chemical probes and as therapeutic agents, development of a PROTAC typically requires synthesis of numerous analogs to thoroughly explore variations on the chemical linker; without extensive trial and error, it is unclear how to link the two protein-recruiting moieties to promote formation of a productive ternary complex. Here, we describe a structure-based computational method for evaluating suitability of a given linker for ternary complex formation. Our method uses Rosetta to dock the protein components, then builds the PROTAC from its component fragments into each binding mode; complete models of the ternary complex are then refined. We apply this approach to retrospectively evaluate multiple PROTACs from the literature, spanning diverse target proteins. We find that modeling ternary complex formation is sufficient to explain both activity and selectivity reported for these PROTACs, implying that other cellular factors are not key determinants of activity in these cases. We further find that interpreting PROTAC activity is best approached using an ensemble of structures of the ternary complex rather than a single static conformation, and that members of a structurally-conserved protein family can be recruited by the same PROTAC through vastly different binding modes. To encourage adoption of these methods and promote further analyses, we disseminate both the computational methods and the models of ternary complexes.Significance StatementRecent years have brought a flood of interest in developing compounds that selectively degrade protein targets in cells, as exemplified by PROTACs. Fully realizing the promise of PROTACs to transform chemical biology by delivering degraders of diverse and undruggable protein targets has been impeded, however, by the fact that designing a suitable chemical linker between the functional moieties requires extensive trial and error. Here, we describe a structure-based computational method to predict PROTAC activity. We envision that this approach will allow design and optimization of PROTACs for efficient target degradation, selection of E3 ligases best suited for pairing with a given target protein, and understanding the basis by which PROTACs can exhibit different target selectivity than their component warheads.

Comparative Labelling and Biokinetic Studies of 99mTc-EDTA(Sn) and 99mTc-DTPA(Sn)

1977 ◽  
Vol 16 (01) ◽  
pp. 30-35 ◽  
Author(s):  
N. Agha ◽  
R. B. R. Persson
Keyword(s):  
Complex Formation ◽  
Significant Influence ◽  
Room Temperature ◽  
Ph Value ◽  
Maximum Activity ◽  
Reduction Step ◽  
Labelling Yield ◽  
Different Temperatures ◽  
Kinetics Of

SummaryGelchromatography column scanning has been used to study the fractions of 99mTc-pertechnetate, 99mTcchelate and reduced hydrolyzed 99mTc in preparations of 99mTc-EDTA(Sn) and 99mTc-DTPA(Sn). The labelling yield of 99mTc-EDTA(Sn) chelate was as high as 90—95% when 100 μmol EDTA · H4 and 0.5 (Amol SnCl2 was incubated with 10 ml 99mTceluate for 30—60 min at room temperature. The study of the influence of the pH-value on the fraction of 99mTc-EDTA shows that pH 2.8—2.9 gave the best labelling yield. In a comparative study of the labelling kinetics of 99mTc-EDTA(Sn) and 99mTc- DTPA(Sn) at different temperatures (7, 22 and 37°C), no significant influence on the reduction step was found. The rate constant for complex formation, however, increased more rapidly with increased temperature for 99mTc-DTPA(Sn). At room temperature only a few minutes was required to achieve a high labelling yield with 99mTc-DTPA(Sn) whereas about 60 min was required for 99mTc-EDTA(Sn). Comparative biokinetic studies in rabbits showed that the maximum activity in kidneys is achieved after 12 min with 99mTc-EDTA(Sn) but already after 6 min with 99mTc-DTPA(Sn). The long-term disappearance of 99mTc-DTPA(Sn) from the kidneys is about five times faster than that for 99mTc-EDTA(Sn).

Inactivation of α- and β-Thrombin by Antithrombin-III and Heparin

1976 ◽  
Vol 36 (03) ◽  
pp. 503-508 ◽  
Author(s):  
Raymund Machovich ◽  
György Blaskó ◽  
Anna Borsodi
Keyword(s):  
Heat Treatment ◽  
Complex Formation ◽  
Blood Circulation ◽  
Antithrombin Iii

SummaryInactivation of α- and β-thrombin by antithrombin-III and heparin was studied, since it had been suggested that two forms of thrombin exist with respect to heparin sensitivity (Machovich 1975b).It was found that the inactivation rates of α- and β-thrombin by antithrombin were different, namely α-thrombin was more sensitive to antithrombin than β-thrombin. Heparin facilitated the complex formation between α-thrombin and antithrombin-III, whereas β-thrombin inactivation was only slightly affected.Furthermore, heparin protected α-thrombin against the inactivating effect of heat, while β-thrombin lost its activity during the heat treatment.These findings suggest that the formation of β-thrombin in blood circulation may have an important role in thrombosis predisposition.

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

Western Blot Analyses of Prekallikrein and its Activation Products in Human Plasma

1991 ◽  
Vol 65 (04) ◽  
pp. 382-388 ◽  
Author(s):  
Dulce Veloso ◽  
Robert W Colman
Keyword(s):  
Complex Formation ◽  
Western Blot ◽  
Human Plasma ◽  
Contact System ◽  
Plasma Activation ◽  
Normal Plasma ◽  
Sds Page ◽  
Activation Products ◽  
Major Antigen ◽  
Formation Of Complexes

SummaryPrekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAl-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G1L. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with CL inhibitor (C1 INH) and α2-rrtzcroglobulin (α2M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-α2M complexes. Complexes of KALantithrombin III (ATIII) and the ratio of KALα2M/ KAL-CL INH were higher in activated CL INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KALα2M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and α2M. Immunoblots of activated plasma also showed bands at the position of KALCL INH and KALATIII complexes. When α1-antitrypsin Pittsburgh (cα1-AT, Pitts) was added to plasma before activation, KAL-α1-ALPitts was the main complex. The non-activated normal plasma revealed only an overloaded PK band. This is the first report of an antibody that recognizes KAL epitope(s) in KAL-α2M, KALATIII and KALa1-α1Pitts complexes and in the 45-kDa fragment(s). Therefore, MAb 13G11 should be useful for studying the structure of these complexes as well as the mechanism of complex formation. In addition, immunoblotting with MAb 13G11 would allow detection of KAl-inhibitor complexes in patient plasmas as indicators of activation of the contact system.

Sign in / Sign up

Export Citation Format

Share Document