scholarly journals Allosteric regulation: Illuminating allosteric conformational change with an environmentally sensitive fluorescent probe

2015 ◽  
Vol 37 (3) ◽  
pp. 54-57
Author(s):  
Robert B. Freedman ◽  
Alan D.B. Malcolm
Keyword(s):  
Glutamate Dehydrogenase ◽  
Fluorescent Probe ◽  
Conformational Change ◽  
Conformational Changes ◽  
Allosteric Regulation ◽  
Structural Data ◽  
Biochemical Journal ◽  
Environmentally Sensitive ◽  
The 1960S ◽  
Classic Paper

Allosteric regulation was a hot topic in the 1960s, but there was very limited structural data on allosteric equilibria, and no solid information on the rates of allosteric conformational changes. In this Biochemical Journal Classic paper from 1969 George Radda and his first D.Phil. student, George Dodd determined the rate of allosteric transition in the regulatory enzyme glutamate dehydrogenase by a method new in the 1960s, the fluorescence of an environmentally sensitive extrinsic probe.

scholarly journals Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride

1993 ◽  
Vol 291 (1) ◽  
pp. 103-107 ◽  
Author(s):  
H M Zhou ◽  
X H Zhang ◽  
Y Yin ◽  
C L Tsou
Keyword(s):  
Creatine Kinase ◽  
Fluorescent Probe ◽  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Emission Intensity ◽  
Enzyme Inactivation ◽  
Amino Groups ◽  
Guanidinium Chloride ◽  
Low Concentrations

It has been previously reported that, during denaturation of creatine kinase by guanidinium chloride (GdmCl) or urea [Tsou (1986), Trends Biochem. Sci. 11, 427-429], inactivation occurs before noticeable conformational change can be detected, and it is suggested that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. In this study, the thiol and amino groups at or near the active site of creatine kinase are labelled with o-phthalaldehyde to form a fluorescent probe. Both the emission intensity and anisotropy decrease during denaturation indicating exposure of this probe and increased mobility of the active site. The above conformational changes take place together with enzyme inactivation at lower GdmCl concentrations than required to bring about intrinsic fluorescence changes of the enzyme. At the same GdmCl concentration, the rate of exposure of the probe is comparable with that of inactivation and is several orders of magnitude faster than that for the unfolding of the molecule as a whole.

scholarly journals Slow conformational changes of a Neurospora glutamate dehydrogenase studied by protein fluorescence

1974 ◽  
Vol 143 (2) ◽  
pp. 317-329 ◽  
Author(s):  
Barrie Ashby ◽  
John C. Wootton ◽  
John R. S. Fincham
Keyword(s):  
Glutamate Dehydrogenase ◽  
Conformational Change ◽  
Conformational Changes ◽  
Fluorescence Emission ◽  
Enzyme Activation ◽  
Protein Fluorescence ◽  
Fluorescence Emission Spectra ◽  
Iodide Ions ◽  
Inactive State ◽  
Ph Values

1. The NADP-dependent glutamate dehydrogenase of Neurospora crassa undergoes slow reversible structural transitions, with half-times in the order of a few minutes, between active and inactive states. The inactive state of the enzyme, which predominates at pH values below 7.0, has an intrinsic tryptophan fluorescence 25% lower than that of the active state, which predominates at pH values above 7.6. The inactive state can be activated either by an increase in pH or by addition of activators such as succinate. 2. The kinetics of the slow transitions that follow activating and inactivating rapid changes in conditions have been monitored by measurements of protein fluorescence. The results show that the slow reversible conformational change detected by the change in fluorescence is the rate-limiting process for enzyme activation and inactivation. 3. In both directions this conformational change follows apparent first-order kinetics and the rate constant is independent of protein concentration. These kinetics and published measurements of molecular weight are indicative of an isomerization process. 4. In both directions the changes show a large energy of activation and a large positive entropy of activation, consistent with a considerable disturbance of conformation in the transition state. 5. Comparisons of the fluorescence emission spectra of the active and inactive states indicate that the difference in fluorescence is produced by quenching, possibly intramolecular, in the inactive conformation. Iodide ions cause similar quenching. 6. In some mutationally altered forms of the enzyme comparable but modified conformational changes can be followed by protein fluorescence.

scholarly journals Studies of Conformational Changes of Tubulin Induced by Interaction with Kinesin Using Atomistic Molecular Dynamics Simulations

2021 ◽  
Vol 22 (13) ◽  
pp. 6709
Author(s):  
Xiao-Xuan Shi ◽  
Peng-Ye Wang ◽  
Hong Chen ◽  
Ping Xie
Keyword(s):  
Molecular Dynamics ◽  
High Resolution ◽  
Binding Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Weak Interactions ◽  
Molecular Motor ◽  
Structural Data ◽  
Calculated Binding Energy ◽  
Dynamics Simulations

The transition between strong and weak interactions of the kinesin head with the microtubule, which is regulated by the change of the nucleotide state of the head, is indispensable for the processive motion of the kinesin molecular motor on the microtubule. Here, using all-atom molecular dynamics simulations, the interactions between the kinesin head and tubulin are studied on the basis of the available high-resolution structural data. We found that the strong interaction can induce rapid large conformational changes of the tubulin, whereas the weak interaction cannot. Furthermore, we found that the large conformational changes of the tubulin have a significant effect on the interaction of the tubulin with the head in the weak-microtubule-binding ADP state. The calculated binding energy of the ADP-bound head to the tubulin with the large conformational changes is only about half that of the tubulin without the conformational changes.

scholarly journals New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

2002 ◽  
Vol 76 (9) ◽  
pp. 4456-4466 ◽  
Author(s):  
Jennifer A. Gruenke ◽  
R. Todd Armstrong ◽  
William W. Newcomb ◽  
Jay C. Brown ◽  
Judith M. White
Keyword(s):  
Influenza Virus ◽  
Conformational Change ◽  
Conformational Changes ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Fusion Peptide ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Target Membrane ◽  
Mutant Forms

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI

2018 ◽  
Vol 118 (02) ◽  
pp. 340-350 ◽  
Author(s):  
Ingrid Stroo ◽  
J. Marquart ◽  
Kamran Bakhtiari ◽  
Tom Plug ◽  
Alexander Meijer ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Catalytic Domain ◽  
Active Form ◽  
Coagulation Factor ◽  
Lysine Residue ◽  
Factor Ix ◽  
Factor Xi ◽  
Lysine Residues ◽  
Factor Xia

AbstractCoagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

scholarly journals The structure and allosteric regulation of mammalian glutamate dehydrogenase

2012 ◽  
Vol 519 (2) ◽  
pp. 69-80 ◽  
Author(s):  
Ming Li ◽  
Changhong Li ◽  
Aron Allen ◽  
Charles A. Stanley ◽  
Thomas J. Smith
Keyword(s):  
Glutamate Dehydrogenase ◽  
Allosteric Regulation
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