Time-Resolved Infrared Spectroscopy of Intermediates and Products from Photolysis of 1-(2-Nitrophenyl)ethyl Phosphates:  Reaction of the 2-Nitrosoacetophenone Byproduct with Thiols

Barth, Andreas; Corrie, John E. T.; Gradwell, Michael J.; Maeda, Yashusi; Mäntele, Werner; Meier, and Tanja; Trentham, David R.

doi:10.1021/ja964430u

Cited by 116 publications

(128 citation statements)

References 23 publications

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“…Note that, as in other vibrational analyses of caged compounds (16,18), construction of double-difference spectra utilizes an interactive subtraction to eliminate the 1525 and 1344 cm -1 lines, assigned to NO 2 stretching vibrations, to remove contributions from the photolytic reaction. The 15 N-nitro isotopomers of caged ATP (19), generously provided by J. Corrie (National Institute for Medical Research, U.K.), downshift vibrational modes in the 1525 cm -1 region (see Results) and permit analysis of any potential protein bands in the 1525 cm -1 region (data not shown).…”

Section: Methodsmentioning

confidence: 99%

Disparity in Allosteric Interactions of Monastrol with Eg5 in the Presence of ADP and ATP: A Difference FT-IR Investigation

et al. 2004

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Eg5 is a kinesin-like motor protein required for mitotic progression in higher eukaryotes. It is thought to cross-link antiparallel microtubules, and provides a force required for the formation of a bipolar spindle. Monastrol causes the catastrophic collapse of the mitotic spindle through the allosteric inhibition of Eg5. Utilizing a truncated Eg5 protein, we employ difference infrared spectroscopy to probe structural changes that occur in the motor protein with monastrol in the presence of either ADP or ATP. Difference FT-IR spectra of Eg5-monastrol-nucleotide complexes demonstrate that there are triggered conformational changes corresponding to an interconversion of secondary structural elements in the motor upon interaction with nucleotides. Notably, conformational changes elicited in the presence of ADP are different from those in the presence of ATP. In Eg5-monastrol complexes, exchange of ADP is associated with a decrease in random structure and an increase in R-helical content. In contrast, formation of the Eg5-monastrol-ATP complex is associated with a decrease in R-helical content and a concomitant increase in -sheet content. One or more carboxylic acid residues in Eg5 undergo unique changes when ATP, but not ADP, interacts with the motor domain in the presence of monastrol. This first direct dissection of inhibitorprotein interactions, using these methods, demonstrates a clear disparity in the structural consequences of monastrol in the presence of ADP versus ATP.Inhibition of mitosis is at the crux of clinical strategies for controlling tumor growth. Mitotic motor proteins, including Eg5, 1 dynein, and C-terminal kinesins, are required for bipolar spindle formation during mitosis. Small molecular inhibitors of the mitotic kinesin Eg5 have been uncovered from chemical screens; these are monastrol (1), terpendole E (2), and HR22C16 (3). These compounds arrest mitosis via reversible, allosteric inhibition of Eg5 and subsequent perturbation of bipolar mitotic spindle formation. It is noteworthy that these compounds do not affect other kinesin family members. Thus, inhibition of kinesin Eg5 provides a novel and specific mechanism for targeting the mitotic spindle, and a possible avenue for conferring general antiproliferative effects on cancerous growth.Eg5 is a member of the BimC/Eg5 (or N-2) class of the kinesin superfamily (4). The native Eg5 molecule is a homotetramer, organized with two sets of antiparallel dimers (5).The resulting pair of motor domains at each end of the tetrameric molecule are thought to cross-link microtubules in the mitotic spindle: the motor domains are attached to antiparallel microtubules and drive the separation of spindle poles by sliding microtubules against each other (6, 7). Kinesins share a mechanism of conformational "switching" for converting small structural changes in their nucleotidebinding sites into larger movements to provide force generation and motion. Responsible for microtubule binding and force generation, the motor domain of kinesins also implement...

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Section: Methodsmentioning

confidence: 99%

Disparity in Allosteric Interactions of Monastrol with Eg5 in the Presence of ADP and ATP: A Difference FT-IR Investigation

et al. 2004

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“…Ras·GTP is formed by a laser flash, and the following hydrolysis reaction is monitored by trFTIR with a resolution of milliseconds. Whereas the frequently used NPE-caged GTP [23,24] forms GTP via an intermediate (the aci-nitro anion), pHP-caged GTP forms GTP directly. In the latter, the cleavage is already performed in the exited state, within 500 ps.…”

Section: Introductionmentioning

confidence: 99%

Surface Change of Ras Enabling Effector Binding Monitored in Real Time at Atomic Resolution

et al. 2007

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Ras, the prototype of the Ras superfamily, acts as a molecular switch for cell growth. External growth signals induce a GDP-to-GTP exchange. This modifies the Ras surface (Ras(on)GTP) and enables effector binding, which then activates signal-transduction pathways. GTP hydrolysis, catalysed by Ras and GAP, returns the signal to "off" (Ras(off)GDP). Oncogenic mutations in Ras prevent this hydrolysis, and thereby cause uncontrolled cell growth. In the Ras(off)-to-Ras(on) transition, the Ras surface is changed by a movement of the switch I loop that controls effector binding. We monitored this surface change at atomic resolution in real time by time-resolved FTIR (trFTIR) spectroscopy. In the transition from Ras(off) to Ras(on) a GTP-bound intermediate is now identified, in which effector binding is still prevented (Ras(off)GTP). The loop movement from Ras(off)GTP to Ras(on)GTP was directly monitored by the C=O vibration of Thr35. The structural change creates a binding site with a rate constant of 5 s(-1) at 260 K. A small molecule that shifted the equilibrium from the Ras(on)GTP state towards the Ras(off)GTP state would prevent effector binding, even if hydrolysis were blocked by oncogenic mutations. We present a spectroscopic fingerprint of both states that can be used as an assay in drug screening for such small molecules.

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“…As the photolysis of caged ATP is a chemical reaction, it reflects in the infrared difference spectra [7,13,14,23]. Most of these changes are instantaneous at the time resolution (60 ms) of our rapid scan experiments, but the side reactions of the photolysis products are slower [12]. Because of the photolysis signals, it is advisable to optimise the concentration of the caged compound in the sample so that just enough compound is released to obtain saturating protein signals.…”

Section: Atp Binding To the Ca 2+ -Atpasementioning

confidence: 99%

Two sides of the same coin: How enzymes distort substrates and vice versa. An infrared spectroscopic view on pyruvate kinase and Ca2+-ATPase

Barth

2016

BSI

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Abstract. This review summarises our infrared spectroscopy and density functional theory studies on the mutual interactions between enzymes and their substrates. We investigated phosphoenolpyruvate bound to pyruvate kinase (EC 2.7.1.40, M1 isozyme), ATP bound to the Ca 2+ -ATPase (SERCA1a), and the aspartylphosphate moiety of the Ca 2+ -ATPase phosphoenzyme E2P. Conformational changes of the enzymes and distortions of substrate structure are discussed. In all cases, the infrared absorption of the substrate in the enzyme environment could be identified by a combination of reaction-induced difference spectroscopy and isotopic labelling. The experimentally-determined vibrational frequencies were interpreted in structural terms using experimental correlations or modelling of the active site in density functional theory calculations. For none of the three systems, a weakening of the bond that is cleaved in the following enzymatic reaction could be detected in the ground state of the enzyme-substrate complex. However, for the dephosphorylation reaction of the Ca 2+ -ATPase phosphoenzyme E2P, a high energy intermediate, not detected in experiments, is the reactant state according to density functional theory calculations.

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Time-Resolved Infrared Spectroscopy of Intermediates and Products from Photolysis of 1-(2-Nitrophenyl)ethyl Phosphates: Reaction of the 2-Nitrosoacetophenone Byproduct with Thiols

Cited by 116 publications

References 23 publications

Disparity in Allosteric Interactions of Monastrol with Eg5 in the Presence of ADP and ATP: A Difference FT-IR Investigation

Disparity in Allosteric Interactions of Monastrol with Eg5 in the Presence of ADP and ATP: A Difference FT-IR Investigation

Surface Change of Ras Enabling Effector Binding Monitored in Real Time at Atomic Resolution

Two sides of the same coin: How enzymes distort substrates and vice versa. An infrared spectroscopic view on pyruvate kinase and Ca2+-ATPase

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