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Burlacu-Miron, Simona; Perrier, Véronique; Gilles, Anne‐Marie; Mispelter, Joël; Bârzu, Octavian; Craescu, Constantin T.

doi:10.1023/a:1008342615045

Cited by 13 publications

(8 citation statements)

References 5 publications

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“…The experimental NMR data were analyzed on Silicon Graphics workstations with the software packages nmrPipe and modelXY. 39 The previously determined assignments of the AKeco 1 H- 15 N correlations, 40 complemented and revised in our earlier study, 15 were used.…”

Section: Methodsmentioning

confidence: 99%

Activation Energy of Catalysis-Related Domain Motion in E. coli Adenylate Kinase

Shapiro

Meirovitch

2006

J. Phys. Chem. B

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Adenylate kinase from E. coli (AKeco), folded into domains CORE, AMPbd, and LID, catalyzes the reaction AMP + ATP <--> 2ADP. Previous X-ray crystallography and optical solution methods showed that the domains AMPbd and LID, and the conserved P-loop, execute large-amplitude catalysis-related motions. We used (15)N NMR spin relaxation methods to find that the simplified model-free (MF) analysis does not, whereas our general Slowly Relaxing Local Structure analysis does, detect catalytic domain motion. SRLS set for the first time the correlation time for domain motion at tau(L)perpendicular = 8.2 ns, to be compared with tau(m) = 15.1 ns for global tumbling. These results were obtained at 303 K. Herein we conduct a temperature-dependent investigation of tau(L)perpendicular and tau(m) in the range of 288-310 K. We found that the activation energy for global tumbling is Ea = 16.9 +/- 0.5 kJ/mol, the hydrodynamic volume of hydrated AKeco is 65.6 +/- 2.1 nm3, its radius is 2.50 +/- 0.03 nm, and the number of hydration layers is 1.77. The average tau(L)perpendicular value decreases from 11 ns at 288 K to 4 ns at 310 K, with activation energies of 29.7 +/- 3.3, 32.1 +/- 4.3, and 30.4 +/- 4.3 kJ/mol for the domains AMPbd and LID, and the catalytic P-loop, respectively. These values are two-to-three times smaller than typical activation energies of enzymatic reactions. Hence kinase catalysis appears not to be controlled by domain motion in the ligand-free enzyme. However, the latter process clearly facilitates important mechanical aspects such as steric recognition and capturing of the AMP and ATP substrates, their proper positioning for phosphorylation, and the release of the ADP product.

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Section: Methodsmentioning

confidence: 99%

Activation Energy of Catalysis-Related Domain Motion in E. coli Adenylate Kinase

Shapiro

Meirovitch

2006

J. Phys. Chem. B

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“…NMR data were analyzed on Silicon Graphics workstations using the software packages nmrPipe and modelXY (37). The previously determined assignments of the AKeco 1 H- 15 N correlations (38), complemented and revised in our earlier study (26), were used. The assignments of the AKeco*AP 5 A 1 H- 15 N correlations were taken from ref 39.…”

Section: Methodsmentioning

confidence: 99%

Domain Flexibility in Ligand-Free and Inhibitor-BoundEscherichia coliAdenylate Kinase Based on a Mode-Coupling Analysis of¹⁵N Spin Relaxation

et al. 2002

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Adenylate kinase from Escherichia coli (AKeco), consisting of a 23.6-kDa polypeptide chain folded into domains CORE, AMPbd, and LID catalyzes the reaction AMP + ATP T 2ADP. The domains AMPbd and LID execute large-amplitude movements during catalysis. Backbone dynamics of ligandfree and AP 5 A-inhibitor-bound AKeco is studied with slowly relaxing local structure (SRLS) 15 N relaxation, an approach particularly suited when the global (τ m ) and the local (τ) motions are likely to be coupled. For AKeco τ m ) 15.1 ns, whereas for AKeco*AP 5 A τ m ) 11.6 ns. The CORE domain of AKeco features an average squared order parameter, , of 0.84 and correlation times τ f ) 5-130 ps. Most of the AKeco*AP 5 A backbone features ) 0.90 and τ f ) 33-193 ps. These data are indicative of relative rigidity. Domains AMPbd and LID of AKeco, and loops 1 /R 1 , R 2 /R 3 , R 4 / 3 , R 5 / 4 , and 8 /R 7 of AKeco*AP 5 A, feature a novel type of protein flexibility consisting of nanosecond peptide plane reorientation about the C i-1 R -C i R axis, with correlation time τ ⊥ ) 5.6-11.3 ns. The other microdynamic parameters underlying this dynamic model include S 2 ) 0.13-0.5, τ || on the ps time scale, and a diffusion tilt MD ranging from 12 to 21°. For the ligand-free enzyme the τ ⊥ mode was shown to represent segmental domain motion, accompanied by conformational exchange contributions R ex e 4.4 s -1 . Loop R 4 / 3 and R 5 / 4 dynamics in AKeco*AP 5 A is related to the "energetic counter-balancing of substrate binding" effect apparently driving kinase catalysis. The other flexible AKeco*AP 5 A loops may relate to domain motion toward product release.The ability to interpret nuclear spin relaxation properties in terms of microdynamic parameters turned NMR into a powerful method for elucidating protein dynamics (1, 2). The amide 15 N spin in proteins is a particularly useful probe, relaxed predominantly by dipolar coupling to the amide proton and 15 N chemical shift anisotropy (CSA) 1 (3). The experimental NMR observables are controlled by the global and local dynamic processes experienced by protein N-H bond vectors, which determine the spectral density function, J(ω). 15 N relaxation data in proteins are commonly analyzed with the model-free (MF) approach, where the global and local motions are assumed to be decoupled (4-6). In a recent study (7), we applied the two-body slowly relaxing local structure (SRLS) approach developed by Freed and coworkers (8, 9) to 15 N relaxation in proteins. SRLS accounts rigorously for dynamical coupling between the local and global motions, and treats the global diffusion, the local diffusion, the local ordering, and the magnetic interactions as tensors that may be tilted relative to one another, providing thereby important information related to protein structure (10-12). The MF spectral density functions constitute asymptotic solutions of the SRLS spectral densities (7,8,13). It was found that currently available experimental 15 N relaxation data are sensitive to the coupling-induce...

~~show abstract~~

“…NMR data were analyzed on Silicon Graphics workstations using the software packages nmrPipe and modelXY. 35 The previously determined assignments of the AKeco 1 H-15 N correlations, 36 complemented and revised in our earlier study, 34 were used.…”

Section: Methodsmentioning
confidence: 99%

Mode-Coupling Analysis of ¹⁵N CSA−¹⁵N-¹H Dipolar Cross-Correlation in Proteins. Rhombic Potentials at the N−H Bond
Meirovitch
¹
,
Shapiro
²
,
Tugarinov
³

et al. 2003
J. Phys. Chem. B
23
3
98
0
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N CSA-15 N-1 H dipolar cross-correlation (η xy ) in proteins has been treated thus far with the model-free (MF) approach, where the global (R C ) and local (R L ) motions are assumed to be decoupled as a consequence of R L . R C . In the context of η xy , it is additionally assumed that the local motion is very fast and highly symmetrical. We have recently applied to auto-correlated 15 N spin relaxation the slowly relaxing local structure (SRLS) approach, which accounts rigorously for mode-coupling. SRLS can analyze η xy for arbitrary time scale separations between R L and R C . Simulations of η xy for slow local motions are presented herein for the first time. Experimental η xy values of RNase and AKeco could not be reproduced from best-fit parameters generated by data fitting that used axial potentials. Calculations showed they are reproducible using rhombic coupling/ordering potentials. The conformational exchange term, R ex , "absorbs" potential rhombicity when axial potentials are used to fit the data. 15 N CSA variability and R C anisotropy are shown to have a small effect on the analysis. The shape of the rhombic potentials detected corresponds to nearly "planar Y M X M ordering" (M denotes the local ordering frame). This potential form is consistent with the known geometry of the peptide plane, the SRLS dynamic model in the R ⊥ L ≈ R C regime, and makes possible associating the local director with the C i-1 R -C i R axis. It is shown that back-calculation of NMR variables from the best-fit parameters and comparison with the experimental counterparts is an effective tool for identifying inappropriate elements of the dynamic model and can thereby help improve it.

~~show abstract~~

Untitled

Cited by 13 publications

References 5 publications

Activation Energy of Catalysis-Related Domain Motion in E. coli Adenylate Kinase

Activation Energy of Catalysis-Related Domain Motion in E. coli Adenylate Kinase

Domain Flexibility in Ligand-Free and Inhibitor-BoundEscherichia coliAdenylate Kinase Based on a Mode-Coupling Analysis of¹⁵N Spin Relaxation

Mode-Coupling Analysis of ¹⁵N CSA−¹⁵N-¹H Dipolar Cross-Correlation in Proteins. Rhombic Potentials at the N−H Bond

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Untitled

Cited by 13 publications

References 5 publications

Activation Energy of Catalysis-Related Domain Motion in E. coli Adenylate Kinase

Activation Energy of Catalysis-Related Domain Motion in E. coli Adenylate Kinase

Domain Flexibility in Ligand-Free and Inhibitor-BoundEscherichia coliAdenylate Kinase Based on a Mode-Coupling Analysis of15N Spin Relaxation

Mode-Coupling Analysis of 15N CSA−15N-1H Dipolar Cross-Correlation in Proteins. Rhombic Potentials at the N−H Bond

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Product

Resources

About

Domain Flexibility in Ligand-Free and Inhibitor-BoundEscherichia coliAdenylate Kinase Based on a Mode-Coupling Analysis of¹⁵N Spin Relaxation

Mode-Coupling Analysis of ¹⁵N CSA−¹⁵N-¹H Dipolar Cross-Correlation in Proteins. Rhombic Potentials at the N−H Bond