Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis.
J Biomol NMR. 2011 Sep;51(1-2):21-34
Authors: Greenwood AI, Rogals MJ, De S, Lu KP, Kovrigin EL, Nicholson LK
Abstract
The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, [Formula: see text] and apparent Michaelis constants, [Formula: see text]. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific (13)C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide (13)C-(1)H constant time HSQC spectra to determine [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis]*=*3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.
Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis
Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis
Abstract The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer,
kcatcis and apparent Michaelis constants,
...
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09-30-2011 08:01 PM
Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis.
Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis.
Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis.
J Biomol NMR. 2011 Sep;51(1-2):21-34
Authors: Greenwood AI, Rogals MJ, De S, Lu KP, Kovrigin EL, Nicholson LK
Abstract
The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation,...
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0
09-30-2011 05:59 AM
Thermodynamic and NMR analysis of inhibitor binding to dihydrofolate reductase.
Thermodynamic and NMR analysis of inhibitor binding to dihydrofolate reductase.
Thermodynamic and NMR analysis of inhibitor binding to dihydrofolate reductase.
Bioorg Med Chem. 2010 Dec 15;18(24):8485-92
Authors: Batruch I, Javasky E, Brown ED, Organ MG, Johnson PE
Isothermal titration calorimetry (ITC) was used to determine the thermodynamic driving force for inhibitor binding to the enzyme dihydrofolate reductase (DHFR) from Escherichia coli. 1,4-Bis-{sulfanylmethyl}-3,6-dimethyl-benzene (1) binds DHFR:NADPH with a K(d) of 13±5 nM while the...
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03-09-2011 02:20 PM
[NMR paper] NMR backbone assignment of a protein kinase catalytic domain by a combination of seve
NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: application to the catalytic subunit of cAMP-dependent protein kinase.
Related Articles NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: application to the catalytic subunit of cAMP-dependent protein kinase.
Chembiochem. 2004 Nov 5;5(11):1508-16
Authors: Langer T, Vogtherr M, Elshorst B, Betz M, Schieborr U, Saxena K, Schwalbe H
Protein phosphorylation is one of the most important mechanisms...
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11-24-2010 10:03 PM
[NMR paper] Interpretation of NMR relaxation properties of Pin1, a two-domain protein, based on B
Interpretation of NMR relaxation properties of Pin1, a two-domain protein, based on Brownian dynamic simulations.
Related Articles Interpretation of NMR relaxation properties of Pin1, a two-domain protein, based on Brownian dynamic simulations.
J Biomol NMR. 2004 May;29(1):21-35
Authors: Bernadó P, Fernandes MX, Jacobs DM, Fiebig K, García de la Torre J, Pons M
Many important proteins contain multiple domains connected by flexible linkers. Inter-domain motion is suggested to play a key role in many processes involving molecular recognition....
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11-24-2010 09:51 PM
[NMR paper] NMR solution structure of the isolated Apo Pin1 WW domain: comparison to the x-ray cr
NMR solution structure of the isolated Apo Pin1 WW domain: comparison to the x-ray crystal structures of Pin1.
Related Articles NMR solution structure of the isolated Apo Pin1 WW domain: comparison to the x-ray crystal structures of Pin1.
Biopolymers. 2002 Feb;63(2):111-21
Authors: Kowalski JA, Liu K, Kelly JW
The NMR solution structure of the isolated Apo Pin1 WW domain (6-39) reveals that it adopts a twisted three-stranded antiparallel beta-sheet conformation, very similar to the structure exhibited by the crystal of this domain in the...
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11-24-2010 08:49 PM
[NMR paper] Three-dimensional modeling of the I-TevI homing endonuclease catalytic domain, a GIY-
Three-dimensional modeling of the I-TevI homing endonuclease catalytic domain, a GIY-YIG superfamily member, using NMR restraints and Monte Carlo dynamics.
Related Articles Three-dimensional modeling of the I-TevI homing endonuclease catalytic domain, a GIY-YIG superfamily member, using NMR restraints and Monte Carlo dynamics.
Protein Eng. 2001 Oct;14(10):717-21
Authors: Bujnicki JM, Rotkiewicz P, Kolinski A, Rychlewski L
Using a recent version of the SICHO algorithm for in silico protein folding, we made a blind prediction of the tertiary...
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11-19-2010 08:44 PM
[NMR paper] 1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides.
1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides.
Related Articles 1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides.
J Biol Chem. 2001 Jul 6;276(27):25150-6
Authors: Wintjens R, Wieruszeski JM, Drobecq H, Rousselot-Pailley P, Buée L, Lippens G, Landrieu I
The recent crystal structure of Pin1 protein bound to a doubly phosphorylated peptide from the C-terminal domain of RNA polymerase II revealed that binding interactions between Pin1 and its substrate take place through its...
Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis.
Linear Mode
