1gsi: Difference between revisions

Revision as of 13:53, 21 February 2008

CRYSTAL STRUCTURE OF MYCOBACTERIUM TUBERCULOSIS THYMIDYLATE KINASE COMPLEXED WITH THYMIDINE MONOPHOSPHATE (TMP)

OverviewOverview

Caged compounds in combination with protein crystallography represent a valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed close to room temperature. Synchronous reaction initiation has only been achieved with enzymes of relatively slow turnover (<0.1 s(-1)) and caged compounds of high quantum yield. Here X-ray crystallography and microspectrophotometry were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds can be photolyzed by UV light at cryotemperatures. NPE-caged ATP in flash-cooled crystals of Mycobacterium tuberculosis thymidylate kinase was photolyzed successfully at 100-150 K as assessed by the structural observation of ATP-dependent enzymatic conversion of TMP to TDP after temporarily warming the crystals to room temperature. A new method is proposed in which cryo-photolysis combined with temperature-controlled protein crystallography can be used to trap reaction intermediates even in some of the fastest enzymes and/or when only compounds of low quantum yield are available. Raising the temperature after cryophotolysis may allow a transition barrier to be passed and an intermediate to accumulate in the crystal. A comparable method has only been used so far with proteins displaying endogenous photosensitivity. The approach described here opens the way to studying the reaction mechanisms of a much larger number of crystalline enzymes. Furthermore, it is shown that X-ray-induced radiolysis of caged compounds occurs if high-intensity synchrotron beamlines are used. This caveat should be taken into account when deriving data-collection protocols. It could also be used potentially as a way to trigger reactions.

About this StructureAbout this Structure

1GSI is a Single protein structure of sequence from Mycobacterium tuberculosis with , , and as ligands. Active as dTMP kinase, with EC number 2.7.4.9 Known structural/functional Site: . Full crystallographic information is available from OCA.

ReferenceReference

Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals., Ursby T, Weik M, Fioravanti E, Delarue M, Goeldner M, Bourgeois D, Acta Crystallogr D Biol Crystallogr. 2002 Apr;58(Pt 4):607-14. Epub 2002, Mar 22. PMID:11914484

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 ==Overview==
-Caged compounds in combination with protein crystallography represent a, valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed close to room, temperature. Synchronous reaction initiation has only been achieved with, enzymes of relatively slow turnover (&lt;0.1 s(-1)) and caged compounds of, high quantum yield. Here X-ray crystallography and microspectrophotometry, were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds, can be photolyzed by UV light at cryotemperatures. NPE-caged ATP in, flash-cooled crystals of Mycobacterium tuberculosis thymidylate kinase was, photolyzed successfully at 100-150 K as assessed by the structural, observation of ATP-dependent enzymatic conversion of TMP to TDP after, temporarily warming the crystals to room temperature. A new method is, proposed in which cryo-photolysis combined with temperature-controlled, protein crystallography can be used to trap reaction intermediates even in, some of the fastest enzymes and/or when only compounds of low quantum, yield are available. Raising the temperature after cryophotolysis may, allow a transition barrier to be passed and an intermediate to accumulate, in the crystal. A comparable method has only been used so far with, proteins displaying endogenous photosensitivity. The approach described, here opens the way to studying the reaction mechanisms of a much larger, number of crystalline enzymes. Furthermore, it is shown that X-ray-induced, radiolysis of caged compounds occurs if high-intensity synchrotron, beamlines are used. This caveat should be taken into account when deriving, data-collection protocols. It could also be used potentially as a way to, trigger reactions.
+Caged compounds in combination with protein crystallography represent a valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed close to room temperature. Synchronous reaction initiation has only been achieved with enzymes of relatively slow turnover (&lt;0.1 s(-1)) and caged compounds of high quantum yield. Here X-ray crystallography and microspectrophotometry were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds can be photolyzed by UV light at cryotemperatures. NPE-caged ATP in flash-cooled crystals of Mycobacterium tuberculosis thymidylate kinase was photolyzed successfully at 100-150 K as assessed by the structural observation of ATP-dependent enzymatic conversion of TMP to TDP after temporarily warming the crystals to room temperature. A new method is proposed in which cryo-photolysis combined with temperature-controlled protein crystallography can be used to trap reaction intermediates even in some of the fastest enzymes and/or when only compounds of low quantum yield are available. Raising the temperature after cryophotolysis may allow a transition barrier to be passed and an intermediate to accumulate in the crystal. A comparable method has only been used so far with proteins displaying endogenous photosensitivity. The approach described here opens the way to studying the reaction mechanisms of a much larger number of crystalline enzymes. Furthermore, it is shown that X-ray-induced radiolysis of caged compounds occurs if high-intensity synchrotron beamlines are used. This caveat should be taken into account when deriving data-collection protocols. It could also be used potentially as a way to trigger reactions.
 ==About this Structure==
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 [[Category: transferase (atp:tmp phosphotransferase)]]
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