Caged-GTP

Recent research focus now on applying FTIR methods to proteins without an intrinsic chromophore. In investigating oncogenic hras p21, for instance, protein activity is stimulated with a photolabile substance such as caged GTP (Fig 6.6-21). A UV laser flash, which separates the head group and releases GTP, triggers the GTPase activity of p21 This new experimental approach offers now a very broad applicability to study chemical reactions by time-resolved FTIR. 

Figure 6.6-20 FTIR-difference spectra of caged GTP-photolysis.

Crystals (P3221 a=5=41.0A, c=164.8A, size about 0.4x0.4x0.2 mm3) of Ha-ras 21 protein (Pai et al 1989) with caged GTP have been used as the starting point for investigating the structural states occurring on a pathway of GTP hydrolysis. The structure of a fairly short-lived complex (p21-GTP) was determined by Laue diffraction methods. 

The use of caged compounds in muscle diffraction had been pioneered by the Heidelberg group. These methods were then applied by these workers to the crystalline state. After photolytic removal (figure 10.14) of the 2-nitrophenyl ethyl group from caged GTP at the active site of p21,... 

A Laue data set was recorded during the first 5 min after photolysis of the crystalline p21 caged GTP complex. The major species present at the active site was GTP as confirmed by the electron density map which showed clearly discernible phosphate peaks (figure 10.15(h)). Around the essential Mg2+ ion an expected water molecule was not apparent. 

Figure 10.15 Electron density maps of the bound nucleotides at the active site of p21c (a) caged GTP, (b) GTP, (c) GTP/GDP, (d) GDP. From Schlichting et al (1990) with permission, copyright 1990 Macmillan Magazines Ltd.

TRIR techniques have also been used to characterize an unexpected decarboxylation that occurs during the photolysis of sulfonamide-protected derivatives of amino acids [148]. In addition, the photochemical release of ATP, GTP, and calcium ions from caged compounds has been used in conjunction with TRIR spectroscopy to gain insight into molecular reaction mechanisms of proteins [149],... 

Derivative 57a was synthesized by reacting the phosphate directly with p-hydroxyacetophenone bromide. Derivative 57b was synthesized from hydrogenolysis of the pHP dibenzyl phosphate ester. For 35 and 57c, the pHP monophosphate (57b) was first protected as the corresponding ketal that was then coupled with ADP or GDP, respectively. The caged ATP and GTP analogs were then obtained by hydrolysis of the ketal. Yields and quantum efficiencies for the disappearance of the pHP phosphate esters are given in Table 69.16. 

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