Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time.

The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Ras was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (P(3)-1-(2-nitro)phenylethyl guanosine 5'-O-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP x Ras x GDP x AlF(x) transition state analog is known, which represents a "snap shot" along the reaction-pathway. As now revealed, binding of GAP to Ras x GTP shifts negative charge from the gamma to beta phosphate. Such a shift was already identified by FTIR in GTP because of Ras binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP x Ras x GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between beta and gamma phosphate is cleaved. In the intermediate, the released P(i) is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Ras. FTIR analysis reveals the release of P(i) from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein-protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.

Ras catalyzes GTP hydrolysis by shifting negative charges from gamma- to beta-phosphate as revealed by time-resolved FTIR difference spectroscopy.

FTIR difference spectroscopy has been used to determine the molecular GTPase mechanism of the small GTP binding protein Ras at the atomic level. The reaction was initiated by the photolysis of caged GTP bound to Ras. The addition of catalytic amounts of the GTPase activating protein (GAP) reduces the measuring time by 2 orders of magnitude but has no influence on the spectra as compared to the intrinsic reaction. The reduced measuring time improves the quality of the data significantly as compared to previously published data [Cepus, V., Scheidig, A., Goody, R. S., and Gerwert, K. (1998) Biochemistry 37, 10263-10271]. The phosphate vibrations are assigned using 18O-labeled caged GTP. In general, there is excellent agreement with the results of Cepus et al., except in the nu(a)(alpha-PO2-) vibration assignments. The assignments reveal that binding of GTP to Ras induces vibrational uncoupling into mainly individual vibrations of the alpha-, beta-, and gamma-phosphate groups. In contrast, for unbound GTP, the phosphate vibrations are highly coupled and the corresponding absorption bands are broader. This result indicates that binding to Ras forces the flexible GTP molecule into a strained conformation and induces a specific charge distribution different from that in the unbound case. The binding causes an unusual frequency downshift of the GTP beta-PO2- phosphate vibration, whereas the alpha-PO2- and gamma-PO3(2-) phosphate vibrations shift to higher wavenumbers. The frequency downshift indicates a lowering of the bond order of the nonbridged P-O bonds of the beta-phosphate group of GTP and GDP. The bond order changes can be explained by a shift of negative charges from the gamma- to the beta-oxygens. Thereby, the GTP charge distribution becomes more like that in GDP. The charge shift appears to be a key factor contributing to catalysis by Ras in addition to the correct positioning of the attacking water. Ras appears to increase the negative charge at the pro-R beta-oxygen mainly by interaction of Mg(2+) and at the pro-S beta-oxygen mainly by interactions of the backbone NHs of Lys 16, Gly 15, and Val 14. The correct positioning of the backbone NHs of Lys 16, Gly 15, and Val 14, and especially the Lys 16 side chain, of the structural highly conserved phosphate binding loop relative to beta-phosphate therefore seems to be important for the catalysis provided by Ras.

Effects of pre-acclimation to aluminium on the physiology and swimming behaviour of juvenile rainbow trout (Oncorhynchus mykiss) during a pulsed exposure.

Anthropogenic acidification of the freshwater environment causes aluminium to be mobilised into the aquatic environment. When pH falls below 5.5, exposure to aluminium concentrations as low as 12.5 microg.l(-1) can cause serious physiological disturbances in freshwater fish. However, under constant laboratory exposures fish can acclimate and recover physiological status within 5-30 days. In reality, fish in the wild are likely to experience chronic sub-lethal exposure, with occasional elevations (pulses) to much higher levels. The experiment described here investigated the effects of an environmentally realistic, 4-day pulse exposure to a high level of aluminium (36 microg.l(-1)) in two groups of juvenile rainbow trout. One group was exposed to a lower level of aluminium (24 microg.l(-1)) for 16 days before and 10 days after the pulse ('aluminium-acclimated' fish). A second group was exposed to pH 5.2 alone for 16 days before and 10 days after the pulse ('aluminium-naïve' fish). A third group exposed to pH 5.2 alone for 30 days (no aluminium added) acted as controls. Triplicate groups of 24 juvenile rainbow trout (2.3-16.7 g) were randomly allocated to one of these three treatments. Swimming behaviour was monitored throughout and samples were taken on days 14, 20, 22, 26 and 30 for assessment of physiological status. No treatment effects were recorded in the control group (pH 5.2 alone). Fish in the 'aluminium-acclimated' treatment became hypo-active upon initiation of the exposure to 24 microg.l(-1) aluminium, but recovered after just 4 days of this exposure. Subsequent challenge on day 16 with the 36 microg.l(-1) aluminium 'pulse' caused these fish to became hypo-active again, but they recovered normal swimming behaviour whilst still subject to the 4-day pulse. The 'aluminium-naïve' fish also became hypo-active during the pulse exposure (36 microg.l(-1) aluminium). However, they did not exhibit any recovery of swimming behaviour, either during the pulse, or even 6 days after the cessation of the pulse, despite a rapid depuration of gill aluminium load (within 2 days of the pulse finishing). Mortality was low in the aluminium-acclimated fish (4%) and significantly higher in the aluminium-naïve fish (26%). Haematological disturbances were most extreme in the aluminium-naïve fish and had not recovered to control levels 6 days after the end of the pulse. This study provides new evidence, using behavioural responses, that previous exposure to low levels of aluminium may be an important factor abating the impact of aluminium on fish in the natural environment.

Fourier transform infrared photolysis studies of caged compounds.

Time-resolved FTIR difference spectroscopy is a powerful tool for investigating molecular reaction mechanisms of proteins. In order to detect, beyond the large background absorbance of the protein and the water, absorbance bands of protein groups that undergo reactions, difference spectra have to be performed between a ground state and an activated state of the sample. Because the absorbance changes are small, the reaction has to be started in situ, in the apparatus, and in thin protein films. The use of caged compounds offers an elegant approach to initiate protein reactions with a nanosecond UV laser flash. Here, time-resolved FTIR and FT-Raman photolysis studies of the commonly used caged compounds, caged Pi, caged ATP, caged GTP, and caged calcium are presented. The use of specific isotopic labels allows us to assign the IR bands to specific groups. Because metal ions play an important role in many biological systems, their influence on FTIR spectra of caged compounds is discussed. The results presented should provide a good basis for further FTIR studies on molecular reaction mechanisms of energy or signal transducing proteins. As an example of such investigations, the time-resolved FTIR studies on the GTPase reaction of H-ras p21 using caged GTP is presented.