ABSTRACT
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.
Subject(s)
Molecular Probes/radiation effects , Photolysis , Spectroscopy, Fourier Transform Infrared/methods , Acetates , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/radiation effects , Calcium/chemistry , Calcium/radiation effects , Cations, Divalent , Chelating Agents , Ethylenediamines , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/radiation effects , Magnesium , Molecular Probes/chemistry , Phosphates/chemistry , Phosphates/radiation effects , Photochemistry/instrumentation , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/radiation effects , Spectrum Analysis, Raman , Thionucleotides/chemistry , Thionucleotides/radiation effectsABSTRACT
FTIR difference spectroscopy has been established as a new tool to study the GTPase reaction of H-ras p21 (Ras) in a time-resolved mode at atomic resolution without crystallization. The phosphate vibrations were analyzed using site specifically 18O-labeled caged GTP isotopomers. One nonbridging oxygen per nucleotide was replaced for an 18O isotope in the alpha-, beta-, or gamma-position of the phosphate chain. In photolysis experiments with free caged GTP, strong vibrational coupling was observed among all phosphate groups. The investigation of Ras*caged GTP photolysis and the subsequent hydrolysis reaction of Ras*GTP showed that the phosphate vibrations are largely decoupled by interaction with the protein in contrast to free GTP. The characteristic isotope shifts allow band assignments to isolated alpha-, beta-, and gamma-phosphate vibrations of caged GTP, GTP, and the liberated inorganic phosphate. The unusually low frequency of the beta (PO2-) vibration of Ras-bound GTP, as compared to free GTP, indicates a large decrease in the P-O bond order. The bond order decrease reveals that the oxygen atoms of the beta (PO2-) group interact much more strongly with the protein environment than the gamma-oxygen atoms. Thereby, electrons are withdrawn from the beta-phosphorus, and thus also from the beta/gamma-bridging oxygen. This leads to partial bond breakage or at least weakening of the bond between the beta/gamma-bridging oxygen and the gamma-phosphorus atom as a putative early step of the GTP hydrolysis. Based on these results, we propose a key role of the beta-phosphate for GTP hydrolysis. The assignments of phosphate bands provide a crucial marker for further time-resolved FTIR studies of the GTPase reaction of Ras.
