Time-resolved FTIR difference spectroscopy can provide a valuable insight into the molecular reaction mechanisms of proteins, especially membrane proteins. Isotopic labeling and site-directed mutagenesis allows an unequivocal assignment of IR absorption bands. Studies are presented which give insight into the proton pump mechanisms of proteins, especially bacteriorhodopsin. H-bonded network proton transfer via internal water molecules seems to be a general feature in proteins, also found in cytochrome c oxidase. Using caged GTP the intrinsic and GAP catalyzed GTPase activity of H-ras p21 is studied. Furthermore, protein folding reactions can be recorded with ns time-resolution. 相似文献
The secondary structure change of the Abeta peptide to beta‐sheet was proposed as an early event in Alzheimer's disease. The transition may be used for diagnostics of this disease in an early state. We present an Attenuated Total Reflection (ATR) sensor modified with a specific antibody to extract minute amounts of Abeta peptide out of a complex fluid. Thereby, the Abeta peptide secondary structure was determined in its physiological aqueous environment by FTIR‐difference‐spectroscopy. The presented results open the door for label‐free Alzheimer diagnostics in cerebrospinal fluid or blood. It can be extended to further neurodegenerative diseases.
An immunologic ATR‐FTIR sensor for Abeta peptide secondary structure analysis in complex fluids is presented. 相似文献
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. 相似文献
Coherent anti-Stokes Raman scattering (CARS) is an emerging tool for label-free characterization of living cells. Here, unsupervised multivariate analysis of CARS datasets was used to visualize the subcellular compartments. In addition, a supervised learning algorithm based on the “random forest” ensemble learning method as a classifier, was trained with CARS spectra using immunofluorescence images as a reference. The supervised classifier was then used, to our knowledge for the first time, to automatically identify lipid droplets, nucleus, nucleoli, and endoplasmic reticulum in datasets that are not used for training. These four subcellular components were simultaneously and label-free monitored instead of using several fluorescent labels. These results open new avenues for label-free time-resolved investigation of subcellular components in different cells, especially cancer cells. 相似文献
Previous time-resolved FTIR measurements suggested the involvement of an intermediary component in the electron transfer step Q(A)- --> Q(B) in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides [Remy and Gerwert (2003) Nat. Struct. Biol. 10, 637]. By a kinetic X-ray absorption experiment at the Fe K-edge we investigated whether oxidation occurs at the ferric non-heme iron located between the two quinones. In isolated reaction centers with a high content of functional Q(B), at a time resolution of 30 micros and at room temperature, no evidence for transient oxidation of Fe was obtained. However, small X-ray transients occurred, in a similar micro- to millisecond time range as in the IR experiments, which may point to changes in the Fe ligand environment due to the charges on Q(A)- and Q(B)-. In addition, VIS measurements agree with the IR data and do not exclude an intermediate in the Q(A)- --> Q(B) transition. 相似文献
The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and P(i) in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and P(i) release back to the diphosphate. Furthermore, the Ras·Mg(2+) complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger. 相似文献
A computational approach to predict structures of rhodopsin-like G protein-coupled receptors (GPCRs) is presented and evaluated by comparison to the X-ray structural models. By combining sequence alignment, the rhodopsin crystal structure, and point mutation data on the beta2 adrenoreceptor (b2ar), we predict a (-)-epinephrine-bound computational model of the beta2 adrenoreceptor. The model is evaluated by molecular dynamics simulations and by comparison with the recent X-ray structures of b2ar. The overall correspondence between the predicted and the X-ray structural model is high. Especially the prediction of the ligand binding site is accurate. This shows that the proposed dynamic homology modelling approach can be used to create reasonable models for the understanding of structure and dynamics of other rhodopsin-like GPCRs. 相似文献
Photosystem II (PSII), an essential component of oxygenic photosynthesis, is a membrane-bound pigment protein complex found in green plants and cyanobacteria. Whereas the molecular structure of cyanobacterial PSII has been resolved with at least medium resolution [Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W. & Orth, P. (2001) Nature (London) 409, 739-743; Kamiya, N. & Shen, J.R. (2003) Proc. Natl Acad. Sci. USA 100, 98-103], the structure of higher plant PSII is only known at low resolution. Therefore Fourier transform infrared (FTIR) difference spectroscopy was used to compare PSII from both Thermosynechococcus elongatus and Synechocystis PCC6803 core complexes with PSII-enriched membranes from spinach (BBY). FTIR difference spectra of T. elongatus core complexes are presented for several different intermediates. As the FTIR difference spectra show close similarities among the three species, the structural arrangement of cofactors in PSII and their interactions with the protein microenvironment during photosynthetic charge separation must be very similar in higher plant PSII and cyanobacterial PSII. A structural model of higher plant PSII can therefore be predicted from the structure of cyanobacterial PSII. 相似文献