TY - JOUR T1 - Calcium and Magnesium Binding in Native and Structurally Perturbed Purple Membrane JF - The Journal of Physical Chemistry Y1 - 1996 A1 - Griffiths, Jennifer A. A1 - King, John A1 - Yang, Difei A1 - Browner, Richard A1 - El-Sayed, Mostafa A AB - The number and identity of the metal cations bound to wild-type bacteriorhodopsin (bR) are determined by using inductively coupled plasma mass spectrometry (ICP-MS) and ICP emission techniques. The results indicate that there at ≈2 total Ca2+ and Mg2+ per bR molecule with a ratio of ≈3:1 Ca2+ to Mg2+. This observed ratio is found to agree with the calculated ratio using previously determined binding constants for the two high affinity sites of Ca2+ to deionized bR (Zhang; et al. Biophys. J. 1992, 61, 1201). This suggests that the high-affinity binding sites in deionized bR are similar to those in native bR. Structural perturbation of the native membrane by cleavage of the C-terminus decreases the number of ions per bR to 1.4. The observed ratio of total ions in this sample to total ions in bR is found to agree with that calculated using known binding constants for each. The results on the number of metal cations/bR and their ratio in bacterio-opsin agrees with the calculated number using previously observed binding constants in deionized bO (Yang; et al. Biophys J., in press) only if one assumes that the second high-affinity site (not the first) is removed by retinal removal. Removal of 75% of the lipids from the purple membrane is found to greatly reduce the number of metal cations from 2 to 0.16. This suggest that if metal cations are in the two high-affinity sites (which are the only type of binding sites evident in our native bR sample), the removal of lipids, known to change the protein tertiary structure, changes also the metal ion binding sites.The number and identity of the metal cations bound to wild-type bacteriorhodopsin (bR) are determined by using inductively coupled plasma mass spectrometry (ICP-MS) and ICP emission techniques. The results indicate that there at ≈2 total Ca2+ and Mg2+ per bR molecule with a ratio of ≈3:1 Ca2+ to Mg2+. This observed ratio is found to agree with the calculated ratio using previously determined binding constants for the two high affinity sites of Ca2+ to deionized bR (Zhang; et al. Biophys. J. 1992, 61, 1201). This suggests that the high-affinity binding sites in deionized bR are similar to those in native bR. Structural perturbation of the native membrane by cleavage of the C-terminus decreases the number of ions per bR to 1.4. The observed ratio of total ions in this sample to total ions in bR is found to agree with that calculated using known binding constants for each. The results on the number of metal cations/bR and their ratio in bacterio-opsin agrees with the calculated number using previously observed binding constants in deionized bO (Yang; et al. Biophys J., in press) only if one assumes that the second high-affinity site (not the first) is removed by retinal removal. Removal of 75% of the lipids from the purple membrane is found to greatly reduce the number of metal cations from 2 to 0.16. This suggest that if metal cations are in the two high-affinity sites (which are the only type of binding sites evident in our native bR sample), the removal of lipids, known to change the protein tertiary structure, changes also the metal ion binding sites. PB - American Chemical Society VL - 100 SN - 0022-3654 UR - http://dx.doi.org/10.1021/jp952951i CP - 3 N1 - doi: 10.1021/jp952951i J1 - J. Phys. Chem. M3 - doi: 10.1021/jp952951i ER - TY - JOUR T1 - Effects of rapid thermal anneal on refractive index and hydrogen content of plasma-enhanced chemical vapor deposited silicon nitride films JF - J. Appl. Phys.Journal of Applied Physics Y1 - 1996 A1 - Cai, L. ED - Rohatgi, A. ED - Yang, Difei ED - El-Sayed, Mostafa A KW - ANNEALING KW - CRYSTAL DEFECTS KW - CVD KW - DESORPTION KW - HYDROGEN KW - IMPURITIES KW - REFRACTIVE INDEX KW - SILICON NITRIDES KW - THICKNESS KW - THIN FILMS AB - The objective of this paper is to understand and quantify the effects of rapid thermal anneal (RTA) on refractive index, thickness, and hydrogen content of plasma‐enhanced, chemical vapor‐deposited (PECVD) silicon nitride films. It is shown that RTA is more effective than identical furnace anneal. A threshold in as‐deposited refractive index value is found, above which the index of a silicon nitride film increases, while the thickness and bonded hydrogen content decreases as result of the RTA. In addition, the magnitude of increase in the index is proportional to the as‐deposited index value. The threshold index value increases with the increase in silicon nitride deposition temperature. A direct correlation is found between the annealing‐induced increase in refractive index and the corresponding decrease in total bonded hydrogen concentration in the PECVD silicon nitride films. Finally, it is shown that the release of bonded hydrogen from the film can passivate defects in the underlying silicon substrate and increase the performance of silicon devices such as solar cells. PB - AIP VL - 80 UR - http://dx.doi.org/10.1063/1.363480 CP - 9 M3 - 10.1063/1.363480 ER - TY - JOUR T1 - The Ca2+ binding to deionized monomerized and to retinal removed bacteriorhodopsin. JF - Biophysical journal Y1 - 1995 A1 - Yang, Difei A1 - El-Sayed, Mostafa A KW - Bacteriorhodopsins KW - Binding Sites KW - Biophysical Phenomena KW - Biophysics KW - Calcium KW - Electrochemistry KW - Halobacterium KW - Ions KW - Kinetics KW - Protein Conformation KW - Retinaldehyde AB - In our continuing effort to characterize the metal cation binding in bacteriorhodopsin (bR) using Ca(2+)-specific electrodes, potentiometric titration was carried out on deionized solubilized bR (containing monomeric units) and deionized bacterioopsin (bR with its retinal removed). Scatchard plots were analyzed. The monomer was found to have plots similar to those of the trimer, suggesting that the binding sites in bR are localized within the protein monomer unit and not between the molecules within the trimer structure. This also supports the previous assumption that the curvature in the Scatchard plot of regenerated bR is not due to cooperativity of metal cation within the trimer, but rather due to multiple sites. Recent studies further support the finding that the curved Scatchard plot is not due to the cooperativity between the metal ions in the two high affinity sites, wherever they are. The results of the analysis of the Scatchard plot for deionized bacterioopsin have shown a change in the binding characteristics of the high affinity but not the low affinity sites from that observed in bR. This result supports previous conclusions that metal cations in the high affinity sites are not far from the retinal cavity. VL - 69 CP - 5 U1 - http://www.ncbi.nlm.nih.gov/pubmed/8580348?dopt=Abstract M3 - 10.1016/S0006-3495(95)80075-5 ER - TY - JOUR T1 - The effect of different metal cation binding on the proton pumping in bacteriorhodopsin JF - Israel journal of chemistry Y1 - 1995 A1 - El-Sayed, Mostafa A A1 - Yang, Difei A1 - Yoo, Seoung-Kyo A1 - Zhang, N. AB - The first section of this paper is a detailed summary of studies made by us and others on metal cations binding to deionized bacteriorhodopsin (dIbR) and its variants. Our studies include the luminescence experiments of Eu3+ binding to dIbR and potentiometric studies of Ca2+ binding to dIbR, to deionized bR mutants, to bacterioopsin, and to dIbR with its C-terminus removed. The results suggest the presence of two classes of binding sites, one class has two high-affinity constants, and one has one low-affinity constant. For Ca2+ binding, there is one metal cation in each of the two high-affinity sites which are coupled to the charged aspartates 85 and 212 (known to be in the retinal cavity) but not coupled to each other. The low-affinity class can accommodate 0-6 Ca2+ ions and most of them are bound to the surface. Mg2+ has a slightly smaller value for its binding constant to the highest-affinity site. Thus, one expects more Ca2+ than Mg2+ bound to the two high-affinity sites. In the second section, we summarize our recent study on the effect of metal cation charge density (Ca2+, Mg2+, Eu3+, Tb3+, Ho3+, Dy3+) on the kinetics of both Schiff base deprotonation and proton transport to the extracellular surface. For all metal cations, the apparent rate constant of the slow components of the deprotonation process is the same as that for the transport process at 22 degrees C. The temperature studies, however, show this apparent equality to be fortuitous and to result from cancellation of the contribution of the energy and entropy of activation. Thus, while the entropy of activation is positive for the deprotonation process, it is negative for the proton transport process. These kinetic parameters depend weakly on the charge density, but in an opposite sense for the two processes. These results suggest that the deprotonation is not the rate-limiting step for the proton transport process. A possible mechanism is proposed in which a hydrated metal cation is used to induce the deprotonation of the protonated Schiff base and to dissociate one of its H2O molecules to donate the proton in the L-->M process. PB - Weizmann VL - 35 SN - 0021-2148 CP - 3-4 ER - TY - JOUR T1 - Fourier-transform infrared spectroscopic comparison of cultured human fibroblast and fibrosarcoma cells JF - Proceedings of SPIE Y1 - 1995 A1 - Yang, Difei ED - Castro, D. ED - El Sayed, I.H. ED - El-Sayed, Mostafa A ED - Saxton, R. ED - Zhang, N. AB - Infrared vibration spectroscopy appears to be a more powerful technique for diagnosis than visible or UV spectroscopy. Advantages of IR spectra include: 1) vibrational motion has a smaller tissue absorption coefficient than electronic motion, 2) scattering of infrared radiation has a lower cross section than visible or UV light, (these two facts allow deeper penetration of IR radiation) and 3) vibration spectra provide a better fingerprint of chemical groups present in cells than the unresolved broad electronic spectrum of biological molecules. In the present work, Fourier-transform IR spectroscopy was used to compare cultured human fibroblast and malignant fibrosarcoma cells. Significant differences were observed by comparing the spectra of the normal cells with that of the cancer cells. the PO2 symmetric stretching mode at 1082cm-1 in the cancer cell is reduced in intensity. These observations are similar to those reported previously by Wong et al in comparing the IR spectra of pairs of normal and cancerous cells from the colon and cervix. However, the observed increase in the relative intensity of the symmetric to antisymmetric CH3 bending mode are only found in fibrosarcoma and basal cell carcinoma. The decrease in intensity of the CH2 bending mode relative to that of CH3 mode was observed only for fibrosarcoma cells. This finding with paired human fibroblast and fibrosarcoma cells suggests that fatty acid chains or side chains of protein in the cancer cells are partially degraded leading to more terminal carbon. It is also possible that changes in the environment upon carcinogenesis induces a change in the relative absorption cross sections for the CH3 and CH2 bending vibrations. PB - SPIE VL - 2389 UR - http://dx.doi.org/10.1117/12.210030 CP - 1 M3 - 10.1117/12.210030 ER - TY - JOUR T1 - A Fourier-transform infrared spectroscopic comparison of cultured human fibroblast and fibrosarcoma cells: a new method for detection of malignancies JF - Journal of clinical laser medicine & surgery Y1 - 1995 A1 - Yang, Difei A1 - Castro, D. A1 - El Sayed, I.H. A1 - El-Sayed, Mostafa A A1 - SAXTON, R.E. A1 - ZHANG, N.Y.I. AB - Infrared vibration spectroscopy appears to be a more powerful technique for tumor diagnosis than visible or UV spectroscopy. In the present work, Fourier-transform infrared (FTIR) spectroscopy was used to compare cultured normal fibroblast and fibrosarcoma cells. Significant differences were observed by comparing the spectra of the normal human cells with that of the cancer cells. The PO2 symmetric stretching mode at 1082 cm-1 is shifted to a higher frequency in the cancer cell and a broad band, whose center is located at 1064 cm-1 in the cancer cell is reduced in intensity. In addition, the decrease in intensity of the CH2 bending mode relative to that of CH3 mode is detectable only in the fibrosarcoma cell. This FTIR difference between fibroblast and fibrosarcoma cells suggests that either fatty acid chains or protein side chains of the cancer cells are partially degraded resulting in more terminal carbon (e.g., CH3). It is also possible that changes in the environment upon carcinogenesis induces a change in the relative absorption cross section for CH3 and CH2 bending vibrations. These results suggest that FTIR spectroscopy may become a promising and sensitive technique for tumor identification. VL - 13 SN - 1044-5471 CP - 2 ER - TY - JOUR T1 - Retinal Isomer Composition in Some Bacteriorhodopsin Mutants under Light and Dark Adaptation Conditions JF - The Journal of Physical Chemistry Y1 - 1995 A1 - Song, Li A1 - Yang, Difei A1 - El-Sayed, Mostafa A A1 - Lanyi, Janos K. AB - View http://dx.doi.org/10.1021/j100024a056 for the article's first page in lieu of an abstract PB - American Chemical Society VL - 99 SN - 0022-3654 UR - http://dx.doi.org/10.1021/j100024a056 CP - 24 N1 - doi: 10.1021/j100024a056 J1 - J. Phys. Chem. M3 - doi: 10.1021/j100024a056 ER - TY - JOUR T1 - The pH dependence of the subpicosecond retinal photoisomerization process in bacteriorhodopsin: evidence for parallel photocycles. JF - Biophysical journal Y1 - 1994 A1 - Song, Li A1 - Logunov, Stephan L. A1 - Yang, Difei A1 - El-Sayed, Mostafa A KW - Bacteriorhodopsins KW - Biophysical Phenomena KW - Biophysics KW - Hydrogen-Ion Concentration KW - Isomerism KW - Kinetics KW - Molecular Structure KW - Photochemistry KW - Retinaldehyde AB - The pH dependence of the subpicosecond decay of the retinal photoexcited state in bacteriorhodopsin (bR) is determined in the pH range 6.8-11.3. A rapid change in the decay rate of the retinal photoexcited state is observed in the pH range 9-10, the same pH range in which a rapid change in the M412 formation kinetics was observed. This observation supports the previously proposed heterogeneity model in which parallel photocycles contribute to the observed pH dependence of the M412 formation kinetics in bR. VL - 67 CP - 5 U1 - http://www.ncbi.nlm.nih.gov/pubmed/7858138?dopt=Abstract M3 - 10.1016/S0006-3495(94)80684-8 ER -