%0 Journal Article %J ISRAPS Bulletin %D 2006 %T Effect of crystallization on the proton pump function of bR %A Sanii, L. S. %A El-Sayed, Mostafa A %B ISRAPS Bulletin %V 18 %P 52-57 %N 1&2 %0 Journal Article %J Photochemistry and Photobiology %D 2005 %T Partial dehydration of the retinal binding pocket and proof for photochemical deprotonation of the retinal Schiff base in bicelle bacteriorhodopsin crystals %A Sanii, L. S. %A El-Sayed, Mostafa A %X In bicelle bacteriorhodopsin (bcbR) crystals, the protein has a different structure from both native bacteriorhodopsin (bR) and in-cubo bR (cbR) crystals. Recently, we studied the ability of bcbR crystals to undergo the photocycle upon laser excitation, characterized by the appearance of the M intermediate by single crystal resonance Raman spectroscopy. Calculation of the M lifetime by flash photolysis experiments demonstrated that in our bchR crystals, the M rise time is much faster than in the native or cbR crystals, with a decay time that is much slower than these other two forms. Although it is now known that the bcbR crystals are capable of photochemical deprotonation, it is not known whether photochemical deprotonation is the only way to create the deprotonated Schiff base in the bcbR crystals. We measured both the visible and Raman spectra of crystals dried under ambient lighting and dried in the dark in order to determine whether the retinal Schiff base is able to thermally deprotonate in the dark. In addition, changes in the visible spectrum of single bcbR crystals under varying degrees of hydration and light exposure were examined to better understand the retinal binding environment. %B Photochemistry and Photobiology %V 81 %P 1356-1360 %8 Nov-Dec %@ 0031-8655 %G eng %M WOS:000233997300014 %R 10.1562/2005-03-09-ra-458 %0 Journal Article %J Biophysical Journal %D 2005 %T The protonation-deprotonation kinetics of the protonated Schiff base in bicelle bacteriorhodopsin crystals %A Sanii, L. S. %A Schill, A. W. %A Moran, C. E. %A El-Sayed, Mostafa A %X In the recently published x-ray crystal structure of the "bicelle" bacteriorhodopsin (bbR) crystal, the protein has quite a different structure from the native and the in cubo bacteriorhodopsin (cbR) crystal. Instead of packing in parallel trimers as do the native membrane and the cbR crystals, in the bbR crystal the protein packs as antiparallel monomers. To date, no functional studies have been performed, to our knowledge, to investigate if the photocycle is observed in this novel protein packing structure. In this study, both Raman and time-resolved transient absorption spectroscopy are used to both confirm the presence of the photocycle and investigate the deprotonation-reprotonation kinetics of the Schiff base proton in the bbR crystal. The observed rates of deprotonation and reprotonation processes of its Schiff base have been compared to those observed for native bR under the same conditions. Unlike the previously observed similarity of the rates of these processes for cbR crystals and those for native bacteriorhodopsin (bR), in bbR crystals the rate of deprotonation has increased by 300%, and the rate of reprotonation has decreased by nearly 700%. These results are discussed in light of the changes observed when native bR is delipidated or monomerized by detergents. Both the change of the hydrophobicity of the environment around the protonated Schiff base and Asp(85) and Asp(96) (which could change the pK(a) values of proton donor-acceptor pairs) and the water structure in the bbR crystal are offered as possible explanations for the different observations. %B Biophysical Journal %V 89 %P 444-451 %8 Jul %@ 0006-3495 %G eng %M WOS:000230114500047 %R 10.1529/biophysj.105.059675 %0 Journal Article %J Applied and Environmental Microbiology %D 2004 %T Model system for growing and quantifying Streptococcus pneumoniae biofilms in situ and in real time %A Donlan, R. M. %A Piede, J. A. %A Heyes, C D %A Sanii, L. S. %A Murga, R. %A Edmonds, P. %A El Sayed, I.H. %A El-Sayed, Mostafa A %X Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation. A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S. pneumoniae in situ over time. A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer. Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods. ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS). Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides. Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h. The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides. The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 10(5) cells per cm(2), while viable counts decreased as the biofilm aged. This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S. pneumoniae over time by using multiple, corroborative techniques. This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance. %B Applied and Environmental Microbiology %V 70 %P 4980-4988 %8 Aug %@ 0099-2240 %G eng %M WOS:000223290100072 %R 10.1128/aem.70.8.4980-4988.2004 %0 Journal Article %J Biophysical journal %D 2002 %T Fourier transform infrared study of the effect of different cations on bacteriorhodopsin protein thermal stability %A Heyes, C D %A Wang, Jianping %A Sanii, L. S. %A El-Sayed, Mostafa A %X The effect of divalent ion binding to deionized bacteriorhodopsin (dI-bR) on the thermal transitions of the protein secondary structure have been studied by using temperature-dependent Fourier transform infrared (FT-IR) spectroscopy. The native metal ions in bR, Ca2+, and Mg2+, which we studied previously, are compared with Mn2+, Hg2+, and a large, synthesized divalent organic cation, ((Et)3N)2Bu2+. It was found that in all cases of ion regeneration, there is a pre-melting, reversible conformational transition in which the amide frequency shifts from 1665 to 1652cm−1. This always occurs at ∼80°C, independent of which cation is used for the regeneration. The irreversible thermal transition (melting), monitored by the appearance of the band at 1623cm−1, is found to occur at a lower temperature than that for the native bR but higher than that for acid blue bR in all cases. However, the temperature for this transition is dependent on the identity of the cation. Furthermore, it is shown that the mechanism of melting of the organic cation regenerated bR is different than for the metal cations, suggesting a difference in the type of binding to the protein (either to different sites or different binding to the same site). These results are used to propose specific direct binding mechanisms of the ions to the protein of deionized bR. %B Biophysical journal %I Elsevier %V 82 %P 1598-1606 %@ 0006-3495 %G eng %U http://dx.doi.org/10.1016/S0006-3495(02)75511-2 %N 3 %R 10.1016/S0006-3495(02)75511-2