TY - JOUR T1 - Eu3+ binding to europium-regenerated bacteriorhodopsin upon delipidation and monomerization JF - Febs Letters Y1 - 2004 A1 - Heyes, C D A1 - Reynolds, K. B. A1 - El-Sayed, Mostafa A AB - We have studied the effect of monomerization of the purple membrane lattice, as well as removal of 75% of the lipids, on the binding properties of Eu3+ ions. We found that delipidation and monomerization do not cause the cations to lose their binding ability to the protein. This suggests that the three most strongly bound Eu3+ cations do not bind to the lipids, but directly bind to the protein. Furthermore, we found that delipidation actually causes a slight increase in the binding affinity. This is likely a result of reduced aggregation of europium-regenerated bacteriorhodopsin (bR) upon lipid removal causing more exposure of the binding sites to the Eu3+ cations. These results, taken with those from our previous publication [Heyes and El-Sayed, Biophys. J. 85 (2003) 426-434], might suggest that the cations remain bound upon delipidation of bR, but have no effect on the function. This is discussed with respect to the role of cations in the function of native bR. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. VL - 562 SN - 0014-5793 N1 - Heyes, CD Reynolds, KB El-Sayed, MA M3 - 10.1016/s0014-5793(04)00182-6 ER - TY - JOUR T1 - Model system for growing and quantifying Streptococcus pneumoniae biofilms in situ and in real time JF - Applied and Environmental Microbiology Y1 - 2004 A1 - Donlan, R. M. A1 - Piede, J. A. A1 - Heyes, C D A1 - Sanii, L. S. A1 - Murga, R. A1 - Edmonds, P. A1 - El Sayed, I.H. A1 - El-Sayed, Mostafa A AB - 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. VL - 70 SN - 0099-2240 N1 - Donlan, RM Piede, JA Heyes, CD Sanii, L Murga, R Edmonds, P El-Sayed, I El-Sayed, MA M3 - 10.1128/aem.70.8.4980-4988.2004 ER - TY - JOUR T1 - Proton transfer reactions in native and deionized bacteriorhodopsin upon delipidation and monomerization JF - Biophysical journal Y1 - 2003 A1 - Heyes, C D A1 - El-Sayed, Mostafa A AB - We have investigated the role of the native lipids on bacteriorhodopsin (bR) proton transfer and their connection with the cation-binding role. We observe that both the efficiency of M formation and the kinetics of M rise and decay depend on the lipids and lattice but, as the lipids are removed, the cation binding is a much less important factor for the proton pumping function. Upon 75% delipidation using 3-[(cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS), the M formation and decay kinetics are much slower than the native, and the efficiency of M formation is ∼30%–40% that of the native. Upon monomerization of bR by Trition X-100, the efficiency of M recovers close to that of the native (depending on pH), M formation is ∼10 times faster, and M decay kinetics are comparable to native at pH 7. The same results on the M intermediate are observed if deionized blue bR (deI bbR) is treated with these detergents (with or without pH buffers present), even though deionized blue bR containing all the lipids has no photocycle. This suggests that the cation(s) has a role in native bR that is different than in delipidated or monomerized bR, even so far as to suggest that the cation(s) becomes unimportant to the function as the lipids are removed. PB - Elsevier VL - 85 SN - 0006-3495 UR - http://dx.doi.org/10.1016/S0006-3495(03)74487-7 CP - 1 M3 - 10.1016/S0006-3495(03)74487-7 ER - TY - JOUR T1 - Thermal properties of bacteriorhodopsin JF - Journal of Physical Chemistry B Y1 - 2003 A1 - Heyes, C D A1 - El-Sayed, Mostafa A AB - In this Feature Article we review the effects of various parameters on the structure and thermal stability of one of the most widely studied membrane proteins, bacteriorhodopsin. This protein has recently been crystallized and its high-resolution structure elucidated to 1.55 Angstrom. This information has proved invaluable in understanding its proton-pumping mechanism. However, the question of why bacteriorhodopsin is so stable over a wide range of conditions, and which factors contribute to this stability, is still largely unanswered. Spectroscopic and calorimetric experiments provide information on the thermodynamics, kinetics and structural changes upon unfolding and refolding the protein under various environmental perturbations. FT-IR spectroscopy has been particularly useful in determining the changes in secondary structure upon heating through its thermal transitions after changing pH and cations, removing and adding lipids and detergents, retinal reduction and removal, and site-directed mutagenesis. These experiments are reviewed, and the information that they have afforded have been brought together to try to understand how nature has controlled the conditions of bR to make it one of the most stable proteins known. VL - 107 SN - 1520-6106 N1 - Heyes, CD El-Sayed, MA M3 - 10.1021/jp035327b ER - TY - JOUR T1 - Comparison of the dynamics of the primary events of bacteriorhodopsin in its trimeric and monomeric states JF - Biophysical Journal Y1 - 2002 A1 - Wang, Jianping A1 - Link, Stephan A1 - Heyes, C D A1 - El-Sayed, Mostafa A AB - In this paper, femtosecond pump-probe spectroscopy in the visible region of the spectrum has been used to examine the ultrafast dynamics of the retinal excited state in both the native trimeric state and the monomeric state of bacteriorhodopsin (bR). It is found that the excited state lifetime (probed at 490 nm) increases only slightly upon the monomerization of bR. No significant kinetic difference is observed in the recovery process of the bR ground state probed at 570 nm nor in the fluorescent state observed at 850 nm. However, an increase in the relative amplitude of the slow component of bR excited state decay is observed in the monomer, which is due to the increase in the concentration of the 13-cis retinal isomer in the ground state of the light-adapted bR monomer. Our data indicate that when the protein packing around the retinal is changed upon bR monomerization, there is only a subtle change in the retinal potential surface, which is dependent on the charge distribution and the dipoles within the retinal-binding cavity. In addition, our results show that 40% of the excited state bR molecules return to the ground state on three different time scales: one-half-picosecond component during the relaxation of the excited state and the formation of the J intermediate, a 3-ps component as the J changes to the K intermediate where retinal photoisomerization occurs, and a subnanosecond component during the photocycle. VL - 83 SN - 0006-3495 N1 - Wang, JP Link, S Heyes, CD El-Sayed, MA ER - TY - JOUR T1 - Fourier transform infrared study of the effect of different cations on bacteriorhodopsin protein thermal stability JF - Biophysical journal Y1 - 2002 A1 - Heyes, C D A1 - Wang, Jianping A1 - Sanii, L. S. A1 - El-Sayed, Mostafa A AB - 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. PB - Elsevier VL - 82 SN - 0006-3495 UR - http://dx.doi.org/10.1016/S0006-3495(02)75511-2 CP - 3 M3 - 10.1016/S0006-3495(02)75511-2 ER - TY - JOUR T1 - Refolding of thermally denatured bacteriorhodopsin in purple membrane JF - Journal of Physical Chemistry B Y1 - 2002 A1 - Wang, Jianping A1 - Heyes, C D A1 - El-Sayed, Mostafa A AB - The change in protein conformational structure and retinal chromophore binding state have been examined by using in situ UV-vis, FTIR, and CD spectroscopies during the thermal denaturation and refolding processes in bacteriorhodopsin (bR) of purple membrane (PM), in its native trimeric and in Triton X-100 solubilized monomeric form. For the trimeric bR, it is found that heating bR through its premelting transition (T > 78 degreesC, T-m') does not cause any permanent damage in the protein secondary structure, and a reversible refolding occurs when it cools back to room temperature. For the monomeric bR, it is found that it is less thermally stable than the trimer. There is a significant change in its protein secondary structure and a complete dissociation of retinal occurs irreversibly at a temperature as low as 66 degreesC. In addition, it is found that heating the trimeric bR through its main molten state (T > 96 degreesC, T-m) changes the protein secondary structure so that bR does not refold fully into its original secondary structure. Upon cooling back to room temperature, about 90% of the bound retinal in native bR recovers after being heated through its premelting transition, whereas only about 12% of bound retinal recovers if bR is heated above its main melting temperature. It is also found that refolded bR molecules with their retinal chromophore rebound have a photocycle and are capable of pumping protons. Our results also suggest that from its molten state, protein secondary structure refolding precedes retinal rebinding to the Schiff base. VL - 106 SN - 1520-6106 N1 - Wang, JP Heyes, CD El-Sayed, MA M3 - 10.1021/jp013131a ER - TY - JOUR T1 - The role of the native lipids and lattice structure in bacteriorhodopsin protein conformation and stability as studied by temperature-dependent Fourier transform-infrared spectroscopy JF - Journal of Biological Chemistry Y1 - 2002 A1 - Heyes, C D A1 - El-Sayed, Mostafa A AB - We report the effect of partial delipidation and monomerization on the protein conformational changes of bacteriorhodopsin (bR) as a function of temperature. Removal of up to 75% of the lipids is known to have the lattice structure of the purple membrane, albeit as a smaller unit cell, whereas treatment by Triton monomerizes bR into micelles. The effects of these modifications on the protein secondary structure is analyzed by monitoring the protein amide I and amide II bands in the Fourier transform-infrared (FT-IR) spectra. It is found that removal of the first 75% of the lipids has only a slight effect on the secondary structure at physiological temperature, whereas monomerizing bR into micelles alters the secondary structure considerably. Upon heating, the bR monomer is found to have a very low thermal stability compared with the native bR with its melting point reduced from 97 to 65 degreesC, and the premelting transition in which the protein changes conformation in native bR at 80 degreesC could not be observed. Also, the N-H to N-D exchange of the amide II band is effectively complete at room temperature, suggesting that there are no hydrophobic regions that are protected from the aqueous medium, possibly explaining the low thermal stability of the monomer. On the other hand, 75% delipidated bR has its melting temperature close to that of the native bR and does have a pre-melting transition, although the pre-melting transition occurs at significantly higher temperature than that of the native bR (91 degreesC compared with 80 degreesC) and is still reversible. Furthermore, we have also observed that the reversibility of this pre-melting transition of both native and partially delipidated bR is time-dependent and becomes irreversible upon holding at 91 degreesC between 10 and 30 min. These results are discussed in terms of the lipid and lattice contribution to the protein thermal stability of native bR. VL - 277 SN - 0021-9258 N1 - Heyes, CD El-Sayed, MA M3 - 10.1074/jbc.M203435200 ER - TY - JOUR T1 - Effect of temperature, pH, and metal ion binding on the secondary structure of bacteriorhodopsin: FT-IR study of the melting and premelting transition temperatures JF - Biochemistry Y1 - 2001 A1 - Heyes, C D A1 - El-Sayed, Mostafa A AB - We have measured the temperature dependence of the FT-IR spectra of bacteriorhodopsin (bR) as a function of the pH and of the divalent cation regeneration with Ca2+ and Mg2+. It has been found that although the irreversible melting transition shows a strong dependence on the pH of the native bR, the premelting reversible transition at 78-80 degreesC shows very little variation over the pH range studied. It is further shown that the acid blue bR shows a red-shifted amide I spectrum at physiological temperature, which shows a more typical alpha -helical frequency component at 1652 cm(-1) and could be the reason for the observed reduction of its melting temperature and lack of an observed premelting transition. Furthermore, the thermal transitions for Ca2+- and Mg2+-regenerated bR (Ca-bR and Mg-bR, respectively) each show a premelting transition at the same 78-80 degreesC temperature as the native purple membrane, but the irreversible melting transition has a slight dependence on the cation identity. The pH dependence of the Ca2+-regenerated bR is studied, and neither transition varies over the pH range studied. These results are discussed in terms of the cation contribution to the secondary structural stability in bR. VL - 40 SN - 0006-2960 N1 - Heyes, CD El-Sayed, MA M3 - 10.1021/bi002594o ER -