Nanoscale electrical conductivity of the purple membrane monolayer.

Nanoscale electron transport through the purple membrane monolayer, a two-dimensional crystal lattice of the transmembrane protein bacteriorhodopsin, is studied by conductive atomic force microscopy. We demonstrate that the purple membrane exhibits nonresonant tunneling transport, with two characteristic tunneling regimes depending on the applied voltage (direct and Fowler-Nordheim). Our results show that the purple membrane can carry significant current density at the nanometer scale, several orders of magnitude larger than previously estimated by macroscale measurements.

Electron transport through supported biomembranes at the nanoscale by conductive atomic force microscopy.

We present a reliable methodology to perform electron transport measurements at the nanoscale on supported biomembranes by conductive atomic force microscopy (C-AFM). It allows measurement of both (a) non-destructive conductive maps and (b) force controlled current-voltage characteristics in wide voltage bias range in a reproducible way. Tests experiments were performed on purple membrane monolayers, a two-dimensional (2D) crystal lattice of the transmembrane protein bacteriorhodopsin. Non-destructive conductive images show uniform conductivity of the membrane with isolated nanometric conduction defects. Current-voltage characteristics under different compression conditions show non-resonant tunneling electron transport properties, with two different conduction regimes as a function of the applied bias, in excellent agreement with theoretical predictions. This methodology opens the possibility for a detailed study of electron transport properties of supported biological membranes, and of soft materials in general.

Applications of environmental scanning electron microscopy (ESEM) in biomaterials field.

Various tasks were undertaken in our laboratory where environmental scanning electron microscopy (ESEM) has been of particular interest within the biomaterials field. The possibility of observing wet samples, as well as the fact that sample preparation is minimal, has improved shorter time scales and lower costs in microscopy. Minimal preparation has also reduced the possibility of introducing artifacts. Examples like cell cultures used for pit resorption assays, calcium phosphate deposition processes, and dissolution of phosphate glasses used as biomaterials are presented. Finally, a servohydraulic testing machine designed for mechanical testing in situ in ESEM has allowed the study of shape memory alloys for orthodontic applications or the behavior of different adhesives used in odontology.

Thr90 is a key residue of the bacteriorhodopsin proton pumping mechanism.

Mutation of Thr90 to Ala has a profound effect on bacteriorhodopsin properties. T90A shows about 20% of the proton pumping efficiency of wild type, once reconstituted into liposomes. Mutation of Thr90 influences greatly the Schiff base/Asp85 environment, as demonstrated by altered lambda(max) of 555 nm and pK(a) of Asp85 (about 1.3 pH units higher than wild type). Hydroxylamine accessibility is increased in both dark and light and differential scanning calorimetry and visible spectrophotometry show decreased thermal stability. These results suggest that Thr90 has an important structural role in both the unphotolysed bacteriorhodopsin and in the proton pumping mechanism.

Contribution of extracellular Glu residues to the structure and function of bacteriorhodopsin. Presence of specific cation-binding sites.

Single and multiple mutants of extracellular Glu side chains of bacteriorhodopsin were analyzed by acid and calcium titration, differential scanning calorimetry, and thermal difference spectrophotometry. Acid titration spectra show that the second group protonating with Asp(85) is revealed in E204Q in the absence of Cl(-) but is not observed in the triple mutant E9Q/E194Q/E204Q or in the quadruple mutant E9Q/E74Q/E194Q/E204Q. The results point to Glu(9) as the second group protonating cooperatively with Asp(85). Comparison of the apparent pK(a) of Asp(85) protonation in water and in the deionized forms and results of calcium titration suggest that cation-binding sites are of low affinity in the multiple Glu mutants. Like for deionized wild type bacteriorhodopsin, differential scanning calorimetry reveals a lack of the pretransition in the multiple mutants, whereas in E9Q it appears at lower temperature and with lower cooperativity. Additionally, at neutral pH the band at 630 nm arising from cation release upon temperature increase is absent for the multiple mutants. Based on these results, we propose the presence of two cation-binding sites in the extracellular region of bacteriorhodopsin having as ligands Glu(9), Glu(194), Glu(204), and water molecules.

The secondary structure of the inhibited mitochondrial ADP/ATP transporter from yeast analyzed by FTIR spectroscopy.

Fourier transform infrared spectroscopy has been applied to the study of the carboxyatractyloside-inhibited mitochondrial ADP/ATP transporter from the yeast Saccharomyces cerevisiae, either solubilized in dodecyl maltoside or reconstituted in phosphatidylcholine liposomes. Its secondary structure has been estimated by means of Fourier self-deconvolution followed by curve fit. A Voigt function was used to fit the components of the deconvoluted spectrum, aiming to account for any distortions introduced by deconvolution. For any of the states analyzed, reconstituted or solubilized, in solution or in dry films, 60-70% of the amino acids are found to adopt alpha-helix plus unordered structures, coherent with the six transmembrane spanning helix model. Moreover, the problem of structure preservation on drying was addressed, and several observations pointed to a maintenance of the protein structure in dry films. Comparison of reconstituted and solubilized samples indicated the presence of both lipid-induced changes in the protein (decrease of the beta-sheets and increase of unordered structures) and protein-induced changes in the lipids (strong hydrogen bonding of lipid C=O groups). To obtain a better discrimination of alpha-helix and unordered structure contributions for the reconstituted form, H/D exchange experiments were performed. Between 35% and 45% of the amino acids were finally assigned to alpha-helix structures, compatible with the existence of five or six transmembrane spanning helices in the transporter. The level of H/D exchange was determined after 15 h of exposure to D(2)O vapor to be 85%, reflecting a high accessibility of the amide hydrogens even for the carboxyatractyloside-inhibited state.

Secondary structure components and properties of the melibiose permease from Escherichia coli: a fourier transform infrared spectroscopy analysis.

The structure of the melibiose permease from Escherichia coli has been investigated by Fourier transform infrared spectroscopy, using the purified transporter either in the solubilized state or reconstituted in E. coli lipids. In both instances, the spectra suggest that the permease secondary structure is dominated by alpha-helical components (up to 50%) and contains beta-structure (20%) and additional components assigned to turns, 3(10) helix, and nonordered structures (30%). Two distinct and strong absorption bands are recorded at 1660 and 1653 cm(-1), i.e., in the usual range of absorption of helices of membrane proteins. Moreover, conditions that preserve the transporter functionality (reconstitution in liposomes or solubilization with dodecyl maltoside) make possible the detection of two separate alpha-helical bands of comparable intensity. In contrast, a single intense band, centered at approximately 1656 cm(-1), is recorded from the inactive permease in Triton X-100, or a merged and broader signal is recorded after the solubilized protein is heated in dodecyl maltoside. It is suggested that in the functional permease, distinct signals at 1660 and 1653 cm(-1) arise from two different populations of alpha-helical domains. Furthermore, the sodium- and/or melibiose-induced changes in amide I line shape, and in particular, in the relative amplitudes of the 1660 and 1653 cm(-1) bands, indicate that the secondary structure is modified during the early step of sugar transport. Finally, the observation that approximately 80% of the backbone amide protons can be exchanged suggests high conformational flexibility and/or a large accessibility of the membrane domains to the aqueous solvent.

New method for the in vitro evaluation of dental alloy bonding systems.

STATEMENT OF PROBLEM: Bonding systems are used in some fixed prosthetic devices with base alloys. However, different studies of the same dental alloy bonding agents, under similar circumstances, have yielded disparate results in bond strength testing. PURPOSE: This study compared directly 2 dental alloy bonding systems through a "duel" type of confrontation, which basically is a 2-way tensile force test. MATERIAL AND METHODS: Ninety Wiron 88 base alloy cylinders (diameter of 8 mm length 15 mm) were sandblasted on both sides with Al(2)0(3) powder (particle size 50 microm) during 10 seconds at an approximate distance of 5 mm, at an air pressure of 60 psi determined before sandblasting procedures. The surface of each cylinder was cleaned from Al(2)0(3) powder with a strong burst of oil-free air from a chairside air syringe. Thirty cylinders were randomly assigned to 1 of 3 groups for direct bond strength comparison: (1) Panavia 21 to Panavia EX, (2) Panavia 21 to Metabond, or (3) Panavia 21 to a combination of a resin bonding agent plus Panavia 21. Each group was composed of 10 specimens that used 3 cylinders for each specimen. Each side of the sample cylinder received the same quantity of cement and 1 cylinder at a time was bonded to it. Cylinder alignment was verified with a Boley gauge during luting procedures. The bonded 3-piece block was held together for 24 hours under a compressive force of 2 kg/cm(2) using a hydraulic press. Excess cement was removed with a brush, and the pertinent air sealant was applied to allow for autocuring of the cement. Specimens were later stored in water at room temperature for 48 hours before thermocycling procedures. Each specimen was thermocycled for 100 cycles with a 5-minute dwelling time in water at 4 degrees C and 60 degrees C. Specimens were subject to tensile force testing until debonding in 1 of the cylinders. RESULTS: The opposing pull duel test (OPDT) showed that the Panavia EX failed (40. 3 MPa) 10 of 10 duels against Panavia 21, whereas Panavia 21 failed (49.7 MPa) 9 of 10 duels against Metabond, and Panavia 1 failed (50. 1 MPa) 10 of 10 duels against Photobond+Panavia 21. ANOVA revealed significant differences (P <.05) between PAN-EX group and MET and PHB+P21 groups. However, no significant differences were found between MET and PHB+P21 groups. CONCLUSION: The opposing pull duel test was a valid method to directly compare bond strengths of 2 bonding systems to dental base alloys. There was a small dispersion of the values even though cement mixing and thickness variables were difficult to control. Duel tensile testing provides meaningful information on the superiority of one bonding system over another in this controlled environment.

Fourier transform infrared evidence for early deprotonation of Asp(85) at alkaline pH in the photocycle of bacteriorhodopsin mutants containing E194Q.

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).

Opening the Schiff base moiety of bacteriorhodopsin by mutation of the four extracellular Glu side chains.

The quadruple bacteriorhodopsin (BR) mutant E9Q+E74Q+E194Q+E204Q shows a lambda(max) of about 500 nm in water at neutral pH and a great influence of pH and salts on the visible absorption spectrum. Accessibility to the Schiff base is strongly increased, as detected by the rapid bleaching effect of hydroxylamine in the dark as well as in light. Both the proton release kinetics and the photocycle are altered, as indicated by a delayed proton release after proton uptake and changed M kinetics. Moreover, affinity of the color-controlling cation(s) is found to be decreased. We suggest that the four Glu side chains are essential elements of the extracellular structure of BR.

Nucleotide and Mg2+ dependency of the thermal denaturation of mitochondrial F1-ATPase.

The influence of adenine nucleotides and Mg2+ on the thermal denaturation of mitochondrial F1-ATPase (MF1) was analyzed. Differential scanning calorimetry in combination with ATPase activity experiments revealed the thermal unfolding of MF1 as an irreversible and kinetically controlled process. Three significant elements were analyzed during the thermal denaturation process: the endothermic calorimetric transition, the loss of ATP hydrolysis activity, and the release of tightly bound nucleotides. All three processes occur in the same temperature range, over a wide variety of conditions. The purified F1-ATPase, which contains three tightly bound nucleotides, denatures at a transition temperature (Tm) of 55 degrees C. The nucleotide and Mg2+ content of MF1 strongly influence the thermal denaturation process. First, further binding of nucleotides and/or Mg2+ to MF1 increases the thermal denaturation temperature, whereas the thermal stability of the enzyme is decreased upon removal of the endogenous nucleotides. Second, the stabilizing effect induced by nucleotides is smaller after hydrolysis of ATP (i.e., in the presence of ADP . Mg2+) than under nonhydrolytical conditions (i.e., absence of Mg2+ or using the nonhydrolyzable analog 5'-adenylyl-imidodiphosphate). Third, whereas the thermal denaturation of MF1 fully loaded with nucleotides follows an apparent two-state kinetic process, denaturation of MF1 with a low nucleotide content follows more complex kinetics. Nucleotide content is therefore an important factor in determining the thermal stability of the MF1 complex, probably by strengthening existing intersubunit interactions or by establishing new ones.

Experimental and theoretical characterization of the high-affinity cation-binding site of the purple membrane.

Binding of Mn2+ or Mg2+ to the high-affinity site of the purple membrane from Halobacterium salinarium has been studied by superconducting quantum interference device magnetometry or by ab initio quantum mechanical calculations, respectively. The binding of Mn2+ cation, in a low-spin state, to the high-affinity site occurs through a major octahedral local symmetry character with a minor rhombic distortion and a coordination number of six. A molecular model of this binding site in the Schiff base vicinity is proposed. In this model, a Mg2+ cation interacts with one oxygen atom of the side chain of Asp85, with both oxygen atoms of Asp212 and with three water molecules. One of these water molecules is hydrogen bonded to both the nitrogen of the protonated Schiff base and the Asp85 oxygen. It could serve as a shuttle for the Schiff base proton to move to Asp85 in the L-M transition.

Effect of nucleotides on the thermal stability and on the deuteration kinetics of the thermophilic F0F1 ATP synthase.

Differential scanning calorimetry has been used to characterize the influence of specific nucleotide binding on the thermal unfolding of the F0F1-type ATP synthase from the thermophilic Bacillus PS3 (TF0F1). The calorimetric trace shows an irreversible and kinetically controlled endothermic transition for TF0F1 in the absence of nucleotides. The thermal denaturation occurs at a transition temperature (t(m)) of 81.7 degrees C. The remarkable thermostability of this enzyme was decreased upon tight binding of Mg2+ x ATP to noncatalytic sites, whereas binding of Mg2+ x ADP increased the temperature at which thermal denaturation occurred. At high temperatures, an exothermic transition due to aggregation processes was also affected by nucleotide binding. With the aim to correlate these thermal effects with possible structural differences among the various forms of TF0F1, Fourier transform infrared spectroscopy was carried out. Hydrogen/deuterium exchange was clearly affected by specific nucleotide occupancy. As illustrated by the total extent of protons exchanged, our results demonstrate that more peptide groups are exposed to the medium in the presence of Mg2+ x ATP than in the presence of Mg2+ x ADP. Therefore, consistent with microcalorimetric data, binding of Mg2+ x ADP induces conformational changes which shield amide protons to more buried hydrogen-bonded structures, whereas binding of Mg2+ x ATP results in a more open or flexible structure.

Scanning calorimetry and Fourier-transform infrared studies into the thermal stability of cleaved bacteriorhodopsin systems.

Differential scanning calorimetry and Fourier-transform infrared spectroscopy have been used to characterize the thermal stability of bacteriorhodopsin (BR) cleaved within different loops connecting the helical rods. The results are compared to those of the native protein. We show that the denaturation temperature and enthalpy of BR cleaved at peptide bond 71-72 or 155-156 are lower than those of the intact protein, and that these values become even lower for the BR cleaved at both peptide bonds. The effect of cleavage on the denaturation temperature and enthalpy values seems to be additive as has been previously suggested [Khan, T. W., Sturtevant, J. M., & Engelman, D. M. (1992) Biochemistry 31, 8829]. The thermal denaturation of all the samples was irreversible and scan-rate dependent. When cleaved at the 71-72 bond BR follows quantitatively the predictions of the two-state kinetic model at pH 9.5, with an activation energy of 374 kJ/mol, similar to that of native BR. Calorimetry experiments with different populations of intact and cleaved BR provide direct evidence for some intermolecular cooperativity upon denaturation. The denatured samples maintain a large proportion of alpha helices and beta structure, a fact which seems to be related to their low denaturation enthalpy as compared to that of water-soluble, globular proteins.

Helical and reverse turn changes in the BR->N transition of bacteriorhodopsin.

Fourier transform infrared deconvoluted spectra of bacteriorhodopsin and the N intermediate were compared with the N/BR infrared difference spectrum. In the amide I, clear changes in the bands at 1666 cm-1, assigned to alpha II helices, 1659 cm-1, assigned to alpha I and alpha II helices, and 1652 cm-1, assigned to both alpha I helices and unordered structures, were found. These changes could arise from conversion of some alpha II and alpha I helices. Variations in the bands at 1692 and 1683 cm-1, corresponding to reverse turns, were also detected. The side chains of Tyr (band at 1517 cm-1) and Phe (band at 1498 cm-1) were found to change in going from BR to N. In the carboxylate region, no band was detected at 1737 cm-1 in the deconvoluted spectra that could correspond to the peak observed in the difference spectrum. It is argued that resolution-enhancement methods used along with difference spectra provide more detailed insights into the conformational changes occurring between photocycle intermediates.

Analysis of conformational changes in bacteriorhodopsin upon retinal removal.

The conformation of bacterioopsin in the apomembrane has been studied by Fourier transform infrared spectroscopy. Resolution enhancement techniques and curve-fitting procedures have been used to determine the secondary structural components from the amide I region. Bacterioopsin contains about 54% helicoidal structure (alpha I and alpha II helices + 3(10) turns), 21% sheets, 16% reverse turns, and 9% unordered structure. Thus, after retinal removal, all of the secondary structural types of bacteriorhodopsin remain present, and only slight quantitative differences appear. On the other hand, H/D exchange studies show that there is a higher degree of exchange for reverse turns and protonated carboxylic lateral chains in bacterioopsin as compared to bacteriorhodopsin. This gives further support to the idea of a more open tertiary structure of bacterioopsin, and to the consideration of the retinal molecule as an important element in complementing the interhelical interactions in bacteriorhodopsin folding.

An extended x-ray absorption fine structure study of the high-affinity cation-binding site in the purple membrane.

The structure of the high-affinity cation-binding site of bacteriorhodopsin was studied using extended x-ray absorption fine structure techniques. The results obtained for Mn2+ in aqueous solution and for the complex BR-Mn2+ (1:1 molar ratio) show great similarities, suggesting that Mn2+, when bound to this site, is coordinated with six atoms of oxygen, forming an octahedral disposition. The interatomic distance between the atoms of oxygen and the Mn2+ was found to be 2.17 A for the complex BR-Mn2+, similar to Mn2+ in solution (2.15 A). In addition, the absence of any other peak at greater distances in the Fourier-transformed spectrum indicates that neither phosphorus nor sulphur atoms are present in the second coordination shell. This suggests that this binding site is located in the protein, discarding the proximity of lipid polar headgroups.

Conformational changes in bacteriorhodopsin associated with protein-protein interactions: a functional alpha I-alpha II helix switch?

Fourier transform infrared spectra of bacteriorhodopsin samples were obtained in conditions in which the aggregation state of the protein (i.e., monomeric or trimeric) was modified by different treatments. Two approaches were followed: (1) renaturation of bacteriorhodopsin starting from bacterioopsin dissolved in SDS and (2) reconstitution of bacterioopsin in Halobacterium lipid liposomes at two different lipid/protein ratios. Concomitant with the gradual recovery of the native interactions between transmembrane helices, we observed clear and gradual changes in the infrared absorption spectra in the amide I band and also in the band at 1741 cm-1. These processes were found to be compatible with the two-state oligomerization model. The whole set of experiments shows that the band at 1665 cm-1 in the deconvoluted spectra appears only when monomers interact forming trimers, even when the lattice is not present. This implies that the trimeric organization of bacteriorhodopsin is responsible for the unique features described in the amide I of purple membrane. The spectroscopic changes detected can be attributed to changes in secondary structure compatible with the interconversion of alpha I and alpha II helices. However, the exact nature and functional relevance of these changes is still unknown.

Influence of nucleotides on the secondary structure and on the thermal stability of mitochondrial F1 visualized by infrared spectroscopy.

We have studied the secondary structure of mitochondrial F1 using infrared spectroscopy. Our results show that in the absence of added nucleotides this complex contains similar percentages of alpha-helices, beta-structures and reverse turns (30%, 28% and 31%, respectively). The influence of ADP and ATP on the different types of secondary structure was determined; when all the nucleotide-binding sites were occupied, small but reproducible changes were observed, corresponding to a decrease in beta-structure and an increase in alpha-helix and reverse turns. The effect of nucleotide binding on the thermal stability of F1 was also studied; the thermal denaturation temperature, 55 degrees C, was increased by 11 degrees C and 7 degrees C by ATP and ADP, respectively. These results indicate that nucleotide binding affects the secondary structure of F1, stabilizing the complex.

Fourier transform infrared spectroscopy indicates a major conformational rearrangement in the activation of rhodopsin.

The study of the structural differences between rhodopsin and its active form (metarhodopsin II) has been carried out by means of deconvolution analysis of infrared spectra. Deconvolution techniques allow the direct identification of the spectral changes that have occurred, which results in a significantly different view of the conformational changes occurring after activation of the receptor as compared with previous difference spectroscopy analysis. Thus, a number of changes in the bands assigned to solvent-exposed domains of the receptor are detected, indicating significant decreases in extended (beta) sequences and in reverse turns, and increases in irregular/aperiodic sequences and in helices with a non-alpha geometry, whereas there is no decrease in alpha-helices. In addition to secondary structure conversions, qualitative alterations within a given secondary structure type are detected. These are seen to occur in both reverse turns and helices. The nature of this spectral change is of great importance, since a clear alteration in the helices bundle core is detected. All these changes indicate that the rhodopsin --> metarhodopsin II transition involves not a minor but a major conformational rearrangement, reconciling the infrared data with the energetics of the activation process.