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H J Butt

Publications and source records attributed to H J Butt.

5 recordsLinked to original sources
PubMed1990

Imaging the membrane protein bacteriorhodopsin with the atomic force microscope.

The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.

Adsorption↗
PubMed1989

Aspartic acids 96 and 85 play a central role in the function of bacteriorhodopsin as a proton pump.

A spectroscopic and functional analysis of two point-mutated bacteriorhodopsins (BRs) from phototrophic negative halobacterial strains is reported. Bacteriorhodopsin from strain 384 contains a glutamic acid instead of an aspartic acid at position 85 and BR from strain 326 contains asparagine instead of aspartic acid at position 96. Compared to wild-type BR, the M formation in BR Asp85---Glu is accwelerated approximately 10-fold, whereas the M decay in BR Asp96---Asn is slowed down approximately 50-fold at pH6. Purple membrane sheets containing the mutated BRs were oriented and immobilized in polyacrylamide gels or adsorbed to planar lipid films. The measured kinetics of the photocurrents under various conditions agree with the observed photocycle kinetics. The ineffectivity of BR Asp85---Glu resides in the dominance of an inactive species absorbing maximally at approximately 610 nm, while BR Asp96---Asn is ineffective due to its slow photocycle. These experimental results suggest that aspartic acid 96 plays a crucial role for the reprotonation of the Schiff base. Both residues are essential for an effective proton pump.

Amino Acid Sequence↗
PubMed1989

A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide.

Addition of azide fully restored the proton pump activity of defective bacteriorhodopsin (BR) mutant protein Asp96----Asn. The decay time of M of BR Asp96----Asn, the longest living intermediate, was decreased from 500 ms at pH 7.0 to approximately 1 ms under conditions of saturating azide concentrations. This decay was faster than the decay of M in the wild-type, where no such azide effect was detectable. Stationary photocurrents, measured with purple membranes immobilized and oriented in a polyacrylamide gel, increased upon addition of azide up to the level of the wild-type. Different small anions of weak acids restored the pump activity with decreasing affinity in the order: cyanate greater than azide greater than nitrite greater than formiate greater than acetate. The activation energy of the M decay in the mutant was higher in the presence (48 kJ/mol) than in the absence (27 kJ/mol) of 100 mM azide even though the absolute rate was dramatically increased by azide. This effect of azide is due to the substitution of a carboxamido group for a carboxylic group at position 96 which removes the internal proton donor and causes an increase in the entropy change of activation for proton transfer which is reversed by azide.

Anions↗
PubMedDate not supplied

Imaging cells with the atomic force microscope.

Different types of cells have been imaged with the atomic force microscope. The morphology of the archaebacterium Halobacterium halobium in its dry state was revealed. On a leaf of the small Indian tree Lagerstroemia subcostata a stoma was imaged. The lower side of a water lily leaf was imaged in water showing features down to 12 nm. Finally, fixed red and white blood cells were imaged in buffer showing features down to 8 nm. The images demonstrate that atomic force microscopy can provide high-resolution images of cell surfaces under physiological conditions.

Animals↗