Nondestructive thickness measurement of biological layers at the nanoscale by simultaneous topography and capacitance imaging

Nanoscale capacitance images of purple membrane layers are obtained simultaneously to topography in a nondestructive manner by operating alternating current sensing atomic force microscopy in jumping mode. Capacitance images show excellent agreement with theoretical modeling and prove to be a noninvasive method for measuring the thickness of purple membrane layers beyond the single monolayer limit with nanoscale lateral spatial resolution. With the ability of spatially resolving the capacitance while preserving the sample from damaging, this technique can be applied for nanoscale thickness measurement of other biological layers and soft materials in general

The substitution of Arg149 with Cys fixes the melibiose transporter in an inward-open conformation

The melibiose transporter from Escherichia coli (MelB) can use the electrochemical energy of either H+, Na+ or Li+ to transport the disaccharide melibiose to the cell interior. By using spectroscopic and biochemical methods, we have analyzed the role of Arg149 by mutagenesis. According to Fourier transform infrared difference and fluorescence spectroscopy studies, R149C, R149Q and R149K all bind substrates in proteoliposomes, where the protein is disposed inside-out. Analysis of right-side-out (RSO) and inside- out (ISO) membrane vesicles showed that the functionally active R149Q and R149K mutants could bind externally added fluorescent sugar analog in both types of vesicles. In contrast, the non-transporting R149C mutant does bind the fluorescent sugar analog as well as melibiose and Na+ in ISO, but not in RSO vesicles. Therefore, the mutation of Arg149 into cysteine restrains the orientation of transporter to an inward-open conformation, with the inherent consequences of a) reducing the frequency of access of outer substrates to the binding sites, and b) impairing active transport. It is concluded that Arg149, most likely located in the inner (cytoplasmic) half of transmembrane helix 5, is critically involved in the reorientation mechanism of the substrate-binding site accessibility in MelB

Conformational changes in bacteriohodopsin associated with protein-protein interactions: a functional alfaI-alfaII helix switch?

Postprint (published version

Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems †

ABSTRACT: 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 R helices and β structure, a fact which seems to be related to their low denaturation enthalpy as compared to that of water-soluble, globular proteins. Thermodynamic-data analysis and energetic characterizatio