Biomedical subjects

D J Müller

Publications and source records attributed to D J Müller.

At least 55 records · Page 3Linked to original sources

PubMed1999

Charting the surfaces of the purple membrane.

The preponderance of structural data of the purple membrane from X-ray diffraction (XRD), electron crystallography (EC), and atomic force microscopy (AFM) allows us to ask questions about the structure of bacteriorhodopsin itself, as well as about the information derived from the different techniques. The transmembrane helices of bacteriorhodopsin are quite similar in both EC and XRD models. In contrast, the loops at the surfaces of the purple membrane show the highest variability between the atomic models, comparable to the height variance measured by AFM. The excellent agreement of the AFM topographs with the atomic models from XRD builds confidence in the results. Small technical difficulties in EC lead to poorer resolution of the loop structures, although the combination of atomic models with AFM surfaces allows clear interpretation of the extent and flexibility of the loop structures. While XRD remains the premier technique to determine very-high-resolution structures, EC offers a method to determine loop structures unhindered by three-dimensional crystal contacts, and AFM provides information about surface structures and their flexibility under physiological conditions.

Amino Acid Motifs↗

PubMed1999

Controlled unzipping of a bacterial surface layer with atomic force microscopy.

We have combined high-resolution atomic force microscopy (AFM) imaging and force spectroscopy to gain insight into the interaction forces between the individual protomers of the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans. After imaging the HPI layer, the AFM stylus was attached to individual protomers by enforced stylus-sample contact to allow force spectroscopy experiments. Imaging of the HPI layer after recording force-extension curves allowed adhesion forces to be correlated with structural alterations. By using this approach, individual protomers of the HPI layer were found to be removed at pulling forces of approximately 300 pN. Furthermore, it was possible to sequentially unzip entire bacterial pores formed by six HPI protomers. The combination of high-resolution AFM imaging of individual proteins with the determination of their intramolecular forces is a method of studying the mechanical stability of supramolecular structures at the level of single molecules.

Gram-Positive Cocci↗

PubMed1999

High resolution AFM topographs of the Escherichia coli water channel aquaporin Z.

Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning alpha-helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli. Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two-dimensional crystals with p42(1)2 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change.

Amino Acid Sequence↗

PubMed1999

Atomic force microscopy: a forceful way with single molecules.

The atomic force microscope (AFM) now routinely provides images that reveal subnanometer surface structures of biomolecules. The sensitivity and precision of AFM provide new opportunities for studying the mechanical properties of biomolecules and their interactions in their native environment.

Microchemistry↗

PubMed1999

Surface structures of native bacteriorhodopsin depend on the molecular packing arrangement in the membrane.

Bacteriorhodopsin is the one of the best-studied models of an ion pump. Five atomic models are now available, yet their comparison reveals differences of some loops connecting the seven transmembrane alpha-helices. In an attempt to resolve this enigma, topographs were recorded in aqueous solution with the atomic force microscope (AFM) to reveal the most native surface structure of bacteriorhodopsin molecules in the purple membrane. Individual peptide loops were observed with a lateral resolution of between 4.5 A and 5.8 A, and a vertical resolution of about 1 A. The AFM images demonstrate for the first time, that the shape, the position, and the flexibility of individual polypeptide loops depend on the packing arrangement of bacteriorhodopsin molecules in the lipid bilayer.

Bacteriorhodopsins↗

PubMed1999

Voltage and pH-induced channel closure of porin OmpF visualized by atomic force microscopy.

Gram-negative bacteria are protected by an outer membrane in which trimeric channels, the porins, facilitate the passage of small solutes. The pores are formed by membrane-spanning antiparallel beta-strands, which are connected by short turns on the periplasmic side and long loops on the extracellular side. Voltage and pH-dependent conformational changes of these extracellular loops have now been visualized by atomic force microscopy of two-dimensional crystals of Escherichia coli porin OmpF. The observed conformational changes accompany the closure of the channel entrance, and suggest that this is a mechanism that the cells have evolved to protect themselves from drastic changes of the environment.

Bacterial Proteins↗

PubMed1999

Tapping-mode atomic force microscopy produces faithful high-resolution images of protein surfaces.

Compared to contact-mode atomic force microscopy (CMAFM), tapping-mode atomic force microscopy (TMAFM) has the advantage of allowing imaging surfaces of macromolecules, even when they are only weakly attached to the support. In this study, TMAFM is applied to two different regular protein layers whose structures are known to great detail, the purple membrane from Halobacterium salinarum and the hexagonally packed intermediate (HPI) layer from Deinococcus radiodurans, to assess the faithfulness of high-resolution TMAFM images. Topographs exhibited a lateral resolution between 1.1 and 1. 5 nm and a vertical resolution of approximately 0.1 nm. For all protein surfaces, TMAFM and CMAFM topographs were in excellent agreement. TMAFM was capable of imaging the fragile polypeptide loop connecting the transmembrane alpha-helices E and F of bacteriorhodopsin in its native extended conformation. The standard deviation (SD) of averages calculated from TMAFM topographs exhibited an enhanced minimum (between 0.1 and 0.9 nm) that can be assigned to the higher noise of the raw data. However, the SD difference, indicating the flexibility of protein subunits, exhibited an excellent agreement between the two imaging modes. This demonstrates that the recently invented imaging-mode TMAFM has the ability to faithfully record high-resolution images and has sufficient sensitivity to contour individual peptide loops without detectable deformations.

Bacterial Proteins↗

PubMed1999

Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope.

To achieve high-resolution topographs of native biological macromolecules in aqueous solution with the atomic force microscope (AFM) interactions between AFM tip and sample need to be considered. Short-range forces produce the submolecular information of high-resolution topographs. In contrast, no significant high-resolution information is provided by the long-range electrostatic double-layer force. However, this force can be adjusted by pH and electrolytes to distribute the force applied to the AFM tip over a large sample area. As demonstrated on fragile biological samples, adjustment of the electrolyte solution results in a local reduction of both vertical and lateral forces between the AFM tip and proteinous substructures. Under such electrostatically balanced conditions, the deformation of the native protein is minimized and the sample surface can be reproducibly contoured at a lateral resolution of 0.6 nm.

Bacterial Proteins↗

PubMed1999

Perceptions of psychosocial adjustment to aphasia: applications of the Code--Müller Protocols.

We review research that has examined people's perceptions of likely psychosocial adjustment of aphasia. People's perceptions differ depending on whether they have aphasia themselves, are related closely to a person with aphasia, or work with the aphasic person in a professional capacity. In addition, people differ in what they perceive are the most important or relevant factors for psychosocial adjustment, and this too depends on whether they have aphasia themselves, are a relative or "significant other" in the aphasic person's life, or are a health professional working with the person with aphasia. Furthermore, there are marked differences in the way individuals predict likely psychosocial adjustment, and these perceptions can change over time. Most of this research has used the Code-Müller Protocols (CMP) to examine these perceptions. This article describes the development and application of the CMP in aphasia.

Adaptation, Psychological↗

PubMed1998

Mapping flexible protein domains at subnanometer resolution with the atomic force microscope.

The mapping of flexible protein domains with the atomic force microscope is reviewed. Examples discussed are the bacteriorhodopsin from Halobacterium salinarum, the head-tail-connector from phage phi29, and the hexagonally packed intermediate layer from Deinococcus radiodurans which all were recorded in physiological buffer solution. All three proteins undergo reversible structural changes that are reflected in standard deviation maps calculated from aligned topographs of individual protein complexes. Depending on the lateral resolution (up to 0.8 nm) flexible surface regions can ultimately be correlated with individual polypeptide loops. In addition, multivariate statistical classification revealed the major conformations of the protein surface.

Bacterial Proteins↗

PubMed1998

Surface analysis of the photosystem I complex by electron and atomic force microscopy.

Two-dimensional (2D) crystals of the photosystem I (PSI) reaction center from Synechococcus sp. OD24 were analyzed by electron and atomic force microscopy. Surface relief reconstructions from electron micrographs of freeze-dried unidirectionally shadowed samples and topographs recorded with the atomic force microscope (AFM) provided a precise definition of the lumenal and stromal PSI surfaces. The lumenal surface was composed of four protrusions that surrounded an indentation. One of the protrusions, the PsaF subunit, was often missing. Removal of the extrinsic proteins with the AFM stylus exposed the stromal side of the PSI core, whose surface structure could then be imaged at a resolution better than 1.4 nm. This interfacial surface between core and extrinsic subunits, had a pseudo-2-fold symmetry and protrusions that correlated with the surface helices e and e' or were at the sites of putative alpha-helix-connecting loops estimated from the 4 A map of the complex. The molecular dissection achieved with the AFM, opens new possibilities to unveil the interfaces between subunits of supramolecular assemblies.

Amino Acid Sequence↗

PubMed1997

The bacteriophage phi29 head-tail connector imaged at high resolution with the atomic force microscope in buffer solution.

The surfaces of two- and three-dimensional phi29 connector crystals were imaged in buffer solution by atomic force microscopy (AFM). Both topographies show a rectangular unit cell with dimensions of 16.5 nm x 16.5 nm. High resolution images of connectors from the two-dimensional crystal surface show two connectors per unit cell confirming the p42(1)2 symmetry. The height of the connector was estimated to be at least 7.6 nm, a value close to that found in previous studies using different techniques. The 12 subunits of the wide connector domain were clearly resolved and showed a right-handed vorticity. The channel running along the connector had a diameter of 3.7 nm in the wide domain, while it was 1.7 nm in the narrow domain end, thus suggesting a tronco-conical channel shape. Moreover, the narrow connector end appears to be rather flexible. When the force applied to the stylus was between 50 and 100 pN, the connector end was fully extended. At forces of approximately 150 pN, these ends were pushed towards the crystal surface. The complementation of the AFM data with the three-dimensional reconstruction obtained from electron microscopy not only confirmed the model proposed, but also offers new insights that may help to explain the role of the connector in DNA packing.

Bacillus Phages↗

PubMed1997

Adsorption of biological molecules to a solid support for scanning probe microscopy.

Scanning probe microscopes are now established tools to study the surface structure of biological macromolecules under physiological conditions. Sample preparation methods for this microscopy all have the objective to attach the specimen firmly to a support. Here we analyse the commonly used method of adsorbing biological specimens to freshly cleaved mica. This is facilitated by adjusting the electrolyte concentration and the pH of the buffer solution. Native macromolecular systems absorbed to mica in this way can be reproducibly imaged at submolecular resolution.

Adsorption↗

PubMed1997

Structural changes in native membrane proteins monitored at subnanometer resolution with the atomic force microscope: a review.

Three membrane proteins, OmpF porin from Escherichia coli, bacteriorhodopsin from Halobacterium salinarium, and the hexagonally packed intermediate (HPI) layer from Deinoccocus radiodurans, were investigated with the atomic force microscope in buffer solution. A resolution of up to 0.8 nm allowed structural differences of individual proteins to be detected. OmpF porin exhibits different static conformations on the outer surface, which possibly represent the two conductive states of the ion channels. Reversible structural changes in the cytoplasmic surface of purple membrane have been induced by changing the force applied to the scanning stylus: doughnut-shaped bacteriorhodopsin trimers transformed into a structure with three pronounced protrusions when the force was reduced from 300 to 100 pN. Furthermore, individual pores of the inner surface of the HPI layer were observed to switch from an "open" to a "closed" state. Together, the structural changes in proteins monitored under physiological conditions suggest that direct observation of function-related conformational changes of biomolecules with the atomic force microscope is feasible.

Bacterial Outer Membrane Proteins↗

PubMed1997

The height of biomolecules measured with the atomic force microscope depends on electrostatic interactions.

In biological applications of atomic force microscopy, the different surface properties of the biological sample and its support become apparent. Observed height differences between the biomolecule and its supporting surface are thus not only of structural origin, but also depend on the different sample-tip and support-tip interactions. This can result in negative or positive contributions to the measured height, effects that are described by the DLVO (Derjaguin, Landau, Verwey, Overbeek) theory. Experimental verification shows that the electrostatic interactions between tip and sample can strongly influence the result obtained. To overcome this problem, pH and electrolyte concentration of the buffer solution have to be adjusted to screen out electrostatic forces. Under these conditions, the tip comes into direct contact with the surface of support and biological system, even when low forces required to prevent sample deformation are applied. In this case, the measured height can be related to the thickness of the native biological structure. The observed height dependence of the macromolecules on electrolyte concentration makes it possible to estimate surface charge densities.

Aquaporin 1↗

PubMed1997

High resolution imaging of native biological sample surfaces using scanning probe microscopy.

The possibility of acquiring high resolution topographs using scanning probe microscopes under physiological conditions allows the observation of biomolecules at work. Progress has recently been made in imaging protein-DNA complexes, individual oligomers and protein arrays. Scanning probe microscopes are now tools that complement X-ray crystallography and electron microscopy.

Bacterial Outer Membrane Proteins↗

PubMed1997

Electron and atomic force microscopy of membrane proteins.

Electron crystallography is becoming a powerful tool for the resolution of membrane protein structures. The past year has seen the production of a bacteriorhodopsin model at 3.5 A and the structure of aquaporin 1 approaching atomic resolution. Determination of surface topographies of 2D crystals using the atomic force microscope is similarly advancing to a level that reveals submolecular details. As the latter is operated in solution, membrane proteins can be observed at work.

Aquaporin 1↗

PubMed1996

Surface topographies at subnanometer-resolution reveal asymmetry and sidedness of aquaporin-1.

Aquaporin-1 (AQP1) is an abundant protein in human erythrocyte membranes which functions as a specific and constitutively active water conducting pore. Solubilized and isolated as tetramer, it forms well-ordered two-dimensional (2D) crystals when reconstituted in the presence of lipids. Several high resolution projection maps of AQP1 have been determined, but information on its three-dimensional (3D) mass distribution is sparse. Here, we present surface reliefs at 0.9 nm resolution that were calculated from freeze-dried unidirectionally metal-shadowed AQP1 crystals as well as surface topographs recorded with the atomic force microscope of native crystals in buffer solution. Our results confirm the 3D map of negatively stained AQP1 crystals, which exhibited tetramers with four major protrusions on one side and a large central cavity on the other side of the membrane. Digestion of AQP1 crystals with carboxypeptidase Y, which cleaves off a 5 kDa intracellular C-terminal fragment, led to a reduction of the major protrusions, suggesting that the central cavity of the tetramer faces the outside of the cell. To interpret the results, sequence based structure predictions served as a guide.

Aquaporin 1↗