Stability of Bacteriorhodopsin α-Helices and Loops Analyzed by Single-Molecule Force Spectroscopy

Müller, Daniel J.; Kessler, Max; Oesterhelt, Filipp; Möller, Clemens; Oesterhelt, Dieter; Gaub, Hermann E.

doi:10.1016/s0006-3495(02)75358-7

Cited by 153 publications

(296 citation statements)

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“…These studies indicate that the force peak originates from intrinsic properties of each TM helix rather than the interhelix interactions. Furthermore, experimental studies (13,16,23,24) suggested that the magnitude and the appearance frequency of the force peaks of the F-D curves were affected by the interhelix interactions, whereas the peak positions were not. Kessler et al (31) performed unfolding and refolding experiments of bR by lifting the AFM tip up and down repeatedly, and observed that the peak positions of the F-D curves for the helix pairs ED and CB were always reproduced whereas the magnitudes of these peaks were sometimes reproduced and sometimes not.…”

Section: Introductionmentioning

confidence: 99%

Forced Unfolding Mechanism of Bacteriorhodopsin as Revealed by Coarse-Grained Molecular Dynamics

Yamada

Yamato

Mitaku

2016

Biophysical Journal

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Developments in atomic force microscopy have opened up a new path toward single-molecular phenomena; in particular, during the process of pulling a membrane protein out of a lipid bilayer. However, the characteristic features of the force-distance (F-D) curve of a bacteriorhodopsin in purple membrane, for instance, have not yet been fully elucidated in terms of physicochemical principles. To address the issue, we performed a computer simulation of bacteriorhodopsin with, to our knowledge, a novel coarse-grained (C-G) model. Peptide planes are represented as rigid spheres, while the surrounding environment consisting of water solvents and lipid bilayers is represented as an implicit continuum. Force-field parameters were determined on the basis of auxiliary simulations and experimental values of transfer free energy of each amino acid from water to membrane. According to Popot's two-stage model, we separated molecular interactions involving membrane proteins into two parts: I) affinity of each amino acid to the membrane and intrahelical hydrogen bonding between main chain peptide bonds; and II) interhelix interactions. Then, only part I was incorporated into the C-G model because we assumed that the part plays a dominant role in the forced unfolding process. As a result, the C-G simulation has successfully reproduced the key features, including peak positions, of the experimental F-D curves in the literature, indicating that the peak positions are essentially determined by the residue-lipid and intrahelix interactions. Furthermore, we investigated the relationships between the energy barrier formation on the forced unfolding pathways and the force peaks of the F-D curves.

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Section: Introductionmentioning

confidence: 99%

Forced Unfolding Mechanism of Bacteriorhodopsin as Revealed by Coarse-Grained Molecular Dynamics

Yamada

Yamato

Mitaku

2016

Biophysical Journal

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“…Thus, the stability of a domain in SMFS experiments is commonly described by its average unfolding force. Force peak heights and positions in the F-D trace allow for directly determining the strength and locating the position of molecular interactions within the protein and scrutinizing individual unfolding pathways of the protein (Müller et al 2002c). In some unfolding pathways, transmembrane α-helices unfold in pairs together with their connecting loop.…”

Section: Probing Molecular Interactions Of Single Membrane Proteinsmentioning

confidence: 99%

“…This suggests the presence of distinct molecular interactions establishing stable structural segments within the membrane protein. These molecular interactions are collectively formed by all aa in a segment and can be related to local structural features, such as single helical domains (Müller et al 2002c), helical kinks ), Pi-interactions (Cisneros et al 2005). It was, however, also possible to detect molecular interactions originating from protein-protein interactions (Sapra et al 2005) and ligand binding and protein activation (Kedrov et al 2005a).…”

Section: Probing Molecular Interactions Of Single Membrane Proteinsmentioning

confidence: 99%

“…Probing mechanical stability of NhaA Many of above insights on molecular interactions being established within membrane proteins have been originally revealed on bacteriorhodopsin (Müller et al 2002c;Oesterhelt et al 2000). At the same time, most of these insights could also be obtained on other membrane proteins, such as halorhodopsin from H. salinarum (Cisneros et al 2005), human aquaporin-1 from red blood cells (Moller et al 2003), bovine rhodopsin from native disc membranes (Sapra et al, submitted), or NhaA from E. coli .…”

Section: Probing Molecular Interactions Of Single Membrane Proteinsmentioning

confidence: 99%

“…Once the force externally applied by the SMFS reaches a certain critical value, all aa within these segments unfold cooperatively. Initial experiments allowed investigation into how environmental variations such as temperature changes (Janovjak et al 2003), pH variations (Müller et al 2002c;Kedrov et al 2005a), or oligomeric assembly (Sapra et al 2005;Janovjak et al 2005a) influence the strength of molecular interactions establishing stable structural segments within the protein. It has become possible to directly observe the various pathways and to determine the folding steps and kinetics of a polypeptide folding into the final antiporter.…”

Section: Introductionmentioning

confidence: 99%

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Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters

Kedrov

Müller

2006

Naunyn Schmied Arch Pharmacol

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Using the example of sodium/proton antiporter from Escherichia coli NhaA, we review the capabilities of single-molecule atomic force microscopy and force spectroscopy to observe structural and functional insights of a membrane protein, which are not attainable by other traditional methods. While atomic force microscopy provides high-resolution topographs of single membrane proteins, their oligomeric state and assembly, single-molecule force spectroscopy experiments detect molecular interactions of the protein. The sensitivity of this method makes it possible to detect and locate interactions that stabilize secondary structures such as transmembrane α-helices, polypeptide loops and segments within them. Controlled refolding experiments using single-molecule force spectroscopy observed individual secondary structure segments folding into the functional protein. Various folding pathways of NhaA were detected, each one exhibiting a certain probability to be taken. Time-lapse refolding experiments enabled determining the folding kinetics and hierarchy of individual secondary structural elements. Recent examples detected and located the ligand binding of an antiporter. Similarly, inhibitor binding and location can be detected which in future may guide towards comparative studies of agonist and antagonist altering the functional state of a membrane protein. We review current and future potentials of these approaches to characterize the action of pharmacological molecules on the antiporter function.

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Fundamentals and Applications of FluidFM Technology in Single‐Cell Studies

Saha

Duanis‐Assaf

Reches

2020

Adv Materials Inter

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various microfluidic devices have been routinely employed. However, in these approaches, there is either a high chance of damaging the cells while detaching the adherent cells from the surfaces, or isolation of individual cells from the adherent cell culture is not at all possible. Table 1 lists the drawbacks and challenges associated with these cell sorting techniques. Recently, atomic force microscope (AFM) has emerged as a significant tool to study microorganisms and mammalian cells in real time. This is due to its compatibility with the physiological conditions of the cells. [3] AFM allows the bioimaging of cells with various degrees of resolution, from whole cells [4] down to individual molecules. [5] Using force-curve based imaging, Alsteens et al. revealed single bacteriophages extruding from the surface of bacteria, [6] whereas our group was able to image nanostructures termed microvilli on the surface of T cells. [7] With the introduction of high-speed AFMs, Yamashita et al. were able to follow the diffusion of single molecules on the surface of living bacterial cells in real time. [8] Valuable biophysical and structural information can be achieved down to a single-cell level by means of high-resolution imaging and force spectroscopy. [9] In force spectroscopy experiments using AFM, a cell is directly attached to the cantilever apex with a biocompatible glue (e.g., polydopamine (PDA)). However, since the cell is irreversibly bound to the AFM probe, each functionalized cantilever can be utilized with only one cell for manipulation. It is, therefore, challenging to perform serial and rapid measurement of a reasonable number of cells; this makes it a time-consuming and elaborate process to address a statistical distribution. For injecting biomolecules such as dyes, enzymes or nanoparticles into single living cells, various approaches such as nano-fountain probes, [10] nanoneedles, [11] and carbon nanotubes [12] have been used. However, these approaches lack precise handling for local dispensing of fluid and are limited to the handling of very small volumes (on the order of attoliters (aLs) up to 50 femtoliters (fLs)). Moreover, using these approaches, a relatively long period of time is required to perform intracellular injection. With the advancement of technologies, it has become relatively easier to address these problems in a more controlled manner. Conventional AFM has been integrated with microfluidics to overcome these limitations. [13] This bioanalytical tool is known as a fluidic force microscope or FluidFM, which was This review describes the potential of FluidFM technology and its implementation in studying the interface between a single cell (prokaryote or eukaryote) and a surface or a surrounding area. A combination of microfluidics with conventional atomic force microscope (AFM) makes this platform efficient to address challenges associated with various biomolecular systems and biophysical activities down to single-cell levels. Upon regulating the pressure through a microchanneled cantilever v...

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Stability of Bacteriorhodopsin α-Helices and Loops Analyzed by Single-Molecule Force Spectroscopy

Cited by 153 publications

References 56 publications

Forced Unfolding Mechanism of Bacteriorhodopsin as Revealed by Coarse-Grained Molecular Dynamics

Forced Unfolding Mechanism of Bacteriorhodopsin as Revealed by Coarse-Grained Molecular Dynamics

Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters

Fundamentals and Applications of FluidFM Technology in Single‐Cell Studies

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