Transmembrane helices have rough energy surfaces.
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Membranes, Artificial↗
Biomedical subjects
Daniel J Muller
Publications and source records attributed to Daniel J Muller.
Aminosulfonate modulated pH-induced conformational changes in connexin26 hemichannels.
Gap junction channels regulate cell-cell communication by passing metabolites, ions, and signaling molecules. Gap junction channel closure in cells by acidification is well documented; however, it is unknown whether acidification affects connexins or modulating proteins or compounds that in turn act on connexins. Protonated aminosulfonates directly inhibit connexin channel activity in an isoform-specific manner as shown in previously published studies. High-resolution atomic force microscopy of force-dissected connexin26 gap junctions revealed that in HEPES buffer, the pore was closed at pH < 6.5 and opened reversibly by increasing the pH to 7.6. This pH effect was not observed in non-aminosulfonate buffers. Increasing the protonated HEPES concentration did not close the pore, indicating that a saturation of the binding sites occurs at 10 mM HEPES. Analysis of the extracellular surface topographs reveals that the pore diameter increases gradually with pH. The outer connexon diameter remains unchanged, and there is a approximately 6.5 degrees rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca2+-induced closure.
Calcium↗
Out and in: simplifying membrane protein studies by AFM.
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Elasticity↗
Differentiating ligand and inhibitor interactions of a single antiporter.
Regulatory mechanisms of ion and solute transporters are in focus of biomedical and biochemical studies and build a key for disease therapies. Inhibition of sodium/proton exchangers efficiently prevents ischemic heart disease and reperfusion development in humans, but molecular mechanisms behind are not clear. Using single-molecule force spectroscopy we observe the binding of the inhibitor 2-aminoperimidine (AP) to sodium/proton antiporters NhaA from Escherichia coli. Deactivating interactions were significantly suppressed at enhanced sodium concentrations of 200 mM as well as in the pH-locked inactive conformation of NhaA. New molecular interactions were quantified and localized within the protein occurring upon a competitive inhibitor binding. The inhibitor, which was targeted and bound to the ligand-binding pocket, altered interactions established at alpha-helix IX. These molecular mechanisms deactivating the antiporter were different to those established upon ligand binding and activation of NhaA.
Binding Sites↗
An approach to prepare membrane proteins for single-molecule imaging.
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Cell Membrane↗
A new technical approach to quantify cell-cell adhesion forces by AFM.
Cell-cell adhesion is a complex process that is involved in the tethering of cells, cell-cell communication, tissue formation, cell migration and the development and metastasis of tumors. Given the heterogeneous and complex nature of cell surfaces it has previously proved difficult to characterize individual cell-cell adhesion events. Force spectroscopy, using an atomic force microscope, is capable of resolving such individual cell-cell binding events, but has previously been limited in its application due to insufficient effective pulling distances. Extended pulling range is critical in studying cell-cell interactions due to the potential for large cell deformations. Here we describe an approach to such experiments, where the sample stage can be moved 100 microm in the z-direction, by closed loop, linearized piezo elements. Such an approach enables an increase in pulling distance sufficient for the observation of long-distance cell-unbinding events without reducing the imaging capabilities of the atomic force microscope. The atomic force microscope head and the piezo-driven sample stage are installed on an inverted optical microscope fitted with a piezo-driven objective, to allow the monitoring of cell morphology by conventional light microscopy, concomitant with force spectroscopy measurements. We have used the example of the WM115 melanoma cell line binding to human umbilical vein endothelial cells to demonstrate the capabilities of this system and the necessity for such an extended pulling range when quantifying cell-cell adhesion events.
Cell Adhesion↗
Analysis assistant for single-molecule force spectroscopy data on membrane proteins--MPTV.
UNLABELLED: Force-distance (F-D) curves of single membrane proteins reveal information on inter- and intramolecular interactions occurring within a protein and between proteins. However, the analysis of single-molecule force spectroscopy data is a time consuming and complex process requiring objective criteria. In most cases the user requires additional information to interpret F-D curves. Therefore we developed a software assistant representing the force or molecular interaction pattern and the topology or the 3D structure of the membrane protein. This representation establishes a basis for detailed interpretation of the protein structure and its underlying molecular interactions. Various integrated bioinformatic features further assist in the interpretation of measured and assigned molecular interactions that determine membrane protein folding, structure, stability and function. Web queries and programs about the topology are directly linked. Motifs, helix types, representation of Venn diagrams and the complete functionality of the program Jmol belong to it. AVAILABILITY: The program MPTV is freely available from the website at http://www.bioforscher.de/mptv.htm/.
Algorithms↗
Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy.
Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.
Animals↗
Bacteriorhodopsin folds into the membrane against an external force.
Despite their crucial importance for cellular function, little is known about the folding mechanisms of membrane proteins. Recently details of the folding energy landscape were elucidated by atomic force microscope (AFM)-based single molecule force spectroscopy. Upon unfolding and extraction of individual membrane proteins energy barriers in structural elements such as loops and helices were mapped and quantified with the precision of a few amino acids. Here we report on the next logical step: controlled refolding of single proteins into the membrane. First individual bacteriorhodopsin monomers were partially unfolded and extracted from the purple membrane by pulling at the C-terminal end with an AFM tip. Then by gradually lowering the tip, the protein was allowed to refold into the membrane while the folding force was recorded. We discovered that upon refolding certain helices are pulled into the membrane against a sizable external force of several tens of picoNewton. From the mechanical work, which the helix performs on the AFM cantilever, we derive an upper limit for the Gibbs free folding energy. Subsequent unfolding allowed us to analyze the pattern of unfolding barriers and corroborate that the protein had refolded into the native state.
Bacteriorhodopsins↗
Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy.
Using single-molecule force spectroscopy we characterized inter- and intramolecular interactions stabilizing structural segments of individual bacteriorhodopsin (BR) molecules assembled into trimers and dimers, and monomers. While the assembly of BR did not vary the location of these structural segments, their intrinsic stability could change up to 70% increasing from monomer to dimer to trimer. Since each stable structural segment established one unfolding barrier, we conclude that the locations of unfolding barriers were determined by intramolecular interactions but that their strengths were strongly influenced by intermolecular interactions. Subtracting the unfolding forces of the BR trimer from that of monomer allowed us to calculate the contribution of inter- and intramolecular interactions to the membrane protein stabilization. Statistical analyses showed that the unfolding pathways of differently assembled BR molecules did not differ in their appearance but in their population. This suggests that in our experiments the membrane protein assembly does not necessarily change the location of unfolding barriers within the protein, but certainly their strengths, and thus alters the probability of a protein to choose certain unfolding pathways.
Bacteriorhodopsins↗
Observing folding pathways and kinetics of a single sodium-proton antiporter from Escherichia coli.
Mechanisms of folding and misfolding of membrane proteins are of interest in cell biology. Recently, we have established single-molecule force spectroscopy to observe directly the stepwise folding of the Na+/H+ antiporter NhaA from Escherichia coli in vitro. Here, we improved this approach significantly to track the folding intermediates of a single NhaA polypeptide forming structural segments such as the Na+-binding site, transmembrane alpha-helices, and helical pairs. The folding rates of structural segments ranged from 0.31 s(-1) to 47 s(-1), providing detailed insight into a distinct folding hierarchy of an unfolded polypeptide into the native membrane protein structure. In some cases, however, the folding chain formed stable and kinetically trapped non-native structures, which could be assigned to misfolding events of the antiporter.
Binding Sites↗
Imaging and detecting molecular interactions of single transmembrane proteins.
Single-molecule atomic force microscopy (AFM) provides novel ways to characterize structure-function relationships of native membrane proteins. High-resolution AFM-topographs allow observing substructures of single membrane proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. Complementary to AFM imaging, single-molecule force spectroscopy experiments allow detecting molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to detect the interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the position of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes. We review current and future potential of these approaches to reveal insights into membrane protein structure, function, and unfolding as we recognize that they could help answering key questions in the molecular basis of certain neuro-pathological dysfunctions.
Animals↗
Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy.
During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.
Animals↗
Locating ligand binding and activation of a single antiporter.
Single-molecule force spectroscopy was applied to unfold individual Na(+)/H(+) antiporters NhaA from membrane patches. The force-extension curves contained detailed information about the strength and location of molecular interactions established within NhaA. Although molecular interactions that stabilize secondary structure elements remained unaffected on switching NhaA into its functional state, those that are assigned to the Na(+)-binding site changed markedly. These interactions were formed only in the presence of Na(+), with their full strength being established at pH approximately 6. This finding is in apparent contrast to measurements that suggest that NhaA is fully active at pH 7. Statistical analysis, however, showed that not all NhaA molecules activated this molecular interaction at pH 6, but at pH 7. This implies that the molecular interactions established on Na(+) binding may represent an early step in NhaA activation. The direct observation of molecular interactions established within an antiporter provides new insights into their activation mechanisms.
Amino Acid Sequence↗
The c15 ring of the Spirulina platensis F-ATP synthase: F1/F0 symmetry mismatch is not obligatory.
The oligomeric c ring of the F-ATP synthase from the alkaliphilic cyanobacterium Spirulina platensis was isolated and characterized. Mass spectroscopy analysis indicated a mass of 8,210 Da, reflecting that of a c monomer. The mass increased by 206 Da after treatment with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD), which indicated modification of the ion-binding carboxylate by DCCD. Atomic force microscopy topographs of c rings from S. platensis showed 15 symmetrically assembled subunits. The c15-mer reported here is the largest c ring that is isolated and does not show the classical c-ring mismatch to the three-fold symmetry of the F1 domain.
Crystallization↗
Structural evidence for a constant c11 ring stoichiometry in the sodium F-ATP synthase.
The Na+-dependent F-ATP synthases of Ilyobacter tartaricus and Propionigenium modestum contain membrane-embedded ring-shaped c subunit assemblies with a stoichiometry of 11. Subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corresponding gene, extracted from the membrane using detergent and then purified. Subsequent analyses by SDS/PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the majority migrated as monomers. The population of rings consisted mainly of c11, but more slowly migrating assemblies were also found, which might reflect other c ring stoichiometries. We show that they consisted of higher aggregates of homogeneous c11 rings and/or assemblies of c11 rings and single c monomers. Atomic force microscopy topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters and that stoichiometries throughout all rings resolved at high resolution. This finding did not depend on whether the rings were assembled into crystalline or densely packed assemblies. Most of these rings represented completely assembled undecameric complexes. Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed. The latter rings may represent the aggregates between c11 and c1, as observed by SDS/PAGE. Our results are congruent with a stable c11 ring stoichiometry that seems to not be influenced by the expression level of subunit c in the bacteria.
Electrophoresis, Polyacrylamide Gel↗
Evidence of association between smoking and alpha7 nicotinic receptor subunit gene in schizophrenia patients.
A number of studies have suggested that the alpha-7-nicotinic receptor D15S1360 polymorphism is associated with schizophrenia and a deficiency in the normal inhibition of the P50 auditory-evoked response. Schizophrenia patients and some of their unaffected relatives show a failure of inhibition in their 50-ms response to the second of a pair of tones. Biochemical studies have suggested that the alpha7 nicotinic acetylcholine receptor is involved in this sensory gating deficit. Furthermore, high-dose nicotine transiently normalizes the abnormality in P50 inhibition in schizophrenic patients and in their relatives. Schizophrenic patients are unusually heavy smokers, and up to 85% of hospitalized schizophrenic patients smoke. This rate is three times higher than the general population, and may represent an attempt to self-medicate through this pathophysiologic mechanism. Despite schizophrenics' extremely heavy nicotine use, nicotinic receptor genes have not been previously investigated in relation to smoking in schizophrenia. In this study, we hypothesized that the D15S1360 marker is associated with smoking in patients with schizophrenia. We found an association between the homozygous 113 bp allele and smoking risk (chi2=10.37, 3 df, p=0.015). Although this novel finding requires replication, it suggests that further study into the relationship between schizophrenia and nicotine system genes is warranted.
Adult↗