Transmembrane helices have rough energy surfaces.
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Membranes, Artificial↗
Biomedical subjects
Harald Janovjak
Publications and source records attributed to Harald Janovjak.
Pulling single bacteriorhodopsin out of a membrane: Comparison of simulation and experiment.
Mechanical unfolding of single bacteriorhodopsins from a membrane bilayer is studied using molecular dynamics simulations. The initial conformation of the lipid membrane is determined through all-atom simulations and then its coarse-grained representation is used in the studies of stretching. A Go-like model with a realistic contact map and with Lennard-Jones contact interactions is applied to model the protein-membrane system. The model qualitatively reproduces the experimentally observed differences between force-extension patterns obtained on bacteriorhodopsin at different temperatures and predicts a lack of symmetry in the choice of the terminus to pull by. It also illustrates the decisive role of the interactions of the protein with the membrane in determining the force pattern and thus the stability of transmembrane proteins.
Bacterial Proteins↗
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↗
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↗
Direct measurement of single-molecule visco-elasticity in atomic force microscope force-extension experiments.
Measuring the visco-elastic properties of biological macromolecules constitutes an important step towards the understanding of dynamic biological processes, such as cell adhesion, muscle function, or plant cell wall stability. Force spectroscopy techniques based on the atomic force microscope (AFM) are increasingly used to study the complex visco-elastic response of (bio-)molecules on a single-molecule level. These experiments either require that the AFM cantilever is actively oscillated or that the molecule is clamped at constant force to monitor thermal cantilever motion. Here we demonstrate that the visco-elasticity of single bio-molecules can readily be extracted from the Brownian cantilever motion during conventional force-extension measurements. It is shown that the characteristics of the cantilever determine the signal-to-noise (S/N) ratio and time resolution. Using a small cantilever, the visco-elastic properties of single dextran molecules were resolved with a time resolution of 8.3 ms. The presented approach can be directly applied to probe the dynamic response of complex bio-molecular systems or proteins in force-extension experiments.
Data Interpretation, Statistical↗
Complex stability of single proteins explored by forced unfolding experiments.
In the last decade atomic force microscopy has been used to measure the mechanical stability of single proteins. These force spectroscopy experiments have shown that many water-soluble and membrane proteins unfold via one or more intermediates. Recently, Li and co-workers found a linear correlation between the unfolding force of the native state and the intermediate in fibronectin, which they suggested indicated the presence of a molecular memory or multiple unfolding pathways (1). Here, we apply two independent methods in combination with Monte Carlo simulations to analyze the unfolding of alpha-helices E and D of bacteriorhodopsin (BR). We show that correlation analysis of unfolding forces is very sensitive to errors in force calibration of the instrument. In contrast, a comparison of relative forces provides a robust measure for the stability of unfolding intermediates. The proposed approach detects three energetically different states of alpha-helices E and D in trimeric BR. These states are not observed for monomeric BR and indicate that substantial information is hidden in forced unfolding experiments of single proteins.
Bacteriorhodopsins↗
Molecular force modulation spectroscopy revealing the dynamic response of single bacteriorhodopsins.
Recent advances in atomic force microscopy allowed globular and membrane proteins to be mechanically unfolded on a single-molecule level. Presented is an extension to the existing force spectroscopy experiments. While unfolding single bacteriorhodopsins from native purple membranes, small oscillation amplitudes (6-9 nm) were supplied to the vertical displacement of the cantilever at a frequency of 3 kHz. The phase and amplitude response of the cantilever-protein system was converted to reveal the elastic (conservative) and viscous (dissipative) contributions to the unfolding process. The elastic response (stiffness) of the extended parts of the protein were in the range of a few tens pN/nm and could be well described by the derivative of the wormlike chain model. Discrete events in the viscous response coincided with the unfolding of single secondary structure elements and were in the range of 1 microNs/m. In addition, these force modulation spectroscopy experiments revealed novel mechanical unfolding intermediates of bacteriorhodopsin. We found that kinks result in a loss of unfolding cooperativity in transmembrane helices. Reconstructing force-distance spectra by the integration of amplitude-distance spectra verified their position, offering a novel approach to detect intermediates during the forced unfolding of single proteins.
Bacteriorhodopsins↗
Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy.
Single-molecule force-spectroscopy was employed to unfold and refold single sodium-proton antiporters (NhaA) of Escherichia coli from membrane patches. Although transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, two helices predominantly unfolded pairwise, thereby acting as one structural unit. Many of the potential barriers were detected unfolding NhaA either from the C-terminal or the N-terminal end. It was found that some molecular interactions stabilizing secondary structural elements were directional, while others were not. Additionally, some interactions appeared to occur between the secondary structural elements. After unfolding ten of the 12 helices, the extracted polypeptide was allowed to refold back into the membrane. After five seconds, the refolded polypeptide established all secondary structure elements of the native protein. One helical pair showed a characteristic spring like "snap in" into its folded conformation, while the refolding process of other helices was not detected in particular. Additionally, individual helices required characteristic periods of time to fold. Correlating these results with the primary structure of NhaA allowed us to obtain the first insights into how potential barriers establish and determine the folding kinetics of the secondary structure elements.
Amino Acid Sequence↗
Hydrodynamic effects in fast AFM single-molecule force measurements.
Atomic force microscopy (AFM) allows the critical forces that unfold single proteins and rupture individual receptor-ligand bonds to be measured. To derive the shape of the energy landscape, the dynamic strength of the system is probed at different force loading rates. This is usually achieved by varying the pulling speed between a few nm/s and a few microm/s, although for a more complete investigation of the kinetic properties higher speeds are desirable. Above 10 microm/s, the hydrodynamic drag force acting on the AFM cantilever reaches the same order of magnitude as the molecular forces. This has limited the maximum pulling speed in AFM single-molecule force spectroscopy experiments. Here, we present an approach for considering these hydrodynamic effects, thereby allowing a correct evaluation of AFM force measurements recorded over an extended range of pulling speeds (and thus loading rates). To support and illustrate our theoretical considerations, we experimentally evaluated the mechanical unfolding of a multi-domain protein recorded at 30 microm/s pulling speed.
Algorithms↗
Probing the energy landscape of the membrane protein bacteriorhodopsin.
The folding and stability of transmembrane proteins is a fundamental and unsolved biological problem. Here, single bacteriorhodopsin molecules were mechanically unfolded from native purple membranes using atomic force microscopy and force spectroscopy. The energy landscape of individual transmembrane alpha helices and polypeptide loops was mapped by monitoring the pulling speed dependence of the unfolding forces and applying Monte Carlo simulations. Single helices formed independently stable units stabilized by a single potential barrier. Mechanical unfolding of the helices was triggered by 3.9-7.7 A extension, while natural unfolding rates were of the order of 10(-3) s(-1). Besides acting as individually stable units, helices associated pairwise, establishing a collective potential barrier. The unfolding pathways of individual proteins reflect distinct pulling speed-dependent unfolding routes in their energy landscapes. These observations support the two-stage model of membrane protein folding in which alpha helices insert into the membrane as stable units and then assemble into the functional protein.
Bacteriorhodopsins↗
Unfolding pathways of native bacteriorhodopsin depend on temperature.
The combination of high-resolution atomic force microscopy (AFM) imaging and single-molecule force-spectroscopy was employed to unfold single bacteriorhodopsins (BR) from native purple membrane patches at various physiologically relevant temperatures. The unfolding spectra reveal detailed insight into the stability of individual structural elements of BR against mechanical unfolding. Intermittent states in the unfolding process are associated with the stepwise unfolding of alpha-helices, whereas other states are associated with the unfolding of polypeptide loops connecting the alpha-helices. It was found that the unfolding forces of the secondary structures considerably decreased upon increasing the temperature from 8 to 52 degrees C. Associated with this effect, the probability of individual unfolding pathways of BR was significantly influenced by the temperature. At lower temperatures, transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, whereas they predominantly unfolded collectively at elevated temperatures. This suggests that increasing the temperature decreases the mechanical stability of secondary structural elements and changes molecular interactions between secondary structures, thereby forcing them to act as grouped structures.
Bacteriorhodopsins↗
Processing of gene expression data generated by quantitative real-time RT-PCR.
Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.
Computational Biology↗
Observing structure, function and assembly of single proteins by AFM.
Single molecule experiments provide insight into the individuality of biological macromolecules, their unique function, reaction pathways, trajectories and molecular interactions. The exceptional signal-to-noise ratio of the atomic force microscope allows individual proteins to be imaged under physiologically relevant conditions at a lateral resolution of 0.5-1nm and a vertical resolution of 0.1-0.2nm. Recently, it has become possible to observe single molecule events using this technique. This capability is reviewed on various water-soluble and membrane proteins. Examples of the observation of function, variability, and assembly of single proteins are discussed. Statistical analysis is important to extend conclusions derived from single molecule experiments to protein species. Such approaches allow the classification of protein conformations and movements. Recent developments of probe microscopy techniques allow simultaneous measurement of multiple signals on individual macromolecules, and greatly extend the range of experiments possible for probing biological systems at the molecular level. Biologists exploring molecular mechanisms will benefit from a burgeoning of scanning probe microscopes and of their future combination with molecular biological experiments.
Biophysical Phenomena↗