Oriented Membrane Protein Reconstitution into Tethered Lipid Membranes for AFM Force Spectroscopy.

Membrane proteins act as a central interface between the extracellular environment and the intracellular response and as such represent one of the most important classes of drug targets. The characterization of the molecular properties of integral membrane proteins, such as topology and interdomain interaction, is key to a fundamental understanding of their function. Atomic force microscopy (AFM) and force spectroscopy have the intrinsic capabilities of investigating these properties in a near-native setting. However, atomic force spectroscopy of membrane proteins is traditionally carried out in a crystalline setup. Alternatively, model membrane systems, such as tethered bilayer membranes, have been developed for surface-dependent techniques. While these setups can provide a more native environment, data analysis may be complicated by the normally found statistical orientation of the reconstituted protein in the model membrane. We have developed a model membrane system that enables the study of membrane proteins in a defined orientation by single-molecule force spectroscopy. Our approach is demonstrated using cell-free expressed bacteriorhodopsin coupled to a quartz glass surface in a defined orientation through a protein anchor and reconstituted inside an artificial membrane system. This approach offers an effective way to study membrane proteins in a planar lipid bilayer. It can be easily transferred to all membrane proteins that possess a suitable tag and can be reconstituted into a lipid bilayer. In this respect, we anticipate that this technique may contribute important information on structure, topology, and intra- and intermolecular interactions of other seven-transmembrane helical receptors.

Cooperative binding of PhoB(DBD) to its cognate DNA sequence-a combined application of single-molecule and ensemble methods.

A combined approach based on isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) experiments, circular dichroism spectroscopy (CD), atomic force microscopy (AFM) dynamic force spectroscopy (DFS), and surface plasmon resonance (SPR) was applied to elucidate the mechanism of protein-DNA complex formation and the impact of protein dimerization of the DNA-binding domain of PhoB (PhoB(DBD)). These insights can be translated to related members of the family of winged helix-turn-helix proteins. One central question was the assembly of the trimeric complex formed by two molecules of PhoB(DBD) and two cognate binding sites of a single oligonucleotide. In addition to the native protein WT-PhoB(DBD), semisynthetic covalently linked dimers with different linker lengths were studied. The ITC, SPR, FRET, and CD results indicate a positive cooperative binding mechanism and a decisive contribution of dimerization on the complex stability. Furthermore, an alanine scan was performed and binding of the corresponding point mutants was analyzed by both techniques to discriminate between different binding types involved in the protein-DNA interaction and to compare the information content of the two methods DFS and SPR. In light of the published crystal structure, four types of contribution to the recognition process of the pho box by the protein PhoB(DBD) could be differentiated and quantified. Consequently, it could be shown that investigating the interactions between DNA and proteins with complementary techniques is necessary to fully understand the corresponding recognition process.

Membrane protein stability depends on the concentration of compatible solutes--a single molecule force spectroscopic study.

Compatible solutes are small, uncharged, zwitter ionic, osmotically active molecules produced and accumulated by microorganisms inside their cell to counteract different kinds of environmental stress. They enhance protein stability without interfering with the metabolic pathways even at molar concentrations. In this paper, we report the stabilizing effects of compatible solutes, ectoine, betaine and taurine on membrane protein bacteriorhodopsin at different concentrations. Using atomic force microscopy based single molecule force spectroscopy the impact of the osmolytes was quantified by measuring the forces required to pull the protein out of the membrane and the change in the persistence lengths of the unfolded polypeptide chain. Increase in unfolding forces were observed, indicating the strengthening of intramolecular interactions, which are vital for protein stability. The decrease in persistence lengths was recorded and showed increasing tendencies of the polypeptide strand to coil up. Interestingly, it was revealed that these molecules have different stabilizing effects on protein unfolding at different concentrations. The results show that the unfolding of single protein provides insight to the structure-dynamic relationship between the protein and compatible solute molecules at sub-nanometer scale. This also helps to understand the molecular mechanism involved in protein stabilization by organic osmolytes.